Polymeric micelle nano-carrier for targeted epidermal delivery of hedgehog pathway inhibitor TAK-441

Abstract

Micellar compositions comprising hedgehog pathway inhibitors and their use in the treatment of skin diseases, skin conditions or skin disorders such as skin cancer, including basal cell cancer, are disclosed.

Description

[0001] background

[0002] Basal cell carcinoma is one of the most common cancers in the world, accounting for approximately 90% of all skin cancers, with an incidence of 100 / 100,000 in the UK and 884 / 100,000 in Australia (Madan et al., 2010; Staples et al., 2006). The main causative agent is UV light (Couvé-Privat et al., 2002; Daya-Grosjean and Sarasin, 2000). UV-B damage causes C to T (or CC to TT) structural mutations in the DNA of epidermal basal cells (Athar et al., 2006).

[0003] The hedgehog (HH) signaling pathway is significantly involved in the progression of BCC. It is highly active during embryonic development but is inactivated in most adult tissues, except for maintaining stem cell populations and regulating the growth of hair follicles and sebaceous glands (Athar et al., 2006). However, mutations in patch protein 1 (PTCH1) and smoothened protein (SMO) lead to loss of function of PTCH1 or gain of function of SMO. These functional changes can lead to activation of the GLI family of transcription factors (Dlugosz et al., 2012), respectively, leading to the hyperproliferation of basal cells observed in BCC (Roewert-Huber et al., 2007; Samarasinghe and Madan, 2012). Inactivation of PTCH1 has been proposed to be a necessary step in the progression of BCC (Gailani and Bale, 1997).

[0004] Patients with locally advanced BCC are not eligible for surgery or radiation therapy (Gould et al., 2014). Therefore, drug therapies involving inhibition of SMO and thus preventing activation of the HH signaling pathway have been developed. Vismodegib is a "first-in-class" SMO synthesis inhibitor (Robarge et al., 2009; Gould et al., 2014), which was approved by the U.S. Food and Drug Administration (FDA) in 2012 for the treatment of metastatic BCC or locally advanced BCC (Dlugosz et al., 2012). Sonidegib is another hedgehog inhibitor approved by the FDA in 2015 for the treatment of locally advanced BCC (Burness, 2015).

[0005] However, mutations in SMO can inhibit its interaction with such drugs, leading to resistance to treatment. Even before vismodegib was approved by the FDA in 2012, the first case of acquired resistance to vismodegib treatment due to a mutation in SMO (SMO-D473H) was reported in 2009 (Yauch et al., 2009). Treatment with sonidegib was ineffective in patients resistant to vismodegib (Jain et al., 2017). In addition, resistance to treatment with sonidegib has also been reported due to mutations in the drug binding sites of SMO (SMO-Q476 and SMO-D473) (Danial et al., 2016; Jain et al., 2017; Nguyen and Cho, 2022).

[0006] Overview

[0007] In some aspects, the presently disclosed subject matter provides a composition comprising a hedgehog pathway inhibitor and a polymeric surfactant. In certain aspects, the hedgehog pathway inhibitor is active against the vismodegib-resistant smoothin receptor D473H mutant.

[0008] In certain aspects, the Hedgehog pathway inhibitor is selected from TAK-441, Vismodegib, Saridegib / Patidegib, Glasdegib, Sonidegib, Taladegib (Env-101), and BMS-833923 (XL-139). In specific aspects, the Hedgehog pathway inhibitor comprises TAK-441.

[0009] In some aspects, the polymeric surfactant is biocompatible and / or biodegradable. In some aspects, the polymeric surfactant is selected from D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), mPEG-dihex-PLA, poloxamer, poly (ε-caprolactone), poly (L-amino acid) and polyvalerolactone. In a particular aspect, the polymeric surfactant comprises D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS).

[0010] In certain aspects, the composition comprising a hedgehog pathway inhibitor and a polymeric surfactant comprises a micellar composition. In certain aspects, the micellar composition comprises spherical micelles having a diameter in the range of from about 10 nm to about 100 nm. In specific aspects, the micellar composition comprises spherical micelles having a diameter in the range of from about 10 nm to about 15 nm.

[0011] In certain aspects, the composition comprises TPGS having a concentration ranging from about 5 mg / mL to about 300 mg / mL. In a specific aspect, the composition comprises TPGS at a concentration of about 10 mg / mL.

[0012] In certain aspects, the composition comprises TAK-441 having TAK-441 in a range from about 100 mg to about 500 mg per gram of TPGS. In specific aspects, the concentration of TAK-441 has TAK-441 in a range from about 100 mg to about 300 mg per gram of TPGS.

[0013] In other aspects, the composition also comprises a hydrogel. In certain aspects, the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), a cellulose-based gel forming agent and a poloxamer-based gelling agent. In a specific aspect, the hydrogel is selected from HPC and HPMC.

[0014] In a particular aspect, the HPMC is selected from: (a) HPMC having a molecular weight of about 26 kDa, having a methoxy content ranging between about 19% to about 24%, and having a hydroxypropoxy content ranging between about 7% to about 12%; (b) HPMC having a molecular weight of about 10 kDa, having a methoxy content ranging between about 28% to about 30%, and having a hydroxypropoxy content between about 7% to about 12%; and (c) combinations thereof.

[0015] In a more specific aspect, 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, having a methoxyl content ranging between about 19% to about 24%, and having a hydroxypropoxyl content ranging between about 7% to about 12%; and about 3% HPMC having a molecular weight of about 10 kDa, having a methoxyl content ranging between about 28% to about 30%, and having a hydroxypropoxyl content between about 7% to about 12%.

[0016] In certain aspects, the composition further comprises a rheology modifier. In certain aspects, the rheology modifier is selected from glycerol, low molecular weight cellulose and high molecular weight cellulose, and sorbitol. In a specific aspect, the rheology modifier comprises glycerol.

[0017] In some aspects, the composition further comprises a preservative. In some aspects, the preservative is selected from sodium metabisulfite, benzyl alcohol, benzalkonium chloride, chlorobutanol, sodium benzoate, potassium sorbate, methylparaben and propylparaben. In a specific aspect, the preservative comprises sodium metabisulfite.

[0018] In certain aspects, the composition retains between about 90% and 100% of the Hedgehog pathway inhibitor content after storage for about 6 months.

[0019] In other aspects, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway, the method comprising administering the presently disclosed composition described above to a subject in need of corresponding treatment.

[0020] In certain aspects, the composition is topically applied. In specific aspects, the composition is delivered dermally. In specific aspects, the composition is delivered to the viable epidermis of a subject. In specific aspects, the composition is delivered to the upper dermis of a subject. In specific aspects, topical application results in negligible percutaneous penetration.

[0021] In certain aspects, a disease, disorder, or condition associated with a Hedgehog (HH) signaling pathway comprises a skin disease, skin disorder, or skin condition. In a specific aspect, a skin disease, skin disorder, or skin condition comprises skin cancer. In a more specific aspect, skin cancer comprises basal cell carcinoma. In yet more specific aspects, the subject suffers from or is suspected of having locally advanced basal cell carcinoma. In yet more specific aspects, the subject suffers from or is suspected of having metastatic basal cell carcinoma. In certain aspects, the subject is not eligible for surgical treatment or radiotherapeutic treatment. In more certain aspects, administering the composition to the subject reduces the progression of basal cell carcinoma. In a specific aspect, the basal cell carcinoma involves the vismodegib-resistant SMO mutant D473H.

