RU2821529C2 - 17a, 21-diesters of cortexolone for use in treating tumours - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: present invention relates to use of cortexolone 17α-valerate for preparing a pharmaceutical composition, which is used to treat tumour diseases, as well as a pharmaceutical composition. Tumour diseases are epithelial tumours selected from prostate carcinoma, breast carcinoma; pancreatic carcinoma and colon carcinoma.

EFFECT: use of a cortexolone derivative for treating various tumour diseases.

15 cl, 18 dwg, 9 tbl, 25 ex

Description

RELATED APPLICATIONS

This application pursuant to 35 USC 119 claims the benefit of European Patent Application No. 14188063.3, entitled “Cortexolone 17α-Monoesters and 17α,21-Diesters for Use in the Treatment of Tumors,” filed October 8, 2014, the contents of which are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention relates to certain cortexolone derivatives of formula (I):

(I)

and said derivatives for use as antitumor active ingredients for the radical, adjuvant, neoadjuvant or palliative treatment of precancerous conditions, dysplasias, metaplasias and tumor diseases, including malignancies and metastases.

Another aspect of the present invention relates to pharmaceutical compositions comprising at least one cortexolone derivative of formula (I) as an active ingredient, along with at least one physiologically acceptable excipient, as well as said pharmaceutical compositions for use as anticancer medicinal products for radical, auxiliary, neoadjuvant or palliative treatment of precancerous conditions, dysplasias, metaplasias and tumor diseases, including malignancies and metastases.

BACKGROUND OF THE INVENTION

A tumor or neoplasm (neoplasia) is defined as a mass of new tissue that continues to exist and grow independently of surrounding structures and that has no physiological benefit to the body (Doreland's Medical Dictionary, 23 ED.1960).

There are several classifications of tumors: for the purposes of this patent application, the most important are epithelial tumors.

Epithelial tumors are neoplasms arising from epithelial cells, i.e. type of cells that line hollow internal organs and body surfaces; This group of diseases includes many of the most common types of cancer, and most of these tumors develop in the breast, prostate, lungs, pancreas and gastrointestinal tract.

In some cases, epithelial tumors may also be characterized by the presence of certain hormone receptors in the tumor cells, which make the tumors responsive to hormones.

Carcinomas, which are malignant tumors arising from epithelial cells, account for approximately 85 out of every 100 cancers (85%).

One example of epithelial carcinoma is pancreatic carcinoma (also called pancreatic cancer).

Pancreatic cancer is one of the most lethal forms of carcinoma. Exocrine and endocrine pancreatic cells form completely different types of tumors. Exocrine pancreatic tumors are the most common type of pancreatic cancer (over 95%). Although benign (noncancerous) cysts and benign tumors (adenomas) can develop in the pancreas, most exocrine pancreatic tumors are malignant.

Pancreatic carcinoma, particularly exocrine pancreatic carcinoma, and more specifically the most common carcinoma, ductal adenocarcinoma, is among the five most common causes of death in men and is the fourth leading cause of death in women. It is one of the tumors with the poorest prognosis, with survival rates of only 5% in men and 6% in women 5 years after diagnosis. The highest incidence is observed in the age range of 60-70 years (AIOM. Linea Guida Carcinoma del Pancreas Esocrino, ed. 2013).

The etiology of exocrine pancreatic carcinoma is unknown. A genetic predisposition (family inheritance) and certain risk factors have been established, such as smoking, fatty foods, type 2 diabetes, chronic pancreatitis, and exposure to environmental factors such as solvents or pesticides.

Exocrine pancreatic carcinoma is asymptomatic at an early stage, which explains the delay in diagnosis, which is usually made when the disease reaches advanced stages, except for incidental discovery during the diagnosis of other abdominal diseases.

Patients diagnosed with pancreatic cancer generally have a poor prognosis, given the diagnostic delay described above, only 15% of cases have tumor limited to the pancreas, while in the remaining cases, approximately 25% of patients have local-regional tumor spread lymph nodes, and in approximately 60% of cases the presence of metastases is detected.

The median survival time from cancer diagnosis is approximately three to six months, with a five-year survival rate well under 5%.

Treatment of pancreatic carcinoma includes surgery, when possible, also for palliative purposes.

Radical pancreatoduodenectomy is currently the only option for cure, especially with minimal spread of the disease.

Medical treatment, also related to radiation therapy, is limited to cases where surgery is not possible, or in the presence of metastases, or as an additional treatment after surgery. Although there are a small number of reports of individual patients who responded to gemcitabine or fluorouracil, or combination regimens using doxorubicin, methotrexate, cisplatin, oxaliplatin, irinotecan, erlotinib, etc., the results of chemotherapy are generally unsatisfactory and often no better than complete lack of treatment (Martindale, 31 ed., page 530).

Theve et al, in 1983, reviewed the possible influence of sex hormones on the pancreas, based on reports of the presence of protein receptors for steroids in pancreatic tissue, high activity of estrogen binding protein in the human pancreas, and the ability of human pancreatic tissue to convert the main peripheral estrogen, i.e. .e. estrone sulfate, to the final biologically active estradiol-17 beta.

Based on these premises, they tried tamokifen (an estrogen receptor antagonist) in patients with unresectable pancreatic adenocarcinoma and obtained some preliminary results similar to those obtained by Wong et al., in 1993.

Subsequent clinical practice did not produce the expected results, but the conclusion was that even if anti-estrogens are not the optimal form of therapy, other types of hormonal treatment for pancreatic cancer should be tried.

In view of the above, there is a significant need for new approaches to the treatment of tumors, in particular, to the treatment of carcinomas and, even more specifically, to the treatment of epithelial tumors, incl. prostate carcinoma or pancreatic carcinoma (preferably exocrine pancreatic carcinoma).

A number of compounds called 17α-monoesters, 21-monoesters and 17α,21-diesters of cortexolone, as well as methods for their preparation, are known in the art. WO 03/014141 describes compounds belonging to the family of steroids structurally related to cortexolone (which is also known as 11-deoxycortisone) as having primarily antiandrogenic activity. These compounds, such as Cortexolone 17α-propionate, work by interfering with the direct action of androgen hormones on the androgen receptor (AR) in tissues.

WO 2007/031349 discloses C ₃ -C ₁₀ 17α-esters of 9,11-dehydrocortexolone, i.e. derivatives structurally related to cortexolone as antigonadotropic agents that can be used to treat disorders closely associated with excess gonadotropin production.

WO 2009/019138 discloses an enzymatic process for the preparation of 17α-monoesters of cortexolone and 9,11-dehydrocortexolone; In addition, the application discloses the presence of several crystalline forms of cortexolone 17α-propionate, namely, crystalline form I, form II, form III and hydrate IV, as well as certain methods for their preparation.

Cyproterone acetate (abbreviated CPA) is a synthetic steroid that has been considered as a standard therapy for the treatment of androgen-sensitive tumors, particularly prostate cancer. Standard therapy with cyproterone acetate has been completely ineffective in tumors with reduced or absent expression of the androgen receptor (Br. J. Cancer (1989), 60,789-792).

It is known in the art that the presence of an ester group at position 17α gives the 17α-esters of cortexolone a broad spectrum of antiandrogenic activity, which has been demonstrated in animals (Celasco et al., Arzneim-Forsch 2005; 5:581-7).

In the present invention, it was unexpectedly discovered that 17α(alpha)-monoesters, 21-monoesters and 17α(alpha),21-diesters of cortexolone have previously unknown antitumor effects in both isolated in vitro cancer cell lines and xenograft models of prostate cancer and pancreas in vivo in animals.

The antitumor effect of the present invention was observed both for carcinoma cells bearing the androgen receptor (AR ⁺ ), for example, for LNCaP prostate cancer cells or Panc1 pancreatic cancer cells and, very surprisingly, for cells with absent or reduced expression of the androgen receptor (AR ^- ), for example, PC3 prostate cancer cells or MiaPaca pancreatic cancer cells. The antitumor effect of the present invention has also been shown for breast carcinomas (AR ^- ) and gastrointestinal tract carcinomas (AR ^- ).

BRIEF DESCRIPTION OF ILLUSTRATIVE MATERIAL

Hereinafter, the present invention will be described with the help of the following non-limiting drawings and examples.

Figure 1: Average fold change in pancreatic tumor volume measured relative to the start of subcutaneous (SC) administration of Cortexolone 17α-benzoate (referred to as “06” and “CB-03-06” in the drawings) at low dosage (230 μM) and high dosage (1150 µM), in an animal model of a nude mouse xenograft (pancreatic cell line MiaPaca). Data are from a vehicle control group of 0.4% (v/v) Tween80 and 0.5% (w/v) carboxymethylcellulose in normal saline. The compound and vehicle were administered subcutaneously (CS) to mice daily for 28 consecutive days. The “end of administration” arrows refer to the day the administration ended.

Figure 2: Average fold change in pancreatic tumor volume measured relative to the start of SC administration of Cortexolone 17α-valerate-21-propionate (referred to in the drawings as “10” and “CB-03-10”) at a low dosage (230 μM) and high dosage (1150 µM), in a nude mouse xenograft animal model (pancreatic cell line MiaPaca). Data are from a vehicle control group of 0.4% (v/v) Tween80 and 0.5% (w/v) carboxymethylcellulose in normal saline. The compound and vehicle were administered subcutaneously (CS) to mice daily for 28 consecutive days. The “end of administration” arrows refer to the day the administration ended.

Figure 3: Average fold change in pancreatic tumor volume measured in a nude mouse animal model (pancreatic cell line MiaPaca) relative to the start of SC administration of cyproterone acetate (referred to as CPA in the drawing), cortexolone 17α-valerate-21-propionate (referred to as drawing "10") and Cortexolone 17α-benzoate (referred to as "06" in drawing) (each compound at low dosage and high dosage) and vehicle (i.e. 0.4% (v/v) Tween80 and 0.5% (wt/vol) carboxymethylcellulose in normal saline) in the control group. Compound and vehicle were administered to all mice subcutaneously (CS) daily for 28 consecutive days (administration days). The “end of administration” arrows refer to the day the administration ended.

Figure 4: Graph showing P values relative to the vehicle control group (i.e., 0.4% (v/v) Tween80 and 0.5% (w/v) carboxymethylcellulose in normal saline), namely the best results from Figure 3. All mice received compound and SC vehicle daily for 28 consecutive days (administration days). The “end of administration” arrows refer to the day the administration ended.

Figure 5: Dose titration to determine the cytotoxicity of cortexolone derivatives in human prostate cancer (a) and pancreatic cancer (b) cell lines.

Figure 6: Androgen receptor expression levels on cancer cell lines.

Figure 7: Antagonistic activity of CB-03-10 and CB-03-05 against glucocorticoids. Mifepristone (a potent glucocorticoid antagonist) was used as a positive control.

Figure 8: Agonistic activity of CB-03-10 and CB-03-05 against glucocorticoids. Dexamethasone (a potent glucocorticoid agonist) was used as a positive control.

Figure 9: Initiation of apoptosis of MiaPaca2 cells by CB-03-10.

Figure 10: Initiation of cell cycle arrest in MiaPaca2 cells by CB-03-10 at various concentrations.

Figure 11: Caspase activation in MiaPaca2 cells over time (8-24-48 hours). 20 µM (striped bars) or 50 µM (solid bars) indicates the concentration of the compound. Gemcitabine, which is a potent cytotoxic drug against pancreatic cancer, was used as a positive control.

Figure 12: Caspase activation in LNCaP prostate cancer cell lines.

Figure 13: In vitro metabolism of CB-03-10 and CB-03-05 in (A) human and (B) rat plasma.

Figure 14: Evaluation of in vivo pharmacokinetics of CB-03-10 and CB-03-05 in mouse plasma after subcutaneous and oral administration.

Figure 15: In vivo antitumor activity of CB-03-10 when administered subcutaneously in a pancreatic cancer xenograft mouse model.

Figure 16: Antitumor activity of CB-03-10 in an in vivo mouse model of prostate cancer when administered by oral force feeding. Enzalutamide (Enza), which is a potent cytotoxic agent against prostate cancer, was used as a positive control. The results obtained showed that CB-03-10 was statistically significantly different from vehicle from day 7 to day 60. In contrast, enzalutamide was statistically significantly different from vehicle only on days 14 and 25. This comparison shows that CB-03-10 achieves high statistical significance against vehicle, whereas enzalutamide does not achieve statistical significance against vehicle.

Figure 17: in vitro inhibition of the initial level of PSA secretion from LNCaP cancer cells under the influence of CB-03-10 and CB-03-05.