[0022] In certain aspects, administering the composition to a subject prevents activation of the HH signaling pathway.

[0023] In certain aspects, the disease, condition or disorder involves a mutation in patch protein 1 (PTCH1). In a specific aspect, the mutation in patch protein 1 (PTCH1) results in a loss of function of the PTCH1 protein. In certain aspects, the disease, condition or disorder involves a mutation in smoothened protein (SMO). In a specific aspect, the mutation in smoothened protein (SMO) involves a gain of function of the SMO protein. In a more specific aspect, 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 yet a more specific aspect, the activation of one or more GLI transcription factors results in an overproliferation of basal cells associated with basal cell carcinoma.

[0024] In certain aspects, administering a composition to a subject inhibits SMO. In certain aspects, mutations in SMO result in resistance to treatment with a hedgehog pathway inhibitor. In specific aspects, resistance to treatment with a hedgehog pathway inhibitor involves mutations in a drug binding site of SMO. In more specific aspects, the drug binding site of SMO is SMO-Q476 and / or SMO-D473.

[0025] Having set forth above certain aspects of the presently disclosed subject matter, which in whole or in part are presented by the presently disclosed subject matter, other aspects will become apparent as the description proceeds when considered in conjunction with the accompanying embodiments and drawings which are best described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0028] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and in which:

[0029] Figure 1 The chemical structure of TAK-441 is shown (MW 576.57 Da; log P 2.61; aqueous solubility 81 μg / mL at pH 6.8) (Ohashi et al., 2012; Ishii et al., 2014);

[0030] Figure 2 is a TEM image of an embodiment of the TAK-441 micellar formulation (3 mg / mL) disclosed in the present invention;

[0031] Figure 3 is a rheological diagram of an embodiment of a TPGS micelle-based 3% HPC gel loaded with TAK-441 disclosed in the present invention;

[0032] Figure 4 A. Figure 4 B and Figure 4 C shows porcine skin deposition and biodistribution of TAK-441 (micellar solution and micellar HPC gel formulations, n=6). Figure 4 A) Porcine skin deposition of TAK-441; ( Figure 4 B) Biodistribution of TAK-441 in porcine skin at unlimited dose; and ( Figure 4 C) Biodistribution of TAK-441 in pig skin at limited doses; (**P<0.05, one-way ANOVA). (mean ± SD);

[0033] Figure 5is a rheological diagram of an embodiment of a TPGS micelle-based 3% HPMC gel loaded with TAK-441 disclosed in the present invention;

[0034] Figure 6 The stability of micellar formulations loaded with TAK-441 is shown. The TAK-441 content in the HPMC gel of formulation E and formulation E packaged in aluminum tubes (Nussbaum Kesswil AG, Switzerland) was quantified at different time points using UHPLC-MS / MS for 6 months (stored at 4°C). After 6 months, the TAK-441 content in the micellar solution was 79.62% of the initial value, while in the micellar gel, the TAK-441 content was 91.86% of the initial amount. It was found that the micelles were intact in the gel formulation; and

[0035] Figure 7 A. Figure 7 B and Figure 7 C shows the human skin deposition and biodistribution of TAK-441 (micelle-based HPMC gel formulation, n=6). Figure 7 A) Human skin deposition of TAK-441; ( Figure 7 B) Human skin biodistribution of TAK-441 at unlimited doses; and ( Figure 7 C) Human skin biodistribution of TAK-441 at limited doses (**P<0.05, one-way ANOVA). (Mean ± SD).

[0036] Details

[0037] The subject matter disclosed in the present invention will now be described more fully below with reference to the accompanying drawings, in which some but not all embodiments of the present invention are shown. The same numbers always refer to the same elements. The subject matter disclosed in the present invention can be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein; rather, these embodiments are provided so that the present disclosure will satisfy applicable legal requirements. In fact, with the benefit of the teachings presented in the previous description and the associated drawings, many modifications and other embodiments of the subject matter disclosed in the present invention set forth herein will be thought of by those skilled in the art to which the subject matter disclosed in the present invention belongs. Therefore, it should be understood that the subject matter disclosed in the present invention is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims.

[0038] More particularly, in some embodiments, the subject matter disclosed herein provides a composition comprising a hedgehog pathway inhibitor and a polymer surfactant. Representative hedgehog pathway inhibitors include, but are not limited to, vismodegib, sonidegib, and other hedgehog pathway inhibitors currently in clinical trials, particularly SMO inhibitors, including but not limited to IPI-926 (saridegib), BMS-833923 / XL139, PF-04449913 (glasdegib) and LY2940680 (taladegib). In certain embodiments, the hedgehog pathway inhibitor is active against the vismodegib-resistant smoothin receptor D473H mutant.

[0039] In certain embodiments, the Hedgehog pathway inhibitor is selected from TAK-441, Vismodegib, Saridegib / Patidegib, Glasdegib, Sonidegib, Taladegib (Env-101), and BMS-833923 (XL-139). In specific embodiments, the Hedgehog pathway inhibitor comprises TAK-441.

[0040] In certain embodiments, the polymeric surfactant is biocompatible and / or biodegradable. In certain embodiments, the polymeric surfactant is selected from D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), mPEG-dihex-PLA, poloxamer, poly (ε-caprolactone), poly (L-amino acid) and polyvalerolactone. In a specific embodiment, the polymeric surfactant comprises D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS).

[0041] In certain embodiments, the composition comprising a Hedgehog pathway inhibitor and a polymeric surfactant comprises a micellar composition.

[0042] As used herein, the term "micelle" refers to an aggregate of surfactant molecules. Micelles are formed only when the concentration of the surfactant is greater than the critical micelle concentration (CMC). Surfactants are amphiphilic chemical substances, i.e., they contain both hydrophobic and hydrophilic groups. Micelles can exist in different shapes, including spherical, cylindrical, and disc-like.

[0043] In certain embodiments, the micellar composition comprises spherical micelles having a diameter in the range of from about 10 nm to about 100 nm, including 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, and 100 nm. In a specific embodiment, the micellar composition comprises spherical micelles having a diameter in the range of from about 10 nm to about 15 nm, including about 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, and 15 nm.

[0044] Polymer micelles can be used as nanocarriers for delivering poorly water-soluble, water-insoluble or hydrophobic medicines, and the medicine can be dissolved in the hydrophobic core of micelles. Therefore, micelles can be used to improve the solubility and bioavailability of multiple hydrophobic drugs. The small size of micelles (usually about 10nm to about 100nm, including about 10nm, 15nm, 20nm, 25nm, 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm and 100nm) allows the relevant active part to be effectively accumulated in the target tissue. Micelles can be formed by one or more polymer nonionic surfactants.

[0045] As provided above, in a particular embodiment, the surfactant comprises tocopherol or a derivative thereof. Tocopherol is a class of methylated phenols, many of which have vitamin E activity. Tocopherol and its derivatives, such as, for example, esters, are widely used in vitamin supplementation and are used as antioxidants in the food industry and many pharmaceutical compositions. Tocopherol includes a series of natural compounds and synthetic compounds. Vitamin E alpha-tocopherol (chemical name: 2,5,7,8-tetramethyl-2-(4',8',12'-trimethyldecyl)-6-chromanol) is the most active and widely distributed in nature, and has been most extensively studied. Other members of this class include beta tocopherol, gamma tocopherol and delta tocopherol. Tocopherol exists in a variety of isomeric forms, and D form and DL form are the most widely available. As used herein, the term "tocopherol" includes all such natural and synthetic tocopherols or vitamin E compounds.

[0046] Any form or isomer of tocopherol and its derivatives such as esters may be used according to the present disclosure. For example, alpha-tocopherol or its esters may be used herein, including but not limited to alpha-tocopheryl acetate, alpha-tocopheryl linoleate, alpha-tocopheryl nicotinate or alpha-tocopheryl hemisuccinate, many of which are commercially available.

[0047] Tocopherol derivatives include chemical derivatives of vitamin E, which have ester bonds and ether bonds of multiple chemical moieties and polyethylene glycols of different lengths. For example, derivatives can include vitamin E tocopherol polyethylene glycol succinate (TPGS) derivatives with a PEG molecular weight between about 500Da and 6000Da, including about 500Da, 1000Da, 1500Da, 2000Da, 2500Da, 3000Da, 3500Da, 4000Da, 4500Da, 5000Da, 5500Da and 6000Da. In a specific embodiment, the vitamin E polymer derivative is D-α-tocopherol polyethylene glycol 1000 succinate (TPGS).

[0048] More specifically, TPGS is a water-soluble derivative of vitamin E in which polyethylene glycol subunits are attached via succinic acid diesters at the ring hydroxyl groups of the vitamin E molecule. TPGS is a nearly odorless waxy amphiphilic substance with a molecular weight of about 1513. TPGS forms stable micelles in aqueous vehicles due to its amphiphilic structure with a hydrophilic / lipophilic balance (HLB) value of 13.2. TPGS has been approved by the U.S. Food and Drug Administration (FDA) as a pharmaceutical excipient.

[0049] The tocopherol surfactants of the present disclosure may 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.

[0050] In certain embodiments, the composition comprises an amount ranging from about 5 mg / mL to about 300 mg / mL, including about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 100 mg / mL, In some embodiments, the composition comprises TPGS at a concentration of about 10 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, and 300 mg / mL. In certain embodiments, the composition comprises TPGS at a concentration of about 10 mg / mL.

[0051] In certain embodiments, the composition comprises TAK-441 having a concentration of TAK-441 in the range of from about 100 mg to about 500 mg per gram of TPGS, including about 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, and 500 mg of TAK- 441. In specific embodiments, the concentration of TAK-441 has a concentration of TAK-441 in the range of from about 100 mg to about 300 mg per gram of TPGS.

[0052] In other embodiments, the composition further comprises a hydrogel. In certain embodiments, the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), a cellulose-based gel forming agent, and a poloxamer-based gelling agent. In a specific embodiment, the hydrogel is selected from HPC and HPMC.

[0053] In a specific embodiment, the HPMC is selected from: (a) HPMC having a molecular weight of about 26 kDa, having a methoxy content ranging from about 19% to about 24% (including 19%, 20%, 21%, 22%, 23% and 24%), and having a hydroxypropoxy content ranging from about 7% to about 12% (including about 7%, 8%, 9%, 10%, 11% and 12%); (b) HPMC having a molecular weight of about 10 kDa, having a methoxy content ranging from about 28% to about 30% (including 28%, 29% and 30%), and having a hydroxypropoxy content of about 7% to about 12% (including about 7%, 8%, 9%, 10%, 11% and 12%); and (c) combinations thereof.

[0054] 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, having a methoxyl content ranging between about 19% to about 24% and having a hydroxypropoxyl content ranging between about 7% to about 12%; and about 3% HPMC having a molecular weight of about 10 kDa, having a methoxyl content ranging between about 28% to about 30% and a hydroxypropoxyl content between about 7% to about 12%.

[0055] In certain embodiments, the composition further comprises a rheology modifier. In certain embodiments, the rheology modifier is selected from glycerol, low molecular weight cellulose and high molecular weight cellulose, and sorbitol. In a specific embodiment, the rheology modifier comprises glycerol.

[0056] In certain embodiments, the composition further comprises a preservative. In certain embodiments, the preservative is selected from sodium metabisulfite, benzyl alcohol, benzalkonium chloride, chlorobutanol, sodium benzoate, potassium sorbate, methylparaben and propylparaben. In a specific embodiment, the preservative comprises sodium metabisulfite. Exemplary preservatives also include, but are not limited to, sorbic acid, benzoic acid, methylparaben, propylparaben, methylchloroisothiazolinone, methylisothiazolinone, diazolidinyl urea, chlorobutanol, triclosan, benzethonium chloride, parabens, chlorhexidine, digluconate, cetyltrimethylammonium bromide, alcohol, benzalkonium chloride, boric acid, bronopol, butylparaben, calcium butyl acetate, calcium chloride, calcium lactate, carbon dioxide, cationic bentonite, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, citric acid monohydrate, cresol, dimethyl ether, ethylparaben, glycerol, hexetidine, imidurea, isopropyl alcohol, lactic acid, monothioglycerol, pentetic acid 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.

[0057] In certain embodiments, the composition retains between 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 storage for about 6 months, including 1, 2, 3, 4, 5, 6, 7, 8 and 9 months.

[0058] In other embodiments, the presently disclosed subject matter provides a method for treating a disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway, the method comprising administering the presently disclosed composition described above to a subject in need of corresponding treatment.

[0059] In certain embodiments, the composition is topically applied. In a specific embodiment, the composition is delivered cutaneously, i.e., related to the skin. In a specific embodiment, the composition is delivered to the viable epidermis of the subject, i.e., the skin layer immediately below the stratum corneum. In a specific embodiment, the composition is delivered to the upper dermis of the subject. The dermis includes the papillary dermis and the reticular dermis, the papillary dermis being the uppermost layer of the dermis, and the reticular dermis being the lower layer of the dermis, present under the papillary dermis. In a specific embodiment, topical application results in negligible transdermal penetration. Typically, transdermal penetration includes that the therapeutic agent penetrates through the stratum corneum and passes through the deeper epidermis and dermis, without drug accumulation in the dermis. Negligible transdermal penetration can include drug accumulation of about 0.001%, 0.01%, 0.1%, and 1% in the dermis.

[0060] In certain embodiments, a disease, disorder, or condition associated with the Hedgehog (HH) signaling pathway comprises a skin disease, skin disorder, or skin condition. In a specific embodiment, a skin disease, skin disorder, or skin condition comprises skin cancer. In a more specific embodiment, skin cancer comprises basal cell carcinoma. In yet more specific embodiments, the subject suffers from or is suspected of having locally advanced basal cell carcinoma. In yet more specific embodiments, the subject suffers from or is suspected of having metastatic basal cell carcinoma. In certain embodiments, the subject is not eligible for surgical treatment or radiotherapeutic treatment. In more certain embodiments, administration of the composition to the subject reduces the progression of basal cell carcinoma. In a specific embodiment, the basal cell carcinoma involves the vismodegib-resistant SMO mutant D473H.

[0061] In certain embodiments, administering the composition to a subject prevents activation of the HH signaling pathway.

[0062] In certain embodiments, the disease, condition or disorder involves a mutation in patch protein 1 (PTCH1). In specific embodiments, the mutation in patch protein 1 (PTCH1) results in a loss of function of the PTCH1 protein. In certain embodiments, the disease, condition or disorder involves a mutation in smoothened protein (SMO). In specific embodiments, the mutation in smoothened protein (SMO) involves 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 yet more specific embodiments, the activation of one or more GLI transcription factors results in excessive proliferation of basal cells associated with basal cell carcinoma.