Figure 18: Expression of androgen and glucocorticoid receptors in cancer cells of various lines.

DEFINITIONS

Unless otherwise indicated, it is understood that all technical terms, conventions and other scientific terminology used in the text of the application have the meanings usually understood by a person skilled in the field of technology to which the invention relates. In some cases, terms having generally accepted meanings are defined throughout the application for clarity and/or reference; therefore, the inclusion of these definitions in the text of the application should not be construed as significantly different from the meaning generally accepted in the art.

In particular, the terms "physiologically acceptable excipient" or "pharmaceutically acceptable excipient" as used herein refer to a substance that does not have any pharmacological action of its own and does not cause adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in the Handbook of Pharmaceutical Excipients, sixth edition (2009), which is incorporated herein by reference.

The term "alkyl" as used herein means a straight or branched chain saturated hydrocarbon.

The term "aryl" as used herein refers to aromatic mono- and poly-carbocyclic systems, wherein the individual carbocycles in polycarbocyclic systems may be fused or linked together by a single bond. Suitable "aryl" groups include, but are not limited to, phenyl, naphthyl, biphenyl, and the like.

The term "heteroaryl" as used herein refers to aromatic mono- and polycarbocyclic systems, wherein the cyclic system comprises at least one heteroatom, said heteroatom being selected from the group consisting of, but not limited to, nitrogen, sulfur, oxygen, and the like. and individual rings in a polycarbocyclic system can be condensed or connected to each other by a simple bond. Suitable "heteroaryl" groups include, but are not limited to, pyridyl, imidazolyl, pyrrolyl, furyl, benzimidazolyl, thiofuranyl, and the like.

The aryl groups may optionally be replaced at at least one of the cyclic carbon atoms by a group selected from lower alkyls, lower alkenyls, lower haloalkyls, lower haloalkenyls, lower alkoxy groups, lower chalkalkenyls, lower alkenyloxy groups, halogens, nitro, cyano, lower alkylthio groups and etc.

The heteroaryl groups may optionally be substituted at at least one of the cyclic carbon atoms or at one of the cyclic heteroatoms with a group selected from lower alkyls, lower haloalkyls, lower haloalkenyls, lower alkoxy groups, lower alkenyls, lower chalkalkenyls, lower alkenyloxy groups, halogens, nitro , cyano, lower alkylthio groups, etc.

The terms “about” and “approximately” as used herein refer to the range of experimental error that may be present in a measurement.

The terms “including,” “having,” and “comprising” should be understood as open-ended terms (i.e., meaning “including but not limited to”) and should be construed to include and/or serve as the basis for such terms , as in “consists mainly of”, “consisting mainly of”, “consists of” or “consisting of”.

The terms “consisting essentially of”, “consisting essentially of” should be understood as semi-closed terms meaning that the composition does not include other ingredients that significantly affect the basic and novel characteristics of the present invention (thus, the composition may include optional excipients).

The terms “consists of” and “consisting of” should be understood as closed terms.

As used herein, the terms “therapeutically effective amount” and “effective amount” refer to an amount sufficient to cause the desired biological response. In the present invention, the desired biological response is to suppress, reduce or attenuate the severity, duration, progression or onset of a disease, disorder or condition, prevent the development, recurrence or progression of a disease, disorder or condition, or a symptom associated with a disease, disorder or condition. The exact amount of compound administered to a subject will depend on the route of administration, the type and severity of the disease, disorder or condition, and on characteristics of the subject, such as general health, age, sex, body weight and drug tolerance. One skilled in the art should be able to determine appropriate dosages depending on these or other factors. Suitable dosages are known for approved pharmaceutical agents and can be adjusted by one skilled in the art according to the condition of the subject, the type of condition(s) being treated, and the amount of the compound described herein used. In cases where the amount is not explicitly stated, it should be assumed that it is an effective amount. For example, for therapeutic treatment, the compounds and pharmaceutical compositions described herein can be administered to a subject in dosages ranging from about 0.01 to 100 mg/kg body weight/day.

As used herein, the terms “treat” and “treatment” refer to therapeutic treatment and include reduction or amelioration of the progression, severity and/or duration of a disease, disorder or condition, or amelioration of one or more symptoms (specifically, one or more significant symptoms) of a disease , disorder or condition, resulting from the administration of one or more therapeutic agents (eg, one or more of the agents such as the compounds or compositions of the present invention). In specific embodiments, the therapeutic treatment includes alleviation of at least one measurable physical parameter of the disease, disorder or condition. In other embodiments, the therapeutic treatment includes suppressing the progression of the condition either physically, by, for example, stabilizing an expressed symptom, or physiologically, by, for example, stabilizing a physical parameter, or both. In other embodiments, therapeutic treatment includes alleviating or stabilizing a disease, disorder, or condition. The term "radical treatment" as used herein refers to treatment that is aimed at curing a disease or improving symptoms associated with a disease.

The term "palliative care" as used herein refers to treatment or therapy that is aimed at alleviating rather than curing a disease.

The term "adjunctive treatment" as used herein refers to a treatment that is prescribed in addition to the primary, primary, or initially administered treatment.

The term “neoadjuvant treatment” as used herein refers to treatment that is given before primary treatment to reduce the size or extent of the tumor, thereby reducing the consequences of more extensive treatment that would have been required if the tumor size or extent had not been reduced.

As indicated in the text of the application, the compounds of the present invention may optionally be replaced by one or more substituents, for example, described below in general terms, or included in specific examples of the molecules of the present invention. It should be understood that the phrase "optionally substituted" is interchangeable with the phrase "substituted or unsubstituted." Generally, the term “substituted,” whether preceded by the term “optionally” or not, refers to the substitution of one or more hydrogen atoms in a given structure with the remainder of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each position of the group available for substitution. If more than one position in a given structure can be replaced by more than one substituent selected from a specified group, the substituents at each position may be the same or different. If the term "optionally substituted" appears before a list, the specified term applies to all subsequent substituent groups in the list. If a substituting radical or structure is not specified or is defined as “optionally substituted,” the substituting radical or structure is unsubstituted.

The particular substituents and combinations of substituents provided by the present invention are those that result in the formation of stable or chemically possible compounds. The term “stable” as used herein refers to compounds that do not undergo significant changes under conditions that allow them to be prepared, detected, and specifically isolated, purified, and used for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that does not undergo significant changes when stored at a temperature of 40° C. or less, in the absence of moisture or other reactive agents, for at least a week. The present invention considers only those substituents and their combinations that lead to the formation of stable structures. These substituents and their combinations will be obvious to one of ordinary skill in the art and can be determined without too much experimental work.

The term “simultaneous, separate or sequential administration” as used herein refers to the administration of a first and a second compound at the same time or in such a manner that the two compounds act in the body of a patient at the same time, or to the administration of one compound after another compound in order to provide a therapeutic effect. In some embodiments, the compounds are taken with food. In other embodiments, the compounds are taken after a meal, for example, 30 minutes or 60 minutes after a meal. In some embodiments, one compound is administered to a patient a certain period of time after administration of the other compound.

As used herein, the terms “subject” and “patient” are used interchangeably. The terms "subject" and "patient" refer to animals (eg, birds such as chicken, partridge or turkey, or mammals), preferably "mammals" including. non-primates (eg, cows, pigs, horses, sheep, rabbits, guinea pigs, rats, cats, dogs and mice) and primates (eg, monkeys, chimpanzees and humans) and, more specifically, humans. In one embodiment, the subject is a human.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, it has been surprisingly discovered that certain cortexolone derivatives have therapeutically interesting antitumor properties and are capable of acting against tumors, preferably epithelial and/or hormone-dependent tumors.

In accordance with this general concept, the invention relates to compounds of formula (I)

(I)

where R is hydrogen or a C(O)-R ₁ fragment, where R ₁ is a linear alkyl chain containing from 2 to 5 carbon atoms, and where R' is a linear alkyl chain containing from 3 to 6 carbon atoms, or optionally a substituted aryl group, or an optionally substituted heteroaryl group.

Preferred compounds of formula (I) are those wherein R is hydrogen or a C(O)-R ₁ moiety wherein R ₁ is CH ₂ CH ₃ and wherein R' is a -(CH ₂ ) ₃ -CH moiety ₃ or phenyl.

The subject of the present invention are compounds of formula (I)

(I)

where R is a C(O)-R ₁ moiety, where R ₁ is hydrogen or a linear alkyl chain of 2 to 5 carbon atoms, and where R' is a linear alkyl chain of 3 to 6 carbon atoms, or optionally a substituted aryl group, or an optionally substituted heteroaryl group, wherein the substituents R ₁ and R' are not the same.

The subject of the present invention are compounds of formula (I)

(I)

where R is a C(O)-R ₁ fragment, where R ₁ is a linear alkyl chain containing from 2 to 5 carbon atoms, and where R' is a linear alkyl chain containing from 3 to 6 carbon atoms, or an optionally substituted aryl chain group, or an optionally substituted heteroaryl group, wherein the substituents R ₁ and R' are not the same.

Preferred compounds of formula (I) are those in which R is a C(O)-R ₁ moiety, wherein R ₁ is hydrogen or CH ₂ CH ₃ and wherein R' is a -(CH ₂ ) ₃ -CH ₃ moiety or phenyl, and the substituents R ₁ and R' are not the same.

Preferred compounds of formula (I) are those in which R is a C(O)-R ₁ moiety, wherein R ₁ is CH ₂ CH ₃ , and wherein R' is a -(CH ₂ ) ₃ -CH ₃ moiety or phenyl , and the substituents R ₁ and R' are not the same. The most preferred compound of formula (I) is a compound wherein R is a C(O)-R ₁ moiety, wherein R ₁ is a CH ₂ CH ₃ moiety, and wherein R' is a -(CH ₂ ) ₃ -CH ₃ moiety. which is Cortexolone 17α-valerate-21-propionate (also referred to herein as “10” or “CB-03-10”), the formula of which is given below:

Cortexolone 17α-valerato-21-propionate

(cortexolone 17α-valerate-21-propionate (CB-03-10))

Unless otherwise indicated, the structures depicted in the text of the application are intended to include all isomeric forms (eg, enantiomeric, diastereomeric, cis-trans, conformational, and rotational) of the structure shown. For example, the present invention includes R and S configurations for each asymmetric center, (Z) and (E) isomers at double bonds, and (Z) and (E) conformational isomers unless only one is specifically depicted of these isomers. As would be appreciated by one of ordinary skill in the art, the substituent is capable of freely rotating about any linkage that allows such rotation. Thus, individual stereochemical isomers, as well as mixtures of enantiomers, diastereomers, cis/trans, conformational and rotational isomers of the compounds of the present invention are included within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are included within the scope of the invention.

In addition, unless otherwise indicated, the structures depicted herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures presented, except for the replacement of hydrogen with deuterium or tritium, or the replacement of carbon with ¹³ C- or ¹⁴ C-rich carbon, are within the scope of the present invention. Such compounds are useful, for example, as assays or probes in biological assays. Such compounds, especially the deuterated analogues, may also have therapeutic applications.

The compounds of the present invention are identified in the text of the application by their chemical structures and/or chemical names. If a compound is referenced by both a structure and a chemical name, and the chemical structure given and the chemical name given conflict, the chemical structure is determinative of the identification of the compound.

Pharmaceutically acceptable salts, solvates, clathrates, prodrugs and other derivatives

The compounds described herein may be in free form or, if appropriate, in the form of salts. Those salts which are pharmaceutically acceptable are of particular interest because they are suitable for administering the compounds described below for medical purposes. Salts that are not pharmaceutically acceptable are useful in manufacturing, for isolation and purification purposes, and in some cases for use in separating stereoisomeric forms of the compounds of the present invention or their intermediates.

As used herein, the term “pharmaceutically acceptable salt” refers to salts of a compound that, according to sound medical judgment, are suitable for use in contact with tissues of humans and lower animals without unacceptable side effects such as toxicity, irritation, allergic reactions, and the like. and satisfy a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which reference is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. These salts can be obtained in situ during the final isolation and purification of the compounds.

It should be understood that the present invention includes mixtures/combinations of various pharmaceutically acceptable salts, as well as mixtures/combinations of free form compounds and pharmaceutically acceptable salts.

In addition to the compounds described herein, pharmaceutically acceptable solvates (eg, hydrates) and clathrates of these compounds may also be used in compositions for the treatment or prevention of the disorders described herein.

The term "pharmaceutically acceptable solvate" as used herein refers to a solvate formed by the association of one or more molecules of a pharmaceutically acceptable solvent with one of the compounds described in the present invention. The term "solvate" includes hydrates (eg, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, etc.).