[0063] In certain embodiments, administering a composition to a subject inhibits SMO. In certain embodiments, mutations in SMO result in resistance to treatment with a hedgehog pathway inhibitor. In specific embodiments, resistance to treatment with a hedgehog pathway inhibitor involves mutations in a drug binding site of SMO. In more specific embodiments, the drug binding site of SMO is SMO-Q476 and / or SMO-D473.

[0064] The compositions disclosed herein can be administered as monotherapy or in combination with other therapies, including photodynamic therapy (PDT).

[0065] The term "combination" is used in its broadest sense, and means that the subject is administered at least two agents or therapies, such as the compositions disclosed in the present invention and at least one other therapeutic agent or therapy. More particularly, the term "combination (in combination)" refers to the concomitant administration of two (or more) active agents, for the treatment of, for example, a single disease state. As used herein, active agents can be combined and administered in a single dosage form, can be administered simultaneously as separate dosage forms, or can be administered as separate dosage forms administered alternately or sequentially on the same or separate dates. In one embodiment of the subject matter disclosed in the present invention, active agents are combined and administered in a single dosage form. In another embodiment, active agents are administered in a separate dosage form (e.g., where it is desirable to change the amount of one rather than another). A single dosage form may include additional active agents for the treatment of a disease state.

[0066] In addition, the compositions disclosed in the present invention can be used alone or in combination with an adjuvant that enhances the stability of the composition, alone or in combination with one or more therapeutic agents (including other active ingredients), which in certain embodiments promote the use of a pharmaceutical composition comprising them, provide increased dissolution or dispersion, increase inhibitory activity, provide adjuvant therapy, and the like. Advantageously, such combination therapy utilizes lower doses of conventional therapeutic agents, thereby avoiding possible toxicity and adverse side effects caused when these agents are used as monotherapy.

[0067] The timing of administration of the compositions disclosed herein and at least one additional therapeutic agent or therapy can be changed, as long as the beneficial effects of the combination of these agents are achieved. Therefore, the wording "combined with..." refers to the administration of the compositions described herein and at least one additional therapeutic agent or therapy simultaneously, sequentially (or a combination thereof). Therefore, the subject to whom the compositions described herein and at least one additional therapeutic agent or therapy combination is administered can receive the compositions disclosed herein and at least one additional therapeutic agent or therapy simultaneously (i.e., simultaneously) or at different times (i.e., sequentially on the same day or on different days in any order), as long as the effect of the combination of the two agents is achieved in the subject.

[0068] When administered sequentially, the doses can be administered within 1 minute, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes or longer of each other. In other embodiments, the doses administered sequentially can be administered within 1 day, 5 days, 10 days, 15 days, 20 days or more of each other. In cases where a compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to a subject as separate pharmaceutical compositions, each comprising the compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both doses.

[0069] When administered in combination, the effective concentration of each of the agents that elicit a particular biological response can be less than the effective concentration of each agent when administered alone, thereby allowing the dose of one or more of the agents to be reduced relative to the dose that would be required if the agent were administered as a single agent. The effects of multiple agents can, but need not be, additive or synergistic. An agent can be administered multiple times.

[0070] In some embodiments, two or more agents can have a synergistic effect when administered in combination. As used herein, the terms "synergy," "synergistic," "synergistically," and derivatives thereof, such as in "synergistic effect," "synergistic combination," or "synergistic composition," refer to situations where 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 the corresponding agents when administered separately.

[0071] Synergy can be expressed as a "synergy index (SI)" which can generally be determined by the method described by FC Kull et al., Applied Microbiology 9, 538 (1961), by the ratio determined by:

[0072] Q a / Q A +B / Q B =Synergy Index (SI)

[0073] in:

[0074] Q A is the concentration of component A that produces an individual effect that results in an endpoint related to component A;

[0075] Qa is the concentration of component A in the mixture that produces the endpoint;

[0076] Q B is the concentration of component B that produces an endpoint associated with component B alone; and

[0077] Q b is the concentration of component B in the mixture that produces the endpoint.

[0078] Usually, when Q a / Q A and Q b / Q B When the sum of the SI is greater than 1, antagonism is indicated. When the sum is equal to 1, additivity is indicated. When the sum is less than 1, synergy is exhibited. The lower the SI, the greater the synergy shown by the particular mixture. Thus, a "synergistic combination" has an activity that is greater than that which would be expected based on the activity observed for the individual components when used alone. In addition, a "synergistically effective amount" of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0079] As used herein, the term "treating" may include reversing the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition, alleviating the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition, inhibiting the progression of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition, preventing the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. "Prevention" refers to preventing a disease, disorder, condition, or a symptom or manifestation of such disease, disorder, or condition, or a worsening of the severity of such disease, disorder, or condition from occurring. Thus, the compounds disclosed herein may be administered prophylactically to prevent or reduce the incidence or recurrence of a disease, disorder, or condition.

[0080] The "subject" treated by the methods disclosed herein in many of its embodiments is desirably a human subject, however it should be understood that the methods described herein are effective for all vertebrate species, all of which are intended to be included in the term "subject." Thus, a "subject" may include a human subject for medical purposes, such as for treatment of an existing condition or disease or for prophylactic treatment to prevent the onset of a condition or disease, or an animal subject for medical, veterinary, or developmental purposes. Suitable animal subjects include mammals, including but not limited to primates, such as humans, monkeys, apes, etc.; bovines, such as cattle, bulls, etc.; ovines, such as sheep, etc.; caprines, such as goats, etc.; porcines, such as pigs, hogs, 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 can be a transgenic animal. In some embodiments, the subject is a human, including but not limited to fetuses, newborns, infants, adolescents, and adult subjects. In addition, a "subject" may include a patient suffering from or suspected of having a condition or disease. Therefore, the terms "subject" and "patient" are used interchangeably herein. The term "subject" also refers to an organism, tissue, cell, or cell collection from a subject.

[0081] Typically, an "effective amount" of an active agent refers to the amount necessary to elicit a desired biological response. As will be appreciated by those of ordinary skill in the art, an effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the drug target, etc.

[0082] Following long-standing patent law convention, the terms "a," "an," and "the" when used in this application, including the claims, mean "one or more." Thus, for example, reference to "a subject" includes more than one subject unless the context clearly indicates otherwise (e.g., more than one subject), etc.

[0083] Throughout the specification and claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense unless the context requires otherwise. Likewise, the term "include" and its grammatical variations are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0084] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, numbers, characteristics, and other numerical values ​​used in the specification and claims are to be understood as being modified in all cases by the term "about", even though the term "about" may not specifically appear with a value, amount, or range. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are not and need not be exact, but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding, measurement errors, etc., and other factors known to those skilled in the art, depending on the desired properties sought to be obtained by the subject matter disclosed herein. For example, the term "about" when used in reference to an index value can be meant to encompass variations from the particular amount of ±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% in some embodiments, and ±0.1% in some embodiments, as such variations are suitable for performing the disclosed methods or using the disclosed compositions.

[0085] In addition, when used in conjunction with one or more numbers or numerical ranges, the term "about" should be understood to refer to all of these numbers, including all numbers within a range, and to modify the range by extending the boundaries above and below the numerical values ​​set forth. Numerical ranges expressed by endpoints include all numbers within the range, such as whole integers, including decimals thereof (e.g., the expression 1 to 5 includes 1, 2, 3, 4, and 5, and decimals thereof, such as 1.5, 2.25, 3.75, 4.1, etc.) and any range within the range. Example

[0086] The following examples have been included to provide guidance to those of ordinary skill in the art for practicing representative embodiments of the subject matter disclosed herein. Based on the present disclosure and the general level of those skilled in the art, the technician will appreciate that the following examples are intended to be exemplary only, and that many variations, modifications, and changes may be employed without departing from the scope of the subject matter disclosed herein. The subsequent comprehensive description and specific examples are intended only for illustrative purposes and should not be construed as limiting in any way the compounds of the present disclosure prepared by other methods.

[0087] Example 1

[0088] Formulation development and skin biodistribution in porcine and human skin

[0089] 1.1 Overview

[0090] TAK-441 is a potent inhibitor of the Hedgehog pathway (IC 50 4.4nM), is active against the vismodegib-resistant smoothin receptor D473H mutant, and is expected to be used to treat basal cell carcinoma. This example describes the development of a micelle-based TAK-441 formulation using D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and studies of its skin delivery and biodistribution. The results showed that the incorporation of TAK-441 into TPGS micelles increased the aqueous solubility by approximately 40 times. One embodiment, an HPMC hydrogel of TPGS micelles loaded with TAK-441, retained approximately 92% of the initial TAK-441 content after storage at 4°C for 6 months. Limited dose experiments using human skin showed that the formulation resulted in significantly greater skin deposition of TAK-441 than the non-micelle control formulation (0.40±0.11μg / cm 2 and 0.05±0.02μg / cm 2 ). In addition, no percutaneous penetration was observed. The skin biodistribution profile showed that TAK-441 was mainly delivered to the viable epidermis and upper dermis. Delivery of the HPMC hydrogel formulation resulted in TAK-441 epidermal concentrations higher than IC 50 Thousands of times higher, with negligible percutaneous penetration, thus reducing the risk of systemic side effects in vivo.

[0091] 1.2 Background

[0092] TAK-441 is a potent inhibitor of the HH pathway (IC 50 4.4 nM; determined by luciferase reporter activity in NIH3T3 cells carrying a stably transfected 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. reported that TAK-441 has an IC of 50 is 79 nM; in comparison, the IC 50 is 7100 nM. TAK-441 has a molecular weight of 576.57 Da ( Figure 1), is moderately lipophilic (log P 2.61), but has very poor aqueous solubility (81 μg / mL at pH 6.8) (Ohashi et al., 2012; Ishii et al., 2014). Phase I clinical trials have studied its oral administration at doses ranging from 50 mg / day up to the maximum feasible dose (MFD) of 1600 mg / day (Goldman et al., 2015). Gli1 expression in skin biopsies was strongly suppressed at all doses, but there were multiple side effects. All patients experienced at least one adverse event (AE); mild to moderate AEs were dysgeusia, fatigue, nausea, muscle cramps, and hyponatremia (Goldman et al., 2015). About 35% of patients experienced severe AEs, including gastrointestinal disturbances, neoplasms (progression of underlying disease), and hepatobiliary disturbances. One death due to cerebral hemorrhage in a patient with pancreatic cancer was assessed by the investigator as related to the study drug.

[0093] Local delivery of TAK-441 can not only improve efficacy by better targeting the disease site, but also increase treatment tolerance by reducing systemic side effects. Compared with oral administration, direct application to the disease site significantly reduces the required dose and reduces systemic toxicity or "off-target" toxicity by definition. However, topical drug therapy using TAK-441 must ensure sufficient skin bioavailability, and more specifically, reach super-therapeutic concentrations in the basal epidermis. In view of the poor water solubility of TAK-441, it is simpler to formulate TAK-441 in a more lipophilic system, in which TAK-441 is more soluble; however, greater solubility and therefore formulation stability is at the expense of lower thermodynamic activity and less favorable distribution to the stratum corneum.

[0094] 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 have previously described how methoxy poly(ethylene glycol)-dihexyl-substituted poly(lactic acid) (mPEGhexPLA) micelles can be used to develop aqueous formulations of several poorly water-soluble therapeutics with 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), and enabled their improved skin delivery compared to existing approved formulations (Lapteva et al., 2014a, 2015, 2019). Furthermore, in another study, mPEGhexPLA micelles were used to develop the first topical formulation of vismodegib, and skin biodistribution methods were used to show that therapeutically relevant amounts of the drug could be delivered to the epidermis and upper dermis (Kandekar et al., 2019).

[0095] In other studies, we have used D-α-tocopheryl polyethylene glycol succinate 1000 (TPGS) as a copolymer (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 been approved by regulatory agencies as an excipient for use in pharmaceutical products (Aggarwal et al., 2012). TPGS has been approved by the FDA as a pharmaceutical ingredient and has been used as an excipient in a variety of marketed products (Zhang et al., 2015; Vadlapudi et al., 2014). TPGS was also approved as an active pharmaceutical ingredient (API) by the European Medicines Agency on July 24, 2009. For the treatment of vitamin E deficiency due to malabsorption in pediatric patients with congenital chronic cholestasis or hereditary chronic cholestasis (Papas, 2021).

[0096] 1.3 Scope

[0097] The objectives of this example were to (i) investigate the feasibility of using TPGS micelles to overcome the inherently poor aqueous solubility of TAK-441 and develop a stable aqueous formulation; (ii) characterize the micelles in terms of drug content, size, and morphology; (iii) develop a user-friendly micelle-based hydrogel formulation for topical application; (iv) investigate the dermal delivery of TAK-441 and determine the dermal biodistribution in porcine skin after application of micelle solutions and micelle-based hydrogel formulations, and compare the results with those obtained with a non-micelle control formulation; and (v) confirm the results using human skin.

[0098] 1.4 Materials and methods

[0099] 1.4.1 Materials

[0100] TAK-441 was kindly provided by Takeda Pharmaceutical Company Ltd, Japan. D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS), formic acid (MS grade), isopentane and Dulbecco's phosphate buffered saline (DPBS), hydroxypropyl methylcellulose (HPMC, about 26 kDa; methoxy content 19%-24% and hydroxypropoxy content 7%-12%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Low molecular weight HPMC-Methocel TM E5 Premium LV (about 10 kDa; methoxy content 28%-30% and hydroxypropoxy content 7%-12%) was purchased from Dow chemicals (Horgen, Switzerland). Hydroxypropyl cellulose (Klucel TM MF Pharm, HPC; MW about 850 kDa) and glycerol were purchased from AG (Herisau, Switzerland). Bovine serum albumin (BSA) was purchased from Axon Laboratories ( 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) filter units were purchased from VWR (Nyon, Switzerland). Ultrapure water (Millipore Milli-Q Gard 1 purification pack resistivity >18 MΩ cm; Zug, Switzerland) was used for formulation development and analysis. All other chemicals were of at least analytical grade.

[0101] 1.4.2 Analysis methods

[0102] Use equipped with Waters Acquity Core for TQ-MS with Tandem Quadrupole Detector The system was used to quantify TAK-441. C18 VanGuard preparatory column (2.1×5 mm, 1.7 μm) connected in series with Acquity Isocratic separation was performed on a BEH C18 column (2.1×50 mm; 1.7 μm). The mobile phase consisted of a mixture of acetonitrile and water (75:25 v / v). The flow rate and injection volume were 0.1 mL / min and 5 μL, respectively. The peak of TAK-441 was obtained at 1.7 min, and the run time was 3.0 min. Mass spectrometry detection was performed with electrospray ionization in positive ion mode using multiple reaction monitoring (MRM). The detection settings for TAK-441 are presented in Table 1. The limits of detection (LOD) and quantification (LOQ) were 1.29 ng / m and 3.29 ng / mL, respectively. The UHPLC-MS / MS method was validated according to the ICH guidelines.