As used herein, the term “hydrate” means a compound described herein or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

As used herein, the term “clathrate” means the compound described herein or a salt thereof in the form of a crystal lattice that contains voids (eg, channels) in which molecules of foreign substances (eg, solvent or water) are trapped.

In addition to the compounds described herein, pharmaceutically acceptable derivatives or prodrugs of these compounds may also be used in compositions for the treatment or prevention of the disorders described in the application.

The phrase "pharmaceutically acceptable derivatives or prodrugs" includes any pharmaceutically acceptable esters, ester salts or other derivatives, or salts thereof, of the compounds of the present invention, which, when administered to a recipient, are capable of forming, directly or indirectly, the compound of the present invention or a metabolite thereof, or residue having inhibitory activity. Particularly preferred derivatives or prodrugs are those products that increase the bioavailability of the compounds when the compounds are administered to a patient (for example, allowing the compound to more easily enter the bloodstream if administered orally) or that improve the delivery of the parent compound to a specific part of the body (for example, the brain or lymphatic system ) relative to the original compound.

Pharmaceutically acceptable prodrugs of the compounds of the present invention include, but are not limited to, esters, amino acid esters, phosphoric acid esters, metal salts and sulfonic acid esters.

Application in medicine

Another subject of the present invention relates to compounds of formula (I) for use as drugs.

Another subject of the present invention is Cortexolone 17α-valerate (also referred to as “05” or “CB-03-05” throughout the application), represented by the following formula:

CB-03-05 (cortexolone 17α-valerate)

for use as a medicine.

In one embodiment, the compounds of the present invention for use as drugs are Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05).

In another embodiment, the present invention relates to compositions and pharmaceutical compositions comprising compounds of formula (I) for use as a medicine.

In one embodiment, the present invention relates to compositions and pharmaceutical compositions comprising compounds such as cortexolone 17α-valerate-21-propionate (CB-03-10) and cortexolone 17α-valerate (CB-03-05), for use as a medicine.

In another aspect, the present invention relates to the specified compounds of formula (I), including Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), for use as glucocorticoid modulators receptor (GR), preferably glucocorticoid antagonists.

In another embodiment, the present invention relates to compositions and pharmaceutical compositions comprising compounds of formula (I) for use as glucocorticoid receptor (GR) modulators, preferably glucocorticoid antagonists.

In another embodiment, the present invention relates to compositions and pharmaceutical compositions comprising Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), for use as glucocorticoid receptor modulators (GR), preferably glucocorticoid antagonists.

In yet another aspect, the present invention relates to said compounds of formula (I) for use in the treatment of glucocorticoid-mediated diseases or disorders. In one embodiment, the compounds of the present invention useful in the treatment of a glucocorticoid-mediated disease or disorder are Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05) .

In a further aspect, the invention relates to said compounds of formula (I), including Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), for use in the treatment of precancerous conditions, dysplasia, metaplasia and tumor diseases, including malignant neoplasms and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative.

In the most preferred embodiment, compounds of formula (I), incl. Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), are intended for use as antineoplastic agents.

In one embodiment, said tumor diseases are solid tumors, preferably epithelial tumors, such as prostate carcinoma, breast carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably colon carcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma, adrenal carcinoma, etc. In a preferred embodiment of the present invention, said epithelial tumors are prostate carcinoma, pancreatic carcinoma (preferably exocrine pancreatic carcinoma), gastrointestinal carcinoma (preferably colon carcinoma) and breast carcinoma (preferably triple negative breast cancer) .

In a preferred embodiment of the present invention, the tumor disease is prostate cancer. In a preferred embodiment of the present invention, the prostate cancer is adenocarcinoma. In a preferred embodiment of the present invention, the tumor disease is prostate cancer with absent or reduced AR expression. In another preferred embodiment of the invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors.

In a most preferred embodiment, the compounds of formula (I), including cortexolone 17α-valerate-21-propionate (CB-03-10) and cortexolone 17α-valerate (CB-03-05), are intended for use as antineoplastic agents, where the tumor disease is prostate cancer with mutated or shortened androgen receptors. One of the particularly preferred uses of compounds of formula (I), incl. Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), is for use in the treatment of prostate cancers that have been or have become refractory to treatment with antiandrogens such as enzalutamide. This embodiment of the present invention is particularly preferred because it is now found that after 6 months of treatment, 30% of prostate cancers become refractory to enzalutamide because the ARs undergo mutation or change. Interestingly, these unresponsive cancer cells are characterized by up-regulation of GR. Compounds of formula (I), incl. Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05) may be used to treat such cancers because their activity is also mediated by GR.

In a preferred embodiment of the present invention, the exocrine pancreatic carcinoma is an adenocarcinoma. In a preferred embodiment, exocrine pancreatic cancer has absent or reduced expression of AR.

In a preferred embodiment of the present invention, said epithelial tumor is a gastrointestinal carcinoma (preferably a colon carcinoma).

In another preferred embodiment of the present invention, said epithelial tumor is breast carcinoma (preferably triple negative breast cancer). Optionally, the subject or patient being treated does not respond to standard treatment or has relapsed after standard treatment.

In a preferred embodiment, the present invention relates to compounds of formula (I) wherein R is C(O)-R ₁ , wherein R ₁ is hydrogen or a CH ₂ CH ₃ group, and R' is a -(CH ₂ ) moiety. ₃ -CH ₃ or a phenyl group, wherein the substituents R ₁ and R' are not the same, for use in the treatment of precancerous diseases, dysplasias, metaplasias and tumor diseases, including malignancies and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative. In the most preferred embodiment, the compound is for use as an antitumor agent.

In a preferred embodiment, the present invention relates to compounds of formula (I) in which R is C(O)-R ₁ , wherein R ₁ is a CH ₂ CH ₃ group, and R' is a -(CH ₂ ) ₃ - moiety. CH ₃ or a phenyl group, wherein the substituents R ₁ and R' are not the same, for use in the treatment of precancerous diseases, dysplasias, metaplasias and tumor diseases, including malignancies and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative. In the most preferred embodiment, the compound is for use as an antitumor agent.

In another embodiment, the present invention provides a compound of formula (I) wherein R is C(O)-R ₁ wherein R ₁ is a CH ₂ CH ₃ group and R' is a -(CH ₂ ) ₃ - moiety. CH ₃ which is cortexolone 17α-valerate-21-propionate (also referred to herein as “10” or “CB-03-10”), for use in the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative. In the most preferred embodiment, said compound is for use as an antitumor agent.

In one embodiment, said tumor diseases are solid tumors, preferably epithelial tumors, such as prostate carcinoma, breast carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably colon carcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma, adrenal carcinoma, etc.

In a preferred embodiment of the present invention, said epithelial tumors are prostate carcinoma, pancreatic carcinoma (preferably exocrine pancreatic carcinoma), gastrointestinal carcinoma (preferably colon carcinoma) and breast carcinoma (preferably triple negative breast cancer) .

In a preferred embodiment of the present invention, the tumor disease is prostate cancer. In a preferred embodiment of the present invention, the prostate cancer is adenocarcinoma. In a preferred embodiment of the present invention, the tumor disease is prostate cancer with mutated AR, absent or reduced AR expression.

In a preferred embodiment of the present invention, the exocrine pancreatic carcinoma is an adenocarcinoma. In a preferred embodiment, exocrine pancreatic cancer is characterized by the absence or decreased expression of AR.

Another subject of the present invention relates to compounds of formula (I) for use in the manufacture of a medicament for the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative. In one embodiment, the compounds of the present invention for the production of a medicament for the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases, are Cortexolone 17α-valerate-21-propionate (CB-03-10) and cortexolone 17α-valerate (CB-03-05). In the most preferred embodiment, the compounds of formula (I) are intended for use in the production of an antitumor agent. In one embodiment, the compounds of the present invention for use in the production of antineoplastic agents are Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05).

In one embodiment, said tumor diseases are solid tumors, preferably epithelial tumors, such as prostate carcinoma, breast carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably colon carcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma, adrenal carcinoma, etc.

In another aspect, the present invention relates to said compounds of formula (I) for use in the manufacture of a medicament for the treatment of a glucocorticoid-mediated disease or disorder.

In one aspect, the present invention relates to a method of treating precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases, wherein the method comprises administering to a subject in need an effective amount of a compound of formula (I):

(I)

where R is a C(O)-R ₁ moiety, where R ₁ is hydrogen or a linear alkyl chain of 2 to 5 carbon atoms, and where R' is a linear alkyl chain of 3 to 6 carbon atoms, or optionally a substituted aryl group, or an optionally substituted heteroaryl group, wherein the substituents R ₁ and R' are not the same. Preferably, said subject is a mammal. Said mammal is preferably human.

In one aspect, the present invention relates to a method of treating precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases, wherein the method comprises administering to a subject in need an effective amount of a compound of formula (I):

(I)

where R is a C(O)-R ₁ fragment, where R ₁ is a linear alkyl chain containing from 2 to 5 carbon atoms, and where R' is a linear alkyl chain containing from 3 to 6 carbon atoms, or an optionally substituted aryl chain group, or an optionally substituted heteroaryl group, wherein the substituents R ₁ and R' are not the same. Preferably, said subject is a mammal. Said mammal is preferably human.

In one embodiment, said tumor diseases are solid tumors, in particular epithelial tumors, such as, for example, prostate carcinoma, breast carcinoma, uterine carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably carcinoma colon), kidney cancer, thyroid carcinoma, adrenal carcinoma, etc.

In one embodiment, said tumor diseases are solid tumors, in particular epithelial tumors, such as, for example, prostate carcinoma, breast carcinoma, uterine carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably carcinoma colon), kidney cancer, thyroid carcinoma, uterine carcinoma, adrenal carcinoma, etc.

In a preferred embodiment of the present invention, said epithelial tumors are prostate carcinoma or pancreatic carcinoma, more preferably exocrine pancreatic carcinoma, or mammary carcinoma, for example, triple negative breast cancer. In a preferred embodiment of the present invention, the tumor disease is prostate cancer with absent or reduced AR expression. In another preferred embodiment of the invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors. In this case, the prostate cancer that can be treated in accordance with the present invention may be or become refractory to antiandrogen therapy, such as enzalutamide.

In a preferred embodiment, the method comprises administering to a mammal in need an effective amount of a compound of formula (I) wherein R is C(O)-R ₁ , wherein R ₁ is hydrogen or a CH ₂ CH ₃ moiety, and R' represents -(CH ₂ ) ₃ -CH ₃ or a phenyl group, where R ₁ and R' are unequal substituents.

In a preferred embodiment, the method comprises administering to a mammal in need an effective amount of a compound of formula (I) wherein R is C(O)-R ₁ , wherein R ₁ is a CH ₂ CH ₃ moiety, and R' is - (CH ₂ ) ₃ -CH ₃ or a phenyl group, where R ₁ and R' are unequal substituents. In a most preferred embodiment, the method comprises administering an effective amount of a compound of formula (I) wherein R is C(O)-R ₁ , R ₁ is a CH ₂ CH ₃ moiety, and R' is -(CH ₂ ) ₃ -CH ₃ , i.e. Cortexolone 17α-valerate-21-propionate (CB-03-10).

In one aspect, the present invention relates to a method of treating precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases, wherein the method comprises administering to a subject in need an effective amount of Cortexolone 17α-valerate (CB-03-05 ). Preferably, said subject is a mammal. Said mammal is preferably a human.

In one embodiment, said tumor diseases are solid tumors, in particular epithelial tumors, such as, for example, prostate carcinoma, breast carcinoma, uterine carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably carcinoma colon), kidney cancer, thyroid carcinoma, adrenal carcinoma, etc.

In a preferred embodiment of the present invention, said epithelial tumors are prostate carcinoma or pancreatic carcinoma, more preferably exocrine pancreatic carcinoma, or mammary carcinoma, for example, triple negative breast cancer. The compounds of the present invention can be used in various therapeutic applications, in particular oncological applications. In more detail, the compounds of the present invention described herein have been found to be particularly effective for the definitive, adjuvant, neoadjuvant or palliative treatment of pancreatic carcinoma, preferably exocrine pancreatic carcinoma, as well as prostate carcinoma. Illustrations of the pharmacological properties of the compounds of the present invention can be found below in the text of the application in the experimental section.