[0103] Table 1: MS / MS settings for detection of TAK-441

[0104]

[0105] 1.4.3 Preparation of micellar preparations

[0106] 1.4.3.1 Micellar solution

[0107] TPGS-based micelles of TAK-441 were prepared by solvent evaporation (Kandekar et al., 2019). Screening of surfactants and their concentrations was done using a micro-scale formulation technique, which involves performing multiple experiments simultaneously with minimal amounts of drug and excipients. This process reduces material costs, the time required for excipient screening and formulation development, and reduces exposure to the drug, which is beneficial when dealing with cytotoxins.

[0108] The preparations listed in the candidate list are then scaled up to laboratory scale batches. In brief, known amounts of TPGS and TAK-441 are dissolved in 2mL acetone to obtain a clear solution. The solution is slowly added to 4mL water under sonication (Branson DigitalSonifier S-450D). Acetone is then slowly removed by using a rotary evaporator (Büchi RE121Rotavapor). Final volume is supplemented with water in a volumetric flask to obtain micellar preparations in which TAK-441 concentration and TPGS concentration are 3mg / mL and 10mg / mL respectively. After balancing overnight, the micellar solution is centrifuged at 10,000rpm for 15min (Eppendorf centrifuge 5804) to remove excess TAK-441, and the supernatant is carefully collected.

[0109] 1.4.3.2 Micellar gel

[0110] In preliminary studies, TAK-441-TPGS micelles were incorporated into 3% HPC gels to study the skin delivery of TAK-441 in a semisolid gel formulation. The formulations were compared to control gels with the same composition except for the polymer surfactant. Based on the preliminary results, it was decided to prepare micelle-based HPMC gels with better formulation properties for clinical applications (see below for full details; Section 1.5.1).

[0111] 1.4.4 Characterization of micellar preparations

[0112] 1.4.4.1 Dimension determination

[0113] The hydrodynamic diameter (Z) of the micelles was measured using dynamic light scattering (DLS) using a Zetasizer HS 3000 (Malvern Instruments Ltd.; Malvern, UK). av ), polydispersity index (PDI), and volume-weighted diameter and number-weighted diameter (d v and d n ). The measurements were carried out at an angle of 90° and a temperature of 25° C. All values ​​were obtained after 3 runs of 10 measurements.

[0114] 1.4.4.2 Morphology

[0115] The micellar morphology was characterized using a negative staining method using a transmission electron microscope (TEM) (FEI Tecnai G2 Sphera, Eindhoven, Netherlands). Briefly, 5 μL of the micellar solution was dropped onto an ionized carbon-coated copper mesh (0.3 Torr, 400 V for 20 s). The mesh was then placed in a 100 μL saturated uranyl acetate aqueous solution droplet for 1 s, and then in a second 100 μL droplet for 30 s. Excess staining solution was removed, and the mesh was dried at room temperature before measurement.

[0116] 1.4.4.3 Determination of TAK-441 content in micelles

[0117] TAK-441 loaded into micelles was quantified by UHPLC-MS / MS. To ensure complete micelle destruction and release of incorporated drug, the formulations were diluted in acetonitrile and analyzed. Drug content, drug loading, and encapsulation efficiency were calculated using Equation 1-Equation 3:

[0118]

[0119] 1.4.4.4 Viscosity measurement

[0120] The viscosity of micellar gels was determined by using Thermo Scientific TM HAAKE TM MARS TM The rheometer was used for the measurement. The measurements were performed at a fixed temperature (25°C) with a rotating plate spindle at different shear rates. TM HAAKE TM RheoWin software for measurement and post-measurement evaluation.

[0121] 1.4.4.5 Evaluation of the stability of micellar formulations

[0122] TAK-441 micellar aqueous formulations and micellar-based HPMC gel formulations were prepared and stored at 4° C. for 6 months. The formulations were assayed at different time points (day 1, then monthly) to determine drug content.

[0123] 1.4.5 In vitro skin delivery and biodistribution studies

[0124] 1.4.5.1 Skin preparation

[0125] Pig ears were purchased from a local slaughterhouse (CARRE; Rolle, Switzerland) shortly after being sacrificed. After washing under running cold water, skin samples with a thickness of about 0.8 mm were carefully harvested from the external area of ​​the ear using Zimmer air dermatome (Münsingen, Switzerland). Hair was removed from the skin surface using scissors. A disc corresponding to the infiltration area was punched out (Berg & Schmid HK 500; Urdorf, Switzerland). The skin samples were frozen at -20 ° C and stored for a maximum period of 3 months. Before the experiment, the skin samples were thawed at room temperature and placed in a 0.9% saline solution for 15 min to rehydrate.

[0126] Human skin samples were obtained from the Department of Plastic, Aesthetic and Reconstructive Surgery, Geneva University Hospital (Geneva, Switzerland) shortly after surgery, adipose tissue was removed and the skin was stored at -20° C. Donation was approved by the Central Committee for Ethics in Research (CER: 08-150 (NAC08-051); Geneva University Hospital).

[0127] 1.4.5.2 Micellar solution

[0128] Use a 2cm 2 The experiments were performed in a standard two-chamber vertical (Franz-type) diffusion cell (Milian SA; Meyrin, Switzerland) with a cross-sectional area of ​​1.5 Å. The receptor compartment consisted of 10-mL Dulbecco's phosphate-buffered saline (DPBS) pH 7.4 containing 1% BSA to maintain sink conditions. The receiver compartment was maintained between 32°C and 34°C. For unlimited dose conditions, 200 μL of TAK-441 micellar formulation (3 mg / mL) was applied to the surface of the skin sample (i.e., 300 μg TAK-441 / cm 2 Skin surface), and for the limited dose, 20 μL of micellar formulation (3 mg / mL) (30 μg TAK-441 / cm 2 Skin surface). A non-micellar formulation containing 3 mg / mL TAK-441 suspended in aqueous 0.05% hydroxypropylcellulose (HPC) was used as a control.

[0129] TAK-441 has minimum solubility in HPC; This characteristic will minimize the risk of interference of the suspending agent to drug delivery. Take out aliquots (1mL) from the receiver compartment at 1h, 4h and 12h, and replace with equal volume of fresh medium. The sample is diluted in acetonitrile to precipitate BSA. After centrifugation at 10,000rpm for 15min, the infiltration sample is analyzed by UHPLC-MS / MS.

[0130] After the experiment was completed, excess preparation was removed from the skin surface using a validated washing method. The skin samples were cut into small pieces and the deposited TAK-441 was extracted by soaking the small pieces in 2 mL of methanol for 4 h at room temperature with continuous stirring. The extraction procedure was validated. Prior to UHPLC-MS / MS analysis, the extracted samples were centrifuged at 10,000 rpm for 15 min, diluted and filtered through a 0.22-μm PTFE filter.

[0131] 1.4.5.3 Micellar gel

[0132] TAK-441 micelles were incorporated into 3% HPC gel to test the skin delivery of TAK-441 from a semisolid gel formulation to pig skin. The composition of the control gel was identical except for the polymer surfactant. The experiment was performed as described above (Section 1.4.5.2). For unlimited doses, 200 mg of micellar gel (2.88 mg TAK-441 / g gel formulation; i.e., a gel containing 0.29% TAK-441) was applied to the skin surface (i.e., 288 μg TAK-441 / cm 2 Skin surface), and for a limited dose, 20 mg of micellar gel (28.8 μg TAK-441 / cm 2 skin surface).

[0133] Similar experimental conditions were used for micelle-based HPMC gels that were tested for delivery to human skin. For unlimited dosing, 200 mg of micelle gel (2.5 mg TAK-441 / g gel formulation; i.e., gel containing 0.25% TAK-441) was applied to the skin surface (i.e., 250 μg TAK-441 / cm 2 Skin surface), and for limited doses, 20 mg of micellar gel (25 μg TAK-441 / cm 2 Skin surface). The composition of the control gel was the same as the HPMC gel except for TPGS.

[0134] After the experiment was completed, the skin samples were separated into two parts using a punch - the surface area was 0.785 cm 2The inner disk has an area of ​​1.215 cm 2 The outer ring is then cut into small pieces and TAK-441 deposited in the tissue is extracted by a validated extraction method (Section 1.4.5.2) and then quantified using UHPLC-MS / MS.

[0135] 0.785-cm 2 The discs were used to determine the biodistribution of TAK-441 as a function of skin depth. The skin discs were snap-frozen in isopentane cooled by liquid nitrogen. For this procedure, the skin samples were mounted on a round piece of cork with OCT, 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 the different regions of the skin. The skin discs were then cryosectioned (ThermoScientific TM CryoStar TM NX70; Reinach, Switzerland) to obtain 50-μm thick sections, starting from the stratum corneum down to a skin depth of 400 μm. These slices enable the determination of the amount of TAK-441 as the position in the skin (including the stratum corneum, epidermis and upper dermis, respectively) changes. Each slice and the remaining dermis were extracted separately in 250-μL methanol for 4 h, and TAK-441 was quantified by UHPLC-MS / MS.

[0136] 1.4.6 Statistical analysis

[0137] 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.

[0138] Results and discussion

[0139] 1.5.1 Micellar formulation development and characterization

[0140] A set of formulations (AH) with a constant TPGS content (10 mg / mL) but different target TAK-441 loadings were prepared: 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg and 500 mg TAK-441 / g TPGS. The drug loading, drug content and incorporation efficiency obtained for each formulation are given in Table 2. The highest drug content (2.97 ± 0.071 mg / mL) was provided by formulation E.

[0141] Table 2: Characterization of micelle formulations with respect to drug content and size.

[0142]

[0143]

[0144] 1.5.1.1 Dimensional characterization

[0145] The TAK-441-loaded TPGS micelles were characterized using DLS to determine their size (Table 2). All TAK-441-loaded micelle preparations exhibited uniform nanosize, with a hydrodynamic diameter (Z av ) is from 12.14nm to 16.92nm. The volume weighted diameter (d v ) were measured in the range from 10.55 nm to 12.29 nm, and the number-weighted diameter (d n ) in the range from 8.74 nm to 10.51 nm. TEM micrographs of the optimized formulation (Formulation E) are Figure 2 , where it is apparent that the shape of the micelles is spherical, with diameters ranging from 10 nm to 15 nm; these sizes were confirmed by DLS (Table 2).

[0146] 1.5.1.2 Development of HPC Micellar Gel

[0147] Formulation E was used to prepare a 2.88 mg TAK-441 / g gel formulation with a final drug content of 3% HPC gel. -1 The shear rate of the gel is 283.5 Pas ( Figure 3 ). As mentioned above, 3% HPC gel with TAK-441 was used as a control - this would ensure that any advantage in skin delivery of TAK-441 in the micellar gel would be due specifically to the action of the micelles.

[0148] 1.5.2 Evaluation of TAK-441 delivery in vitro

[0149] 1.5.2.1 Dermal delivery of TAK-441 in micellar solution in porcine skin

[0150] This study was conducted to compare the skin deposition and percutaneous penetration of TAK-441 in TPGS micellar solutions and control formulations. In these initial experiments, porcine skin was used to study TAK-441 delivery because it is one of the best substitutes 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 receptor compartment was below the LOD of the UHPLC-MS / MS method—equivalent to a cumulative permeability of <0.1 pg / cm after the formulation was applied for 12 h 2 .like Figure 4 As shown in A, higher skin deposition was found in the micellar solution groups (unlimited dose and limited dose) compared to the control formulation. The amount of TAK-441 deposited in pig skin in the micellar solution and the control formulation was 1.44 ± 0.27 μg / cm2, respectively, under unlimited dose conditions. 2 and 0.41±0.09μg / cm 2 (p = 0.015, one-way ANOVA; n = 6), and for the limiting dose, were found to be 0.61 ± 0.11 μg / cm 2 and 0.19±0.052μg / cm 2 (p = 0.029, one-way ANOVA; n = 6). Under unlimited dose conditions, the concentration corresponding to the total amount of TAK-441 deposited in the entire skin sample after application of the micellar solution was greater than its IC 50 4.4 nM is more than 7,100-fold higher (Ohashi et al., 2012), and more than 3000-fold higher at the limiting dose.

[0151] 1.5.2.2 Dermal delivery of TAK-441 in micelle-HPC gel to porcine skin

[0152] The amount of TAK-441 deposited in porcine skin from micellar HPC gel and control HPC gel formulations was 0.74 ± 0.19 μg / cm under infinite dose conditions, respectively. 2 and 0.12±0.05μg / cm 2 (p = 0.002, one-way ANOVA; n = 6), and for the limiting dose, were found to be 0.32 ± 0.08 μg / cm 2 and 0.03±0.01μg / cm 2 (p=0.002, one-way ANOVA; n=6). The concentration of TAK-441 quantified in skin samples after 12 h of delivery was higher in the micelle-treated groups (solution and gel) compared to the control formulation in porcine skin.

[0153] Biodistribution studies made it possible to determine the amount of TAK-441 deposited as a function of depth. Biodistribution at infinite and finite doses revealed that a relatively large amount of TAK-441 was present primarily at the target site, the epidermal region ( Figure 4 B and Figure 4 C). Given the amount of TAK-441 present in these smaller skin volumes, the estimated concentrations are higher than those estimated for the entire skin sample. Thus, in the first slice from 0 μm to 50 μm, the TAK-441 concentrations obtained after application of the micelle-HPC gel at unlimited and limited doses were higher than IC 50 for the 50 μm-100 μm region, the corresponding values ​​are greater than 6,800 times and greater than 3,400 times higher, respectively.

[0154] 1.5.2.3 Dermal delivery of TAK-441 using micellar-HPMC gel in human skin

[0155] 1.5.2.3.1 Development of Micellar-HPMC Gel

[0156] After promising results in porcine skin, it was decided to conduct delivery studies using a micellar gel formulation of TAK-441 and human skin. However, since HPC precipitates at relatively low temperatures (cloud point: approximately 39°C), and given that this property may cause stability issues (Greiderer et al., 2011), it was decided to develop a micellar gel formulation based on HPMC (Table 3). The drug content was 2.5 mg TAK-441 / g gel preparation. It was found that the viscosity of the gel was 0.01s -1 The shear rate is 646.9Pas( Figure 5 ), have shear-thinning behavior, which will be beneficial for easy application due to their better ductility ( Brummer and Godersky, 1999 ; Kwak et al., 2015 ).