The compounds of formula (I) can be prepared by any known method, for example, the method disclosed in WO 03/014141 and WO 2009/019138, the contents of each of these documents being incorporated herein in their entirety by reference. According to one embodiment of the present invention, Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05) can be prepared by the method disclosed in Example 11.

Pharmaceutical compositions

The compounds described herein can be formulated into pharmaceutical compositions that further contain a pharmaceutically acceptable carrier, diluent, adjuvant or excipient. In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of the present invention described herein, as well as a pharmaceutically acceptable carrier, diluent, adjuvant or excipient. In one embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable carrier, diluent, adjuvant or excipient. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers appropriately selected for the intended form of administration and in accordance with standard pharmaceutical practice.

Another subject of the present invention relates to a pharmaceutical composition comprising, as an active ingredient, at least one compound of formula (I), in which R is C(O)-R ₁ , where R ₁ is hydrogen or a linear alkyl chain containing from 2 up to 5 carbon atoms, and where R' means a linear alkyl chain containing from 3 to 6 carbon atoms or an optionally substituted aryl group, or an optionally substituted heteroaryl group, where R ₁ and R' are unequal substituents, in combination with at least one physiologically acceptable excipient.

Another subject of the present invention relates to a pharmaceutical composition comprising, as an active ingredient, at least one compound of formula (I), in which R is C(O)-R ₁ , where R ₁ is a linear alkyl chain containing from 2 to 5 carbon atoms, and where R' means a linear alkyl chain containing from 3 to 6 carbon atoms or an optionally substituted aryl group, or an optionally substituted heteroaryl group, where R ₁ and R' are unequal substituents, in combination with at least one physiologically acceptable excipient . According to a preferred embodiment of the present invention, said pharmaceutical composition comprises, as an active ingredient, at least one compound of formula (I), in which R is C(O)-R ₁ , where R ₁ is hydrogen or a -CH ₂ CH ₃ moiety, and R' represents -(CH ₂ ) ₃ -CH ₃ or a phenyl group, and R ₁ and R' are not the same substituents.

According to a preferred embodiment of the present invention, said pharmaceutical composition comprises, as an active ingredient, at least one compound of formula (I) in which R is C(O)-R ₁ , wherein R ₁ is a -CH ₂ CH ₃ moiety, and R ' represents -(CH ₂ ) ₃ -CH ₃ or a phenyl group, where R ₁ and R' are unequal substituents. According to a most preferred embodiment, said pharmaceutical composition comprises 17α-valerate-21-propionate (CB-03-10) as an active ingredient of cortexolone in combination with at least one physiologically acceptable excipient. Another subject of the present invention is a pharmaceutical composition comprising, as an active ingredient, cortexolone 17α-valerate (CB-03-05) in combination with at least one physiologically acceptable excipient.

In another embodiment, the compounds and pharmaceutical compositions of the present invention are intended for use as a medicine, preferably in the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases; in another aspect, such treatment may be definitive, adjuvant, neoadjuvant, or palliative. In this case, they are used as antitumor agents. Preferably, said tumor diseases are solid tumors. More preferably, said solid tumors are epithelial tumors, such as, for example, prostate carcinoma, breast carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably colon carcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma , adrenal carcinoma, etc. In a preferred embodiment of the present invention, said epithelial tumors are prostate carcinoma and pancreatic carcinoma, more preferably exocrine pancreatic carcinoma, or mammary carcinoma, for example, triple negative breast cancer. In another preferred embodiment of the present invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors. In the most preferred embodiment, pharmaceutical compositions comprising compounds of formula (I), incl. Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), are intended for use as antineoplastic agents where the tumor diseases are prostate cancer with mutated or truncated androgen receptors. In this case, the prostate cancer that can be treated according to the present invention may be or become refractory to antiandrogen therapy, for example, enzalutamide.

As stated above, in another embodiment, the invention relates to pharmaceutical compositions comprising compounds of formula (I), incl. Cortexolone 17α-valerate-21-propionate (CB-03-10) and Cortexolone 17α-valerate (CB-03-05), for use as a glucocorticoid receptor (GR) modulator, preferably a glucocorticoid antagonist.

The pharmaceutical compositions of the present invention may be in solid form, such as the form of powders, freeze-dried powders, granules, dragees, tablets or capsules. If desired, certain sweetening, flavoring or coloring agents may be added. The compounds of the present invention may also be in microencapsulated form with one or more excipients. Solid dosage forms, such as tablets, capsules, pills and granules, can be prepared with coatings and coatings, for example, enteric coatings and other coatings well known in the pharmaceutical formulation art. Excipients suitable for solid pharmaceutical compositions may be selected, without any limitation, from categories known to one skilled in the art, such as adsorbents, fillers, surfactants, compression aids, binders, lubricants, disintegrants, diluents, disintegrants, flow aids, freeze-drying aids, glidants, lyophilization aids, film-forming agents, dyes, antioxidants, etc. By way of example, suitable excipients for solid pharmaceutical compositions may be selected from, but not limited to, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose and its derivatives, polyvinylpyrrolidone, coating agents, dyes and waxes . Any mixture of these excipients may be suitable for use in the present invention.

According to the present invention, solid pharmaceutical compositions, for example, tablets, granules, dragees, capsules and the like, can be prepared in the form of immediate release forms, delayed release forms, controlled release forms, sustained release forms or sustained release, and also suitable for oral, sublingual or implant administration.

Controlled-release, sustained-release or extended-release compositions can be prepared using any known method or system, for example according to WO 00/76478, incorporated herein by reference in its entirety.

The pharmaceutical compositions of the present invention may also be in liquid form, for example, in the form of solutions, emulsions, suspensions or syrups. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art. In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents and fragrances. Suitable excipients for liquid pharmaceutical compositions can be selected, without any limitation, from classes of components well known to one skilled in the art, for example, solvents, co-solvents, oil carriers, buffering agents, surfactants, emulsifying agents, solubility enhancing agents, suspending agents agents, solubilizing agents, chelating agents, acidifying agents, alkalizing agents, antioxidants, preservatives, osmotic agents, tonic agents, viscosity adjusting agents, and the like. By way of example, suitable pharmaceutical excipients for liquid preparations may be selected from water for injection, organic solvents or cosolvents such as ethanol, glycols, glycerol and mixtures thereof, natural oils such as soybean oil, medium chain triglycerides, polyoxyl 15- hydroxystearate, polysorbate 80, polyoxyl 35-castor oil, sodium chloride, sodium phosphate, potassium phosphate, etc. According to the present invention, said liquid pharmaceutical compositions may be sterile or non-sterile. In one embodiment, the liquid pharmaceutical compositions are terminally sterilized using techniques well known to one skilled in the art, such as dry heat sterilization, moist heat sterilization, gamma irradiation, electron beam sterilization, and the like. In another embodiment, the liquid pharmaceutical compositions are sterilized by sterilizing filtration and placed under aseptic conditions in final primary packaging containers. The liquid pharmaceutical compositions of the present invention can be used for injection, infusion or perfusion, for example, for intravenous, intramuscular, intraperitoneal, subcutaneous or intratumoral administration.

Routes of administration

The compounds and compositions of the present invention can be administered orally, parenterally, by inhalation sprays, topically, rectally, nasally, buccally, vaginally or by implants containing a certain amount of the active substance. The term “parenteral” as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusions.

Injectable preparations, for example, sterile or oleaginous injectable suspensions, can be prepared according to techniques known in the art, using suitable dispersing or wetting agents, as well as suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in a non-toxic diluent or solvent suitable for parenteral use. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents into sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

Sterile injectable forms of the compounds and compositions of the present invention may be aqueous or oily suspensions. These suspensions can be prepared by methods known in the art, using suitable dispersing or wetting agents, as well as suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in a non-toxic diluent or solvent suitable for parenteral use.

Compounds for use in the methods of the present invention may be included in dosage forms. The term "dosage form" refers to physically discrete units suitable as unitary doses for subjects being treated, where each of these units contains a predetermined amount of active ingredient calculated to achieve the desired therapeutic effect, optionally in combination with a suitable pharmaceutical carrier . The dosage form may be designed to administer a full daily dose or one of several portions of a daily dose (eg, about 1 to 4 or more times per day). If multiple doses per day are used, the dosage forms may be the same or different for each of these administrations.

According to the present invention, the compounds of formula (I) or pharmaceutical compositions comprising these compounds are preferably administered by intravenous injection, more preferably using an infusion bag, syringe or pump catheter, or intramuscular injection, or subcutaneous injection, or orally (orally). in the form of tablets or capsules.

According to one embodiment, said pharmaceutical composition is a liquid dosage form suitable for injection and comprises a cortexolone derivative of formula (I) in an amount of from 0.1% to 50% weight to volume (w/v), preferably from 0.25 % to 25% w/v, more preferably 0.5% to 10% w/v, even more preferably 1% to 5% w/v.

According to another embodiment, said pharmaceutical composition is in solid form and comprises a cortexolone derivative of formula (I) in an amount of from 0.1% to 50% weight by weight (w/w), preferably from 0.5% to 40% by weight /wt, more preferably from 1% to 30% wt/wt.

The amount of at least one compound of formula (I) in said pharmaceutical composition is such that an effective dosage level can be obtained when administered to a mammal suffering from precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases. The compounds of formula (I) and pharmaceutical compositions containing them as active ingredients, intended for use in the definitive, adjuvant, neoadjuvant or palliative treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases, are preferably administered to mammals, wherein said mammals are humans or animals, preferably humans.

Combination therapy

According to another embodiment, the compounds, compositions and pharmaceutical compositions may contain at least one other active ingredient, preferably a chemotherapeutic active ingredient, as a combination for simultaneous, separate or sequential administration.

In some embodiments, compounds of formula (I) and pharmaceutical compositions comprising at least one compound of formula (I) and at least one physiologically acceptable excipient of the present invention can be used in combination therapy with at least one other drug, in particular chemotherapy drug. In some embodiments, the compounds of the present invention can be administered simultaneously with the administration of another drug, in particular a chemotherapy drug. In some embodiments, the compounds of the present invention can be administered before or after the administration of another drug, in particular a chemotherapy drug. The at least one other drug, in particular a chemotherapy drug, may be effective for treating the same or another disease, disorder or condition. The methods of the present invention include the administration of one or more compounds of formula (I) or pharmaceutical compositions comprising at least one compound of formula (I) of the present invention and at least one other drug, preferably a chemotherapy drug, provided that the combined administration does not weaken therapeutic efficacy of said one or more compounds of the present invention and/or does not cause unacceptable undesirable combination effects.

Cortexolone 17α-valerate (also referred to herein as “05” or “CB-03-05”)

As stated above, another subject of the present invention is Cortexolone 17α-valerate (also referred to herein as "05" or "CB-03-05") represented by the following formula:

CB-03-05 (cortexolone 17α-valerate)

for use as a medicine.

In the most preferred embodiment, Cortexolone 17α-valerate is intended for use in the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, including malignancies and metastases. Preferably, Cortexolone 17α-valerate is intended for use as an antitumor agent. Preferably, the tumor diseases are solid tumors, preferably epithelial tumors. Epithelial tumors can be selected from prostate carcinoma; breast carcinoma; pancreatic carcinoma (preferably exocrine pancreatic carcinoma); lung carcinoma; carcinomas of the gastrointestinal tract, such as colon carcinoma; kidney cancer; thyroid carcinoma; uterine carcinoma; and adrenal carcinoma.

In one embodiment, the epithelial tumor is prostate carcinoma. In another preferred embodiment of the invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors. In this case, the prostate cancer that can be treated according to the present invention may be or become refractory to antiandrogen therapy, for example, enzalutamide.

In another embodiment, the epithelial tumor is a pancreatic carcinoma, preferably an exocrine pancreatic carcinoma.

In one embodiment, the epithelial tumor is a breast carcinoma, preferably a triple negative breast cancer (TNBC). In one embodiment, the breast carcinoma is triple negative breast cancer and the disease is recurrent or unresponsive to conventional therapy.

In another embodiment, the epithelial tumor is a carcinoma of the gastrointestinal tract, such as colon carcinoma.

In another embodiment, Cortexolone 17α-valerate is for use as a glucocorticoid receptor (GR) modulator, preferably a glucocorticoid antagonist.

According to another aspect, the invention relates to a pharmaceutical composition comprising a compound of the following structural formula:

and at least one physiologically acceptable excipient, for use as a medicament, preferably in the treatment of precancerous diseases, dysplasias, metaplasias and neoplastic diseases, optionally including malignancies and metastases. Preferably, said tumor diseases are solid tumors, preferably epithelial tumors, for example prostate carcinoma; breast carcinoma; pancreatic carcinoma; lung carcinoma; carcinoma of the gastrointestinal tract, for example, colon carcinoma; kidney cancer; thyroid carcinoma; uterine carcinoma; and adrenal carcinoma.

According to another embodiment, said epithelial tumor is prostate carcinoma. In another preferred embodiment of the invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors. In this case, the prostate cancer that can be treated according to the present invention may be or become refractory to antiandrogen therapy, for example, enzalutamide.

In another embodiment, the epithelial tumor is a pancreatic carcinoma, preferably an exocrine pancreatic carcinoma.

In another embodiment, the epithelial tumor is a breast carcinoma, preferably a triple negative breast cancer (TNBC). In one embodiment, the breast carcinoma is triple negative breast cancer and the disease is recurrent or unresponsive to conventional therapy.

In yet another embodiment, the epithelial tumor is a carcinoma of the gastrointestinal tract, such as colon carcinoma.

The pharmaceutical composition may also include at least one other active ingredient, preferably a chemotherapeutic active ingredient, for simultaneous, separate or sequential administration.

According to another aspect, the invention relates to a pharmaceutical composition comprising a compound of the following structural formula:

and at least one physiologically acceptable excipient for use as a glucocorticoid receptor (GR) modulator, preferably a glucocorticoid antagonist.

In another aspect, the invention relates to a method of treating precancerous diseases, dysplasias, metaplasias and neoplastic diseases in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a compound of the following structural formula:

or a pharmaceutical composition comprising said compound.

According to one embodiment, the tumor diseases are malignant neoplasms or metastases.

Preferably, the subject is a mammal. In the most preferred embodiment, the mammal is human.

In one embodiment, the tumor diseases are solid tumors. Optionally, solid tumors are epithelial tumors. The epithelial tumors may be selected from prostate carcinoma, breast carcinoma, uterine carcinoma, pancreatic carcinoma, lung carcinoma, gastrointestinal carcinoma (preferably colon carcinoma), kidney cancer, thyroid carcinoma, uterine carcinoma, adrenal carcinoma, etc. .P.

In another embodiment, the epithelial tumors are prostate carcinoma, pancreatic carcinoma, exocrine pancreatic carcinoma, or breast carcinoma.

According to another embodiment, said epithelial tumor is prostate carcinoma. In another preferred embodiment of the invention, the tumor disease is prostate cancer with mutated or truncated androgen receptors. In this case, the prostate cancer that can be treated according to the present invention may be or become refractory to antiandrogen therapy, for example, enzalutamide.

In another embodiment, the epithelial tumor is a pancreatic carcinoma, preferably an exocrine pancreatic carcinoma.

In another embodiment, the breast carcinoma is a triple negative breast cancer. In one embodiment, the breast carcinoma is triple negative breast cancer and the disease is recurrent or unresponsive to conventional therapy.

In another embodiment, the epithelial tumor is a carcinoma of the gastrointestinal tract, such as colon carcinoma.

According to yet another aspect, the invention relates to a method of treating a glucocorticoid-mediated disease or disorder in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of cortexolone 17α-valerate or a pharmaceutical composition comprising cortexolone 17α-valerate.

EXAMPLES

Example 1: In vitro antitumor activity of cortexolone 17α-benzoate (CB-03-06) against prostate cancer cell lines

This experiment was conducted to investigate and determine the in vitro antitumor activity of cortexolone 17α-benzoate against prostate cancer cell lines LNCaP (AR ⁺ ) and PC3 (AR ^- ), positive or negative for androgen receptor (AR) expression, respectively.

The experimental procedure was as follows:

1. 3000 cancer cells were seeded in 96-well flat-bottomed plates in complete medium containing 2% bovine serum purified on activated carbon.

2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or without antiandrogenic compounds or DMSO vehicle (negative control) was added to the culture.

3. After 3 days, the number of surviving cells was counted using an ATP-dependent proliferation assay.

The purpose of the described test was to determine the concentration at which each compound kills 50% of cancer cells (IC ₅₀ ) for the potential use of the compound in an in vivo animal test.

Data from Experiment 1 were fitted to sigmoidal dose-response curves and analyzed using the Prizm statistical analysis program. Data from Experiment 2 were analyzed by curve fitting using nonlinear least squares regression in the statistical analysis program Prizm.

The IC ₅₀ values determined for each cell line are placed in the table below in comparison with well known standards, i.e. The most effective antiandrogen steroids, namely CPA and enzalutamide, are oral androgen receptor inhibitors that can prolong the survival of men with metastatic castration-resistant prostate cancer used to treat prostate cancer. The results of 2 series of experiments are presented.

Experiment 1

The results were fitted with sigmoidal dose-response curves using the statistical analysis program Prizm.

Tumor cell lines IC ₅₀ (microM)
CB-03-06 (cortexolone 17α-benzoate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide LNCaP 12 29 40 PC3 29 98 208

The IC ₅₀ values demonstrate that the antitumor activity of cortexolone 17α-benzoate, although tending to be weakly correlated, could not be considered strictly dependent on androgen receptor expression, unlike the reference compounds.

Experiment 2

The results below were obtained from experiments complementary to those described in Experiment 1. The results were analyzed using nonlinear least squares regression curve fitting in the Prizm statistical analysis program.

Tumor cell lines IC ₅₀ (microM)
CB-03-06 (cortexolone 17α-benzoate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide LNCaP 12 22 38 PC3 28 90 180

Example 2: In vitro antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) against prostate cancer cell lines

This experiment was conducted to study and determine the in vitro antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) against representatives of prostate cancer cell lines LNCaP (AR ⁺ ) and PC3 (AR ^- ), positive or negative for expression AR, respectively.

The experimental procedure was as follows:

1. 3000 cancer cells were seeded in 96-well flat-bottomed plates in complete medium containing 2% bovine serum purified on activated carbon.

2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or without antiandrogenic compounds or DMSO vehicle (negative control) was added to the culture.

3. After 3 days, the number of surviving cells was counted using an ATP-dependent proliferation assay.

The purpose of the described test was to determine the concentration at which each compound kills 50% of cancer cells (IC ₅₀ ) for the potential use of the compound in an in vivo animal test.

Data from Experiment 1 were fitted to sigmoidal dose-response curves and analyzed using the Prizm statistical analysis program. Data from Experiment 2 were analyzed by curve fitting using nonlinear least squares regression in the statistical analysis program Prizm.

The IC ₅₀ values determined for each cell line are listed in the table below, in comparison with well known standards, i.e. The most effective antiandrogen steroids, namely CPA and enzalutamide, are oral androgen receptor inhibitors that can prolong the survival of men with metastatic castration-resistant prostate cancer.

Experiment 1

The results were fitted with sigmoidal dose-response curves using the statistical analysis program Prizm.

Tumor cell lines IC ₅₀ (microM)
CB-03-10 (cortexolone 17α-valerate-21-propionate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide LNCaP 13 29 40 PC3 55 98 208

Experiment 2

The results below were obtained from experiments complementary to those described in Experiment 1. The results were analyzed using nonlinear least squares regression curve fitting in the Prizm statistical analysis program.

Tumor cell lines IC ₅₀ (microM)
CB-03-10 (cortexolone 17α-valerate-21-propionate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide LNCaP 10 22 38 PC3 50 90 180

IC ₅₀ values demonstrate that the antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) is possibly correlated with androgen receptor expression in cell lines.

Example 3: In vitro antitumor activity of cortexolone 17α-benzoate (CB-03-06) against pancreatic cancer cell lines

This experiment was performed to investigate and determine the in vitro antitumor activity of cortexolone 17α-benzoate against two pancreatic tumor cell lines Panc1 (AR ⁺ ) and MiaPaca2 (low AR expression), i.e. representatives of pancreatic cancer cell lines.

These lines were also classified as positive (AR ⁺ ) or negative/low expression (AR ^+/- ) according to the presence and expression of androgen receptors.

The experimental procedure was as follows:

1. 3000 cancer cells were seeded in 96-well flat-bottomed plates in complete medium containing 2% bovine serum purified on activated carbon.

2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or without antiandrogenic compounds or DMSO vehicle (negative control) was added to the culture.

3. After 3 days, the number of surviving cells was counted using an ATP-dependent proliferation assay.

The purpose of the described test was to determine the concentration at which each compound kills 50% of cancer cells (IC ₅₀ ) for the potential use of the compound in an in vivo animal test.

Data from Experiment 1 were fitted to sigmoidal dose-response curves and analyzed using the Prizm statistical analysis program. Data from Experiment 2 were analyzed by curve fitting using nonlinear least squares regression in the statistical analysis program Prizm.

The IC ₅₀ values determined for each cell line are placed in the table below in comparison with well known standards, i.e. The most effective antiandrogenic steroids are CPA and enzalutamide, both potent oral AR antagonists.

Experiment 1

The results were fitted with sigmoidal dose-response curves using the statistical analysis program Prizm.

Tumor cell lines IC ₅₀ (microM)
CB-03-06 (cortexolone 17α-benzoate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide Panc1 thirty 54 156 MiaPaca2 23 46 77

Experiment 2

The results below were obtained from experiments complementary to those described in Experiment 1. The results were analyzed using nonlinear least squares regression curve fitting in the Prizm statistical analysis program.

Tumor cell lines IC ₅₀ (microM)
CB-03-06 (cortexolone 17α-benzoate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide Panc1 28 46 111 MiaPaca2 20 39 65

The IC ₅₀ values demonstrate that the antitumor activity of cortexolone 17α-benzoate is at least twice that of the reference compounds (CPA and enzalutamide). Because MiaPaca2 cells have low/null AR expression, the anticancer activity of the compound is not directly correlated with androgen receptor expression in cancer cell lines.

Example 4: In vitro antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) against pancreatic cancer cell lines

This experiment was conducted to investigate and determine the in vitro antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) against pancreatic tumor cell lines, namely Panc1 (AR ⁺ ) and MiaPaca2 (low AR expression), those. representatives of pancreatic cancer cell lines.

These lines were also classified as positive (AR ⁺ ) or negative/low expression (AR ^+/- ) based on the presence and expression of androgen receptors.

The experimental procedure was as follows:

1. 3000 cancer cells were seeded in 96-well flat-bottomed plates in complete medium containing 2% bovine serum purified on activated carbon.

2. After 24 hours, 10 nM DHT (dihydrotestosterone) with or without antiandrogenic compounds or DMSO vehicle (negative control) was added to the culture.

3. After 3 days, the number of surviving cells was counted using an ATP-dependent proliferation assay.

The purpose of the described test was to determine the concentration at which each compound kills 50% of cancer cells (IC ₅₀ ) for the potential use of the compound in an in vivo animal test.

Data from Experiment 1 were fitted to sigmoidal dose-response curves and analyzed using the Prizm statistical analysis program. Data from Experiment 2 were analyzed by curve fitting using nonlinear least squares regression in the statistical analysis program Prizm.

The IC ₅₀ values determined for each cell line are placed in the table below in comparison with well known standards, i.e. The most effective antiandrogenic steroids are CPA and enzalutamide, oral AR antagonists.

Experiment 1

The results were fitted with sigmoidal dose-response curves using the statistical analysis program Prizm.

Tumor cell lines IC ₅₀ (microM)
CB-03-10 (cortexolone 17α-valerate-21-propionate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide Panc1 66 54 156 MiaPaca2 43 46 77

Experiment 2

The results below were obtained from experiments complementary to those described in Experiment 1. The results were analyzed using nonlinear least squares regression curve fitting in the Prizm statistical analysis program.

Tumor cell lines IC ₅₀ (microM)
CB-03-10 (cortexolone 17α-valerate-21-propionate) IC ₅₀ (microM)
Cyproterone acetate IC ₅₀ (microM)
Enzalutamide Panc1 60 46 111 MiaPaca2 37 39 65

IC ₅₀ values demonstrate that the antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) does not correlate with androgen receptor expression in pancreatic cancer cell lines.

Example 5: in vivo human pancreatic tumor xenograft in mice

The effect of cortexolone 17α-benzoate (CB-03-06) on pancreatic tumor xenograft growth in male nude mice was assessed in comparison with the most effective antiandrogenic steroid cyproterone acetate (CPA).

Cortexolone 17α-benzoate and cyproterone acetate were separately diluted in DMSO/2-hydroxypropyl β-cyclodextrin (vehicle).

The study compared the antitumor activity of cortexolone 17α-benzoate at two different dosages (8.0 mg/kg, corresponding to approximately 230 μM, and 40 mg/kg, corresponding to approximately 1150 μM), with the activity of the vehicle (i.e. 0 .4% (v/v) tween 80 and 0.5% (w/v) carboxymethylcellulose in normal saline) and with the activity of the comparator cyproterone acetate at two different dosages (7.4 mg/kg and 37 mg/kg) .

1x10 ⁶ MiaPaca-2 cells suspended in Matrigel were administered by subcutaneous injection to 6-week-old athymic nude mice.

Administration of test compounds, vehicle, and reference compounds began when the tumor volume after transplantation reached 50 mm ³ . All compounds were injected in a volume of 100 μl/mouse low dose solution (approximately 230 μM) or 100 μl/mouse high dose solution (approximately 1150 μM) cortexolone 17α-benzoate, vehicle, and cyproterone acetate, respectively. Compounds and control solutions were administered subcutaneously on a daily basis for 28 days.

Tumors were measured every 4 days using a digital caliper.

The results are shown in figure 1 as the average change in tumor volume relative to the start of drug administration. Tumor volume was calculated using the formula 0.5236(r ₁ ) ² (r ₂ ), where r ₁ <r ₂ .

Error bars represent SEM for 7–10 animals per group. P values were calculated according to Student's t test.

A high dose of Cortexolone 17α-benzoate was able to keep pancreatic tumor volume to less than a 5-fold increase relative to the tumor volume at the beginning of administration. In contrast, the mean tumor volume in the vehicle and cyproterone acetate groups increased up to 12-fold. From the data obtained, the antitumor activity of the compound of the present invention, namely cortexolone 17α-benzoate, is obvious.

Example 6: in vivo human pancreatic tumor xenograft in mice

The activity of cortexolone 17α-valerate-21-propionate (CB-03-10) in a pancreatic tumor xenograft model in male nude mice was evaluated in comparison with the antiandrogenic steroid cyproterone acetate (CPA).

Cortexolone 17α-valerate-21-propionate (CB-03-10) and cyproterone acetate were separately diluted in DMSO/2-hydroxypropyl β-cyclodextrin (vehicle).

The study compared the antitumor activity of cortexolone 17α-valerate-21-propionate (CB-03-10) at two different dosages (approximately 8.6 mg/kg and 43 mg/kg) with that of the vehicle (i.e., 0. 4% (v/v) tween 80 and 0.5% (w/v) carboxymethylcellulose in normal saline) and with the activity of the comparator cyproterone acetate at two different dosages (7.4 mg/kg and 37 mg/kg).

1x10 ⁶ MiaPaca-2 cells suspended in Matrigel were administered by subcutaneous injection to 6-week-old athymic nude mice.

Administration of test compounds, vehicle, and reference compound began when the tumor volume after transplantation reached 50 mm ³ by subcutaneous injection of 100 μl/mouse of the low-dose solution (approximately 230 μM) or 100 μl/mouse of the high-dose solution ( approximately 1150 µM) cortexolone 17α-valerate-21-propionate (CB-03-10), vehicle and cyproterone acetate, respectively. Compounds and control solutions were administered subcutaneously on a daily basis for 28 days.

Tumors were measured every 4 days using a digital caliper.

The results are displayed in figure 2 as the average change in tumor volume relative to the start of drug administration. Tumor volume was calculated using the formula 0.5236(r ₁ ) ² (r ₂ ), where r ₁ <r ₂ .

Error bars represent SEM for 7–10 animals per group. P values were calculated according to Student's t test.

A high dose of Cortexolone 17α-valerate-21-propionate (CB-03-10) was able to maintain pancreatic tumor volume to less than a 5-fold increase relative to tumor volume at baseline. In addition, when the drugs were stopped, there was a tendency for tumor volume to re-expand, but at a slower rate and to a smaller size. In contrast, the mean tumor volume in the vehicle and cyproterone acetate groups increased by up to 12-fold or more, necessitating the sacrifice of some animals in these groups for ethical reasons. From the data obtained, the antitumor activity of the compound of the present invention, namely cortexolone 17α-valerate-21-propionate (CB-03-10), is evident.

Data from Examples 5 and 6 confirmed the in vivo activity of Cortexolone 17α-benzoate and Cortexolone 17α-valerate-21-propionate (CB-03-10) against pancreatic tumors, both of which had antitumor activity superior to that of cyproterone acetate in the same animal. models (see Figure 3).

Example 7: In Vitro Therapeutic Index for Pancreatic Cancer Cell Lines

To assess the safety of compounds intended to be tested in cell line survival experiments, all factors affecting cell survival and viability must be taken into account. In this regard, assessing the intrinsic and reference toxicity of compounds is really important. The ratio of the IC ₅₀ of compounds for peripheral blood mononuclear cells (PBMC) and the IC ₅₀ for cancer cell lines represents the therapeutic index, and it indicates how safe the test compound is.

The IC ₅₀ value for PBMC was determined in two different activation states:

activated - actively dividing cells

quiescent - cells in a state of rest, not dividing.

The results are shown in the tables below, which relate to activated PBMC and quiescent PBMC, respectively:

IC ₅₀ (microM) for activated PBMC

Experiment 1

Cell line CB-03-06 (cortexolone 17α-benzoate)
IC ₅₀ (microM) CB-03-10 (cortexolone 17α-valerate-21-propionate)
IC ₅₀ (microM) Cyproterone acetate
IC ₅₀ (microM) Enzalutamide
IC ₅₀ (microM) Panc1 23 68 52 159 MiaPaca2 17 34 39 79 PBMC 113 106 63 52

Experiment 2

Cell line CB-03-06 (cortexolone 17α-benzoate)
IC ₅₀ (microM) CB-03-10 (cortexolone 17α-valerate-21-propionate)
IC ₅₀ (microM) Cyproterone acetate
IC ₅₀ (microM) Enzalutamide
IC ₅₀ (microM) Panc1 28 60 46 110 MiaPaca2 20 37 39 65 PBMC 97 94 62 90

In parallel, we repeated the same experiments on quiescent PBMCs and obtained the results shown in the tables below.

IC ₅₀ (microM) for quiescent PBMC

Experiment 1

Cell line CB-03-06 (cortexolone 17α-benzoate)
IC ₅₀ (microM) CB-03-10 (cortexolone 17α-valerate-21-propionate)
IC ₅₀ (microM) Cyproterone acetate
IC ₅₀ (microM) Panc1 23 68 52 MiaPaca2 17 34 39 PBMC 100 114 18

Experiment 2

Cell line CB-03-06 (cortexolone 17α-benzoate)
IC ₅₀ (microM) CB-03-10 (cortexolone 17α-valerate-21-propionate)
IC ₅₀ (microM) Cyproterone acetate
IC ₅₀ (microM) Panc1 28 60 46 MiaPaca2 20 37 39 PBMC 85 120 84

The resulting therapeutic index (TI) values calculated for activated PBMC are shown in the tables below:

TI for activated PBMCs

Experiment 1

Cell line CB-03-06 (cortexolone 17α-benzoate)
T.I. CB-03-10 (cortexolone 17α-valerate-21-propionate)
T.I. Cyproterone acetate
T.I. Enzalutamide
T.I. Panc1 5 2 1 0 MiaPaca2 7 3 2 1

Experiment 2

Cell line CB-03-06 (cortexolone 17α-benzoate)
T.I. CB-03-10 (cortexolone 17α-valerate-21-propionate)
T.I. Cyproterone acetate
T.I. Enzalutamide
T.I. Panc1 3 2 1 1 MiaPaca2 5 3 2 1

and the resulting values of therapeutic indices calculated for resting PBMC are given in the tables below:

TI for quiescent PBMCs

Experiment 1

Cell line CB-03-06 (cortexolone 17α-benzoate)
T.I. CB-03-10 (cortexolone 17α-valerate-21-propionate)
T.I. Cyproterone acetate
T.I. Panc1 4 2 0 MiaPaca2 6 3 0

Experiment 2

Cell line CB-03-06 (cortexolone 17α-benzoate)
T.I. CB-03-10 (cortexolone 17α-valerate-21-propionate)
T.I. Cyproterone acetate
T.I. Panc1 3 2 4 MiaPaca2 4 3 1

In these tables, a value of 0 indicates higher toxicity for PBMC than for cancer cell lines.

Example 8: In vitro antitumor activity of Cortexolone 17α-benzoate and Cortexolone 17α-valerate-21-propionate (CB-03-10) against epithelial colon cancer cell lines

This experiment was conducted to investigate and determine the in vitro anticancer activity of cortexolone 17α-benzoate and cortexolone 17α-valerate-21-propionate against epithelial intestinal tumor cell lines, namely HT29. The experimental procedure was as follows:

1. A monolayer of HT-29 cells was placed in 96-well plates at a density of 2×10 ⁴ cells/ml. After distribution, cells were stored at 37°C in 5% _CO2 and allowed to adhere to the walls for 24 hours.

2. The cells were then incubated for 72 hours with the test compounds at concentrations of each of them 0.16, 0.8, 4, 20, 100 and 500 mM.

3. After 72 hours, the MTT colorimetric assay was performed.

The purpose of this test was to determine the concentration at which each compound kills 50% of cancer cells (IC ₅₀ ), due to the compound's potential use in an in vivo animal test.

Data were analyzed using curve fitting using nonlinear least squares regression in the statistical analysis program Prizm.

The IC ₅₀ values found for each cell line are given in the table below.

Inhibition(%) at different micromolar concentrations for two compounds in the HT29 cell line

Concentration in micromoles CB-03-10 (cortexolone 17α-valerate-21-propionate) CB-03-06 (cortexolone 17α-benzoate) 0.8 0.44% -1.55% 4 14.23% 20.40% 20 25.49% 53.60% 100 89.77% 92.24% 500 92.10% 92.31%

The IC ₅₀ values calculated for the two compounds (shown below) indicate that both compounds exhibit clear anticancer activity against HT29 cells.

Calculated IC ₅₀ values (concentration in micromoles)

CB-03-06 15.97 CB-03-10 34.16

Example 9: In Vitro Therapeutic Index for Epithelial Colon Cancer Cell Lines

To assess the safety of compounds intended to be tested in cell line survival experiments, all factors affecting cell survival and viability must be taken into account. In this regard, assessing the intrinsic and reference toxicity of compounds is really important. The ratio of the IC ₅₀ of compounds for peripheral blood mononuclear cells (PBMC) and IC ₅₀ for cancer cell lines represents the therapeutic index, i.e. a parameter important for determining the effectiveness of a product under safe conditions.

The IC ₅₀ value for PBMC was determined in two different activation states:

activated - actively dividing cells

quiescent - cells in a state of rest, not dividing.

The resulting values of therapeutic indices calculated for activated and resting PBMC are given in the tables below:

Experiment 1

Product CB-03-06 (cortexolone 17α-benzoate) (TI) CB-03-10 (Cortexolone 17α-valerate-21-propionate) (TI) Activated 7 3 Resting 6 3

Experiment 2

Product CB-03-06 (cortexolone 17α-benzoate) (TI) CB-03-10 (Cortexolone 17α-valerate-21-propionate) (TI) Activated 6 3 Resting 5 4

The data presented for the compound of the present invention, namely cortexolone 17α-valerate-21-propionate, confirmed its safety and antitumor activity against epithelial colon cancer cell lines.

Example 10: Synthesis of Cortexolone 17α-benzoate

Cortexolone 17α-benzoate was prepared according to a synthesis scheme including the following steps:

In step 1, cortexolone was dissolved in a suitable solvent (eg ethyl acetate). Pyridinium tosylate or p-toluenesulfonic acid (1-10 mol%) was added in a catalytic amount, followed by trialkyl orthobenzoate (R=methyl or R=ethyl). The reaction mixture was heated to 80°C for 3-6 hours. After removal of the solvent and crystallization in an alcohol solvent, cortexolone orthobenzoate 1 was obtained as a solid.

In step 2, cortexolone orthobenzoate 1 (R=methyl or R=ethyl) was dissolved in an alcoholic solvent (eg, methanol) and treated with 0.1 N acetic buffer at reflux. After removal of the solvent, the residue was purified by treatment with demineralized water and cortexolone 17α-benzoate was isolated as a solid.

Example 11: Synthesis of Cortexolone 17α-valerate-21-propionate (CB-03-10) (3) and Cortexolone 17α-valerate (CB-03-05)(2)

Cortexolone 17α-valerate-21-propionate (CB-03-10) was prepared according to the synthesis scheme below:

Step 1: Cortexolone was dissolved in a suitable solvent (eg ethyl acetate). Pyridinium tosylate or p-toluenesulfonic acid (1-10 mol%) was added in a catalytic amount, followed by trialkyl orthovalerate (R=methyl or R=ethyl). The reaction mixture was heated to 80°C for 3-5 hours, and after removal of the solvent and crystallization in an alcohol solvent, cortexolone orthovalerate 1 was obtained.

In step 2, cortexolone orthovalerate 1 (R=methyl or R=ethyl) was dissolved in an alcohol solvent (eg, methanol) and treated with 0.1 N acetic buffer (pH 3-3.9) at reflux. After removal of the solvent and subsequent treatment with purified water, cortexolone 17α-valerate 2 was isolated as a solid.

In step 3, cortexolone 17α-valerate 2 was dissolved in pyridine and 1 equivalent of propionyl chloride was added. At the end of the reaction, the mixture was diluted with water, product 3 was isolated as a solid and purified by crystallization from alcohols.

Example 12: In vitro study of anticancer activity of cortexolone derivative CB-03-10

The ability of CB-03-10 to suppress the growth of cancer cell lines in vitro was studied.

Cancer cell lines were seeded in an amount of 3000 cells into 96-well flat-bottomed plates in complete medium containing 2% bovine serum purified on activated carbon. After 24 hours, test compounds or DMSO/vehicle were added (0.1% final concentration as negative control). CPA and enzalutamide were used as positive controls for cell cytotoxicity, i.e. two powerful well-known antiandrogens. After 3 days, the number of surviving cells was determined using an ATP-dependent cell survival assay (Promega Cell Titer Glo). Figure 5 shows the results of determining the cytotoxicity of cortexolone derivatives depending on the dose for cell lines of human prostate and pancreatic tumors.

The concentration at which each compound kills 50% of cancer cells (IC ₅₀ ) was determined, which characterizes the ability of CB-03-10 and other compounds to inhibit the growth of cancer cells. The concentration of each compound was varied from 3 μM to 200 μM. After 3 days, the number of surviving cells was determined using an ATP-dependent proliferation assay. The data shown in Table I were analyzed using nonlinear least squares regression curve fitting in the statistical analysis program Prizm.

Table I: IC50 for CB-03-10 when tested in vitro on prostate and pancreatic cancer cell lines

Fabric type Naimenov. cellular lines CB-03-01 C17 prop. CB-03-03 C17.21 bottles. CB-03-04 9 dehydr 17 bottles. CB-03-05 C17 shaft. CB-03-06 C17 petrol. CB-03-10 C17, 21, shaft. Enza-lutamide CPA Prostate cancer LNCaP 33 16 46 32 12 10 38 22 PC3 190 53 140 170 28 53 180 90 Pancreatic cancer. glands Panc1 490 70 340 74 28 60 110 46 MiaPaca2 110 thirty 160 59 20 37 65 39

From the data shown in Figure 5 and Table I, it is clear that cortexolone derivatives kill cancer cells at different concentrations and with different IC ₅₀ . CB-03-10 killed prostate cancer cells (panel a) better than the powerful antiandrogen CPA. More importantly, CB-03-10 inhibits the growth of prostate cancer cells in vitro better than enzalutamide, i.e. a new and potent antiandrogen drug currently used in clinical practice as the first choice drug for androgen-dependent prostate cancer.

Interestingly, CB-03-10 inhibited the growth of fast-growing pancreatic cancer cell lines (panel b), which are known to express very low levels of the androgen receptor (AR). These data suggest an independent mechanism of action associated with cytotoxicity other than antiandrogenic activity.

Example 13: Study of Androgen Receptor Expression in Cancer Cell Lines Tested

FACS analysis of the prostate and pancreatic cancer cell lines shown in Table I was performed to better understand the relationship between AR expression on cancer cell lines and the ability of CB-03-10 to inhibit cancer cell growth.

Figure 6 shows AR expression levels on the cancer cells examined. As expected, the results of FACS analysis of AR expression in prostate and pancreatic cancer cell lines are consistent with published expression levels: LNCaP>Panc1>PC3=MiaPaca2.

To better identify the correlation between AR and IC ₅₀ , Table I is supplemented with data on AR expression in the cancer cell lines tested (Table II).

Table II: AR expression on prostate and pancreatic cancer cell lines and IC ₅₀ of cortexolone derivatives

Naimenov. cellular lines CB-03-01 C17 prop. CB-03-03 C17.21 bottles. CB-03-04 9 dehydr 17 bottles. CB-03-05 C17 shaft. CB-03-06 C17 petrol. CB-03-10 C17, 21, shaft. Enza-lutamide CPA Expression
AR LNCaP 33 16 46 32 12 10 38 22 9 PC3 190 53 140 170 28 53 180 90 1 Panc1 490 70 340 74 28 60 110 46 4 MiaPaca2 110 thirty 160 59 20 37 65 39 1

As expected, the growth inhibition demonstrated by the potent antiandrogens CPA and enzalutamide correlates with AR expression on prostate cancer cells. The inhibitory activity of CB-03-10 also correlates (less strongly) with AR expression on prostate cancer cells. However, for pancreatic cancer cells, there is an inverse correlation between AR expression and inhibitory effect. All compounds tested were more active against MiaPaca2 (AR+/-) cells with lower AR expression compared to Panc1 (AR+) cells. This result suggests a possible AR-independent mechanism of action in pancreatic cancer. CB-03-10 is one of the most effective compounds in the series of cortexolone derivatives. Importantly, CB-03-10 is more effective than enzalutamide against prostate cancer cell lines.

Example 14: In vitro study of the anticancer activity of cortexolone derivatives, incl. CB-03-10, on a broader sample of cancer cell lines derived from solid tumors

Because the cytotoxic activity of CB-03-10 did not appear to correlate with AR expression, a broader set of solid tumor samples was tested. MCF7 were added to the previous set of cells, i.e. a breast cancer cell line (AR ^+/− ), another pancreatic cell line with higher AR expression (BxPC3), and a colon cancer cell line (HT29) ( ^AR− ). The results are displayed in Table III.

Table III: IC ₅₀ CB-03-10 for cancer cell lines characterized by AR and GR expression

IC ₅₀ for in vitro proliferation (µM) Genotype Fabric type Naimenov. cell line CB-03-05
C17 shaft. CB-03-10 C17.21 shaft. Enzalut amide AR protein expression is related. PC3 GR protein expression is related. LNCaP Prostate cancer LNCaP 32 10 38 9 1 PC3 170 53 180 1 2 22Rv1 18 positive according to literary data negative according to literary data Pancreatic cancer. glands Panc1 74 60 110 4 positive according to literary data MiaPaca2 59 37 65 1 4 BxPC3 thirty 127 3 positive according to literary data Breast cancer MCF7 50 28 129 1 2 MDA-MB-231 inactive 106 200 1 5 Colon cancer guts HT29 530 14 150 1 2 Healthy lymphocytes PBMC resting 120 120 nd positive according to literary data PBMC activated 130 94 90 nd positive according to literary data

CB-03-10 significantly impairs cell survival in a wide range of cancer cell lines derived from epithelial tissues of various organs. The cytotoxic activity of the compounds does not correlate with AR expression. Additionally, in all cancer cell lines tested, CB-03-10 was more effective than enzalutamide.

Example 15: Therapeutic Index of Cortexolone Derivatives for Various Cancer Cell Lines

The therapeutic index (TI) (also called the therapeutic window or safety window or, in some cases, the therapeutic ratio) is the ratio of the amount of a therapeutic agent that produces a therapeutic effect to the amount that exhibits a toxic effect. The IC ₅₀ of compounds for fresh cells isolated from human blood (PBMC) was determined. The toxicity of compounds was determined using the following formula:

Therapeutic Index=Safety/Efficacy=IC ₅₀ for activated PBMC/IC ₅₀ for cancer cells

The results are shown in Table IV.

Table IV: Therapeutic indices of cortexolone derivatives for a range of cancer cell lines

IC ₅₀ for in vitro proliferation (microM) Fabric type Name cellular lines CB-03-01 C17 prop. CB-03-03 C17, C21 bottle. CB-03-04 9 dehydr. 17 bottles CB-03-05 C17 shaft. CB-03-06 C17 bin. CB-03-10 C17.21 shaft. Enza-lut-amide CPA Prostate cancer LNCaP 33 16 46 32 12 10 38 22 PC3 190 53 140 170 28 53 180 90 The cancer has grown. glands Panc1 490 70 340 74 28 60 110 46 MiaPaca2 110 thirty 160 59 20 37 65 39 BxPC3 28 thirty 127 Breast cancer MCF7 121 32 88 50 25 28 129 64 Cancer rim. guts HT29 51 thirty 10 14 150 Healthy. lymphocytes activated PBMC 0.1 140 360 130 97 94 90 62

Therapeutic index=IC ₅₀ for quiescent PBMC/IC ₅₀ for cancer cells Fabric type Name cellular lines CB-03-01 C17 prop. CB-03-03 C17, C21 bottle. CB-03-04 9 dehydr. 17 bottles CB-03-05 C17 shaft. CB-03-06 C17 bin. CB-03-10 C17.21 shaft. Enza-lut-amide CPA Prostate cancer LNCaP 0 9 8 4 8 9 2 3 PC3 0 3 3 1 3 2 1 1 The cancer has grown. glands Panc1 0 2 1 2 3 2 1 1 MiaPaca2 0 5 2 2 5 3 1 2 BxPC3 3 3 1 Breast cancer MCF7 0 4 4 3 4 3 1 1 Cancer rim. guts HT29 7 4 6 3 Healthy. lymphocytes activated PBMC 1 1 1 1 1 1 1 1

All Cortexolone derivatives demonstrate a stable safety profile. CB-03-10 demonstrates a high therapeutic index. These data indicate that CB-03-10 has a safer profile compared with CPA and enzalutamide.

Example 16: Androgen Receptor Binding Affinity of CB-03-10

Previous experiments have demonstrated significant cytotoxic activity of CB-03-10 against cancer cell lines derived from tumors of various origins. Cytotoxic activity does not completely correlate with androgen receptor expression on the cancer cells tested. From these data, assays were designed to determine the compound's affinity for AR. To determine the relative binding affinity of CB-03-10 to wild-type AR, a competition assay was performed using the Polar Screen kit from Life Technologies. Briefly, AR was added to a fluorescent androgen ligand (Fluormone ^TM AL Green) to form an AR-LBD complex. Competing substances replaced the fluorescent ligand Fluormone ^TM AL Green in the AR-LBD complex, which led to a rapid loss of stability of the fluorescent ligand during the fluorescence lifetime, resulting in a low polarization value. Non-competing substances did not replace the fluorescent ligand in the complex, resulting in the magnitude of polarization remaining high. The change in polarization magnitude in the presence of test compounds was used to determine the relative affinity of the test compounds for AR-LBD.

The affinity of CB-03-10 for the AR receptor was 1.1E-06 (IC ₅₀ mol); in a similar assay, the affinity of dihydrotestosterone (which has a potent ability to bind to the AR receptor) was 1.1E-08.

The affinity of CB-03-10 for binding to the AR receptor is low compared to DHT and the obtained affinity value characterizes CB-03-10 as a substance potentially capable of binding to AR.

Example 17: Transcriptional effect of CB-03-10 on the glucocorticoid receptor

Androgens and glucocorticoid hormones cause diverse and often opposing effects in animal cells, tissues and organisms. A wide range of physiological and molecular biological data suggests that the receptors that mediate these effects, i.e. androgen and glucocorticoid receptors (AR and GR, respectively) influence each other's transcriptional activity. The antagonistic and agonistic activities of CB-03-10 against GR were examined in an in vitro assay. Briefly, human kidney epithelial cells were transfected with a DNA construct comprising GR binding sites coupled to a luminescent reporter molecule. After 24 hours, cells were treated under antagonistic or agonistic conditions. After another 24 hours, luminescence was measured, which was proportional to the transcriptional activity of the GR agonist.

The antagonistic activity assay was based on dexamethasone (Dex)-induced luminescence inhibition.

The antagonistic activity of CB-03-10 was compared with a known GR antagonist, mifepristone (also referred to as RU486), as shown in Figure 7.

The agonist activity assay was based on the initiation of luminescence by CB-03-10. The agonist activity of CB-03-10 was compared with RU486, which is not known to have agonist activity. The results are shown in Figure 8.

As can be seen in Figure 7, CB-03-10 is a potent antagonist (10 times weaker than RU486). In contrast, CB-03-10 is virtually ineffective as a GR agonist because very high concentrations are required to provide activity that is 20% of that of 50 nM Dex.

Example 18: Initiation of Apoptosis and Cell Cycle Arrest CB-03-10

Most cytotoxic anticancer drugs currently used have been shown to induce apoptosis in susceptible cells. The fact that a wide variety of agents that interact with different targets cause cell death with some common features suggests that cytotoxicity is determined by the ability of cells to resist so-called. "programmed" cell death. The effect of CB-03-10 was studied to determine whether the mechanism of cytotoxic action on cancer cells is mediated by apoptosis and cell cycle arrest. Cancer cell lines were seeded into 6-well flat-bottomed plates. After 24 hours, test compounds or DMSO vehicle (negative control) were added. After another 24 hours, cells were scraped, stained with fluorescein-conjugated Annexin V and propidium iodide, and analyzed by flow cytometry.

Figure 9 clearly shows that CB-03-10 is able to induce apoptosis of pancreatic cancer cell lines. CB-03-10 induces apoptosis in a total of 28% of cells (early and late apoptosis) versus only 11% in the control.

Apoptosis can occur at the G1/S or G2/M transition points of the cell cycle. Cells were treated with CB-03-10 for 24 hours, then fixed with paraformaldehyde and stained with propidium iodide. The data in Figure 10 show that CB-03-10 causes S-phase blocking at lower concentrations, followed by G2/M blocking at higher concentrations. The absence of G1 blocking indicates no effect on the p53 protein. Blocking S and G2/M may indicate an effect on molecules associated with cell cycle checkpoints. In the case of S phase, a possible target is cyclin-dependent kinase 2 (CDK2). Gemzar and cisplatin are examples of drugs that act in the S phase. In the case of G2 phase, CDK1 is a possible target.

Example 19: Caspase stimulation assay by CB-03-10

Previous experiments using Annexin V staining of MiaPaca2 cells showed that CB-03-10 induces apoptosis. To more thoroughly investigate this phenomenon, the enzymatic activities of caspase 8 (initiator caspase for the extrinsic apoptosis pathway) and caspase 9 (initiator caspase for the intrinsic apoptosis pathway), as well as caspases 3 and 7 (effector caspases), were measured.

For this purpose, MiaPaca2 cells were seeded in 96-well flat-bottom culture plates. After 24 hours, test compounds were added to the wells, gemcitabine (a known chemotherapeutic agent for the treatment of pancreatic cancer) and DMSO were used as positive and negative controls, respectively. After 8, 24, and 48 hours of incubation with test compounds, cells were lysed in a buffer containing caspase 3/7, 8, or 9 substrate and stable luciferase in their own buffers. Lysates were transferred to white opaque plates and then luminescence was measured on a Tecan Safire instrument. Plates containing parallel experiments treated identically were used to determine cell survival. The activity of all caspases was adjusted according to the number of surviving cells. The results are shown in Figure 11.

The activity of caspases 8 and 9 (panels A and B) was stimulated by CB-03-10. This stimulation was rapid, dose-dependent and evident after 8 hours, and resulted in a 7-fold increase in activity relative to control.

Gemcitabine (a known chemotherapeutic agent used to treat pancreatic cancer) also induced caspase 8 and 9 activity, but with a delayed and less potent response compared to CB-03-10. The 2.3-fold increase in caspase 8 and 9 activity was not observed until the 48-hour mark.

Stimulation of caspase 3/7 by CB-03-10 was also observed, in this case as early as 8 hours, and at a really high level after 48 hours of incubation. It is interesting to note that CB-03-05 did not show a good caspase activation profile. No increase in caspase 3/7 activity by gemcitabine was observed until the 48-hour mark.

Similar analyzes were repeated using the LNCaP prostate cancer cell line (Figure 12). In this case, the positive control was enzalutamide, an effective and novel antiandrogen currently used in clinical practice to treat patients with prostate cancer. The results are shown in Fig. 12 after 24 hours of incubation, when caspase activity reached its maximum.

The experimental results clearly show that CB-03-10-induced initiator (8 and 9) and effector (3/7) caspase activities are superior to those of enzalutamide (which was used as a positive control). These results demonstrated a strong stimulation of caspase activity by CB-03-10 in prostate cancer cell lines, affecting both intrinsic and extrinsic apoptotic pathways, consistent with the inhibition observed in the MiaPaca2 cell line.

Example 20: In Vitro Metabolism of CB-03-10 in Rat and Human Plasma

A specific assay was developed to gain some insight into the metabolism of CB-03-10 in human and rat plasma. Briefly, the compound was incubated for various periods of time in human and rat plasma at 37°C. After incubation, samples were tested for the presence of non-metabolized compound using liquid chromatography. The change in concentration over time is shown in Fig. 13.

The result demonstrates that CB-03-10 is rapidly degraded to CB-03-05 in human plasma, and it is degraded faster in rat plasma compared to human plasma.

Example 21: In vivo pharmacokinetics study of CB-03-10 in an animal model (mouse)

The pharmacokinetics of CB-03-10 in mouse plasma after intravenous (IV), subcutaneous (SC) and oral (PO) administration were assessed.

Mice (3 animals per group) were administered the doses of the active substance indicated below and blood was collected at the indicated time points. Plasma samples were analyzed using HPLC-MS/MS.

Group Route of administration Blood sampling time points 1 iv (20 mg/kg) 10 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours 2 SC (40 mg/kg) 30 min, 1 h, 2 h, 4 h, 8 h, 24 h 3 PO (40 mg/kg) 30 min, 1 h, 2 h, 4 h, 8 h, 24 h

Table V

CB-03-10 was not detectable in plasma even after 1 hour, regardless of route of administration. However, CB-03-10 was metabolized to CB-03-05 with body exposures of 189 (SC) and 47 (PO) h/ng/ml (Figure 14).

Example 22: In Vivo Testing of CB-03-10 in a Human Pancreatic Cancer Xenograft Mouse Model (MiaPaca2 Cell Line)

Previous studies have found that CB-03-10 potently inhibits the growth of the pancreatic cancer cell line MiaPaca2 (AR ^+/- ) in vitro. A study was conducted to determine whether this result could be transferred to an in vivo xenograft model of pancreatic cancer. Briefly, 1 x 10 ⁶ MiaPaca2 cells suspended in Matrigel were administered by subcutaneous injection (SC) to 6-week-old nude mice. Tumor sizes were measured every 4 days using digital calipers. Tumor volume was calculated using the formula 0.5236(r ₁ ) ² (r ₂ ), where r ₁ <r ₂ . Administration of CB-03-10 and control compounds began after the tumor reached a volume of 50 mm ³ . Compounds diluted in DMSO/2-hydroxypropyl β-cyclodextrin (vehicle) were administered by subcutaneous injection (SC) daily (100 μl/mouse) at a concentration of 40 mg/kg daily for 28 consecutive days. Figure 15 shows the average tumor growth in an in vivo xenograft model following SC injection of CB-03-10 compared to vehicle.

As can be seen from FIG. 15, CB-03-10 exhibits strong and statistically significant activity against pancreatic tumors compared to the control. In addition, the experiments also showed significant antitumor activity (p<0.5) when compared with vehicle or CPA alone (results not shown).

During administration, CB-03-10 maintained less than a 5-fold increase in tumor size relative to baseline. In contrast, the mean size in the vehicle or CPA groups increased 12-fold. In addition to suppressing tumor growth, CB-03-10 had a beneficial effect on mouse survival. Importantly, 14 days after cessation of administration, mice treated with CB-03-10 still had significantly smaller tumor sizes compared to the vehicle-only group (6-fold versus 14-fold, respectively).

Median survival was 70 days for mice treated with CB-03-10 compared with 60 days for mice treated with vehicle or 40 days for CPA. This difference is significant, with the risk of death being 2 to 4 times higher in the vehicle group.

Example 23: In vivo study of the effect of CB-03-10 when administered orally in a mouse xenograft model of human prostate cancer (LNCaP cells)

Previous studies have found that CB-03-10 is also effective in inhibiting the growth of prostate cancer cell lines in vitro. A study was conducted to determine whether this result could be transferred to an in vivo xenograft model of prostate cancer. 3x10 ⁶ LNCaP cells suspended in Matrigel were injected subcutaneously (in the right flank) into 6-week-old male nude mice. Tumor sizes were measured as described above. Administration of CB-03-10 and control compounds was started after tumor size reached 50 mm ³ . Administration formulations were prepared in a mixture of 15% vitamin E - TPGS and 65% 0.5% w/v CMC solution in 20 mM citrate buffer (pH 4). Oral administration was carried out daily (100 mg/kg in a volume of 200 ml/mouse) for 28 consecutive days. The results were plotted as the average change in tumor volume relative to the start of injection. Figure 16 shows the results obtained for oral administration of CB-03-10 in an in vivo xenograft model of prostate cancer. Enzalutamide, which is a new and effective antiandrogen, was used as a positive control.

Oral administration of CB-03-10 demonstrated greater antitumor activity than enzalutamide over a 28-day administration period. Tumor volume increased only 2-fold with the administration of CB-03-10 and 3-fold with the administration of enzalutamide, compared with a 10-fold increase in the negative control group.

CB-03-10 was also more effective than enzalutamide in maintaining a small increase in tumor size after administration was stopped (5-fold versus 8-fold change). Even 6 weeks after cessation of administration, the mean tumor volume in the CB-03-10 group was 3.6 times less than in the vehicle group.

Example 24: In vitro inhibition of prostate-specific antigen (PSA) secretion from LNCaP prostate cancer cells by CB-03-10

Prostate-specific antigen or PSA is a protein produced by prostate cells. The PSA test measures the level of PSA in a man's blood. PSA levels are often elevated in men with prostate cancer and it is used as a surrogate marker to determine the prevalence of prostate cancer in the population. Following evidence that CB-03-10 was able to inhibit the growth of prostate cancer in vivo, the ability of this compound to inhibit PSA secretion from cancer cells in vitro was determined. LNCaP cells were seeded in 96-well flat-bottom culture plates in activated charcoal-stripped serum with or without 10 nM DHT. After 24 hours, test compounds were added to the cells using DMSO as vehicle and negative control, and enzalutamide as positive control. After 48 hours of incubation with test compounds, supernatants were collected and tested by PSA ELISA, and the cells were lysed to assess cell survival.

As expected, the pure antiandrogen, namely enzalutamide, effectively inhibits PSA secretion with an IC ₅₀ <3 μM; CB-03-10 is also a potent PSA inhibitor (IC ₅₀ 9 µM). However, the activity of enzalutamide does not increase with increasing dosage in the same way as CB-03-10. It is noteworthy that cortexolone, i.e. the parent compound and final metabolite of all compounds tested, has virtually no effect on PSA secretion (IC _50,612 µM). When examining the survival of these cells, enzalutamide showed an IC ₅₀ value of 61 μM and CB-03-10 showed an IC ₅₀ value of 11 μM. This confirms the strong ability of both compounds to inhibit cell growth. It is important and interesting to note that cortexolone, i.e. the parent compound and final metabolite of all compounds tested, inhibited LNCaP cell survival only at very high concentrations (IC ₅₀ 153 μM) and was virtually inactive as a cytotoxic compound in cancer cell lines.

Example 25: In Vitro Anticancer Activity Study of CB-03-10 on Breast Cancer Cell Lines

Triple negative breast cancer (TNBC) accounts for approximately 20% of newly diagnosed invasive breast cancers. This subtype of cancer is not supported by hormones such as estrogen and progesterone, nor by the presence of significant amounts of HER2 receptors, and for this reason patients do not respond to traditional treatments (such as tamoxifen or Herceptin). Consequently, this type of cancer is characterized by resistance to chemotherapy and low patient survival.

There is a correlation between the resistance of these cancers to chemotherapy and high expression of GR (Cancer Therapy 2013). Clinical trials are underway testing a GR antagonist (mifepristone/RU486) in combination with chemotherapy agents for the treatment of TNBC. However, the clinical use of mifepristone is compromised by controversial pharmacological actions associated with progesterone receptor (PR) antagonism. To evaluate whether CB-03-10 could be used as a potential treatment for breast cancer, and in particular TNBC, cytotoxicity studies were performed using breast cancer cell lines characterized by differential expression of hormone receptors.

The following breast cancer cell lines were selected for the study:

breast cancer cells MCF7 (ER ⁺ PR ⁺ Her2 ⁺ ,GR ^+/- )

TBNC cells MDA-MB-231 (ER ^- PR ^- Her2 ^- ,GR ⁺⁺ )

Prior to the cell growth inhibition assay, cells were characterized for AR and GR receptor expression using FACS