[0157] Table 3. HPMC gel composition based on TAK-441-TPGS micelles

[0158]

[0159] 1.5.2.3.2 Determination of Dermal Delivery and Biodistribution of TAK-441 in Micellar-HPMC Gels in Human Skin

[0160] As observed for the porcine skin experiments, after 12 h of application time, the amount of TAK-441 that permeated through human skin was again below the LOD of the UHPLC-MS / MS method. More skin deposition of TAK-441 was observed for the micelle-HPMC gel ( Figure 7A). The amount deposited from micelle-HPMC gel and control HPMC gel formulations was 1.17 ± 0.21 μg / cm, respectively, under infinite dose conditions. 2 and 0.22±0.07μg / cm 2 (p = 0.002, one-way ANOVA; n = 6), and for limited dosing, 0.40 ± 0.11 μg / cm 2 and 0.05±0.02μg / cm 2 (p=0.002, one-way ANOVA; n=6).

[0161] Biodistribution at both the infinite and finite doses revealed a profile similar to that observed in porcine skin. Again, a relatively large amount of TAK-441 was present in the epidermal region ( Figure 7 B and Figure 7 C). For example, based on the amount of TAK-441 delivered by micelle-HPMC gel at infinite and finite doses, the estimated TAK-441 concentrations achieved in the 0 μm-50 μm region were 2.1% and 1.2% higher than the IC 50 for the 50 μm-100 μm region, the corresponding values ​​are greater than 9,400 times and greater than 3,100 times higher, respectively.

[0162] In all skin delivery experiments performed, micelle-HPMC gel shows advantages over HPMC control gel. The distribution of TAK-441 in HPMC gel is most likely more uniform than that of HPMC control gel. The TPGS micelles containing TAK-441 in the lipophilic interior of the micelles produce drug depots at the skin surface, and particularly promote the accumulation of TAK-441 in the intercluster region and hair follicles (Kandekar et al., 2018; Lapteva et al., 2015, 2014b). TPGS micelles will most likely disaggregate after contact with the lipophilic stratum corneum, thereby releasing dissolved TAK-441 and making it available as a molecular dispersion.

[0163] In view of its poor water solubility, this availability will lead to local (super) saturation of TAK-441. High thermodynamic activity is conducive to distribution from the aqueous environment of the formulation into the skin (Hadgraft, 1999; Moser et al., 2001; Schwarb et al., 1999). The increased concentration of TAK-441 present in the stratum corneum leads to an increased concentration gradient across the transport limiting membrane and thus leads to increased flux. This increased flux is manifested at the macroscopic level by the greater amount of TAK-441 measured at each skin depth in the skin biodistribution profile.

[0164] Another important factor is the evaporation of water from the formulation on the skin surface. This factor will be more important under limited dose conditions and will contribute to the formation of a supersaturated solution of TAK-441 (Cilurzo et al., 2015). Since nanocarriers have been shown to accumulate in and around hair follicles, the hair follicle pathway may play an enhanced role in the skin penetration of drugs applied using such a delivery system (Kandekar et al., 2018; Lapteva et al., 2015; Papakostas et al., 2011). It is also conceivable that the surfactant in the micellar formulation can act as a penetration enhancer; in fact, we have used MS imaging to show that TPGS can penetrate into the epidermis (Quartier et al., 2021a, 2021b).

[0165] 1.6 Conclusion

[0166] The results demonstrate that incorporation of TAK-441 into TPGS micelles is feasible and that the micelle-HPMC gel formulation of TAK-441 enables skin delivery in the epidermal region at concentrations several orders of magnitude greater than the IC50 for inhibition of the HH pathway. Given that one of the factors limiting the use of HH inhibitors in general and limiting the clinical development of TAK-441 is the incidence of side effects, it is important to note that the concentrations in the permeate samples were significantly below the LOD of the sensitive UHPLC-MS / MS method (<1.29 ng / mL, corresponding to a cumulative permeation of <0.1 pg / cm 2 ) This minimal penetration of TAK-441 through the skin should help reduce the incidence of systemic side effects. Obviously, the skin delivery of TAK-441 in micelles to diseased human skin may differ from that observed in healthy tissue and depend on the type of lesion, and this will require further in vivo studies.

[0167] 1.7 Abbreviations

[0168] AE side effects

[0169] BCC Basal Cell Carcinoma

[0170] BSA bovine serum albumin

[0171] cAMP Cyclic adenosine monophosphate

[0172] DPBS Dulbecco's Phosphate Buffered Saline

[0173] GLI glioma-associated oncogene homolog

[0174] GPR161 G protein-coupled receptor 161

[0175] HH Hedgehog

[0176] HPC Hydroxypropyl Cellulose

[0177] HPMC Hydroxypropyl Methylcellulose

[0178] KIF7 kinesin family member 7 protein

[0179] LOD Limit of Detection

[0180] LOQ Limit of quantitation

[0181] MFD Maximum feasible dose

[0182] NR Nile Red

[0183] p53 Tumor suppressor protein p53

[0184] PC primary cilia

[0185] PDI Polydispersity Index

[0186] PKA Protein kinase A

[0187] PTCH1 patch protein 1

[0188] SMO smoothin receptor

[0189] SUFU Suppressor of Fusion Homolog

[0190] TEM Transmission electron microscopy

[0191] TPGS D-α-Tocopheryl polyethylene glycol 1000 succinate

[0192] UHPLC Ultra-High Performance Liquid Chromatography

[0193] UV

[0194] References

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[0243] 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 polymer surfactant.

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

3. The composition of claim 1 or claim 2, wherein the hedgehog pathway inhibitor is selected from TAK-441, Vismodegib, Saridegib / 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.

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

6. The composition of any one of claims 1-5, 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 a diameter ranging from about 10 nm to about 100 nm.

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

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

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

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

12. The composition of claim 11, wherein the hydrogel is selected from hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), cellulose-based gel formers and poloxamer-based gelling agents.

13. The composition according to claim 12, wherein the HPMC is selected from: (a) HPMC having a molecular weight of about 26 kDa, having a methoxy content ranging between about 19% to about 24%, and having a hydroxypropoxy content ranging between about 7% to about 12%; (a) HPMC having a molecular weight of about 10 kDa, having a methoxy content ranging between about 28% to about 30% and a hydroxypropoxy content between about 7% to about 12%; and (c) any combination thereof.

14. A composition according to any one of claims 11 to 13, comprising: 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, having a methoxy content ranging between about 19% to about 24%, and having a hydroxypropoxy content ranging between about 7% to about 12%; and About 3% HPMC having a molecular weight of about 10 kDa, having a methoxy content ranging between about 28% to about 30% and a hydroxypropoxy content between about 7% to about 12%.

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

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

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

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

19. The composition of any one of claims 1-18, wherein the composition retains between about 90% and 100% of the Hedgehog pathway inhibitor content after storage for about 6 months.

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

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

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

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

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

25. The method of claim 21, wherein the topical administration results in negligible percutaneous penetration.

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

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

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

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

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

31. The method of claim 29 or claim 30, wherein the subject is not eligible for surgical treatment or radiotherapeutic treatment.

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

33. The method of claim 28, wherein the basal cell carcinoma involves the vismodegib-resistant SMO mutant D473H.

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

35. The method of claim 20, wherein the disease, condition, or disorder involves a mutation in patch protein 1 (PTCH1).

36. The method of claim 35, wherein the mutation of patch protein 1 (PTCH1) results in loss of function of the PTCH1 protein.

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

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

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

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

42. The method of claim 37, wherein the mutation in SMO results in resistance to treatment with the Hedgehog pathway inhibitor.

43. The method of claim 42, wherein resistance to treatment with the Hedgehog pathway inhibitor involves mutations 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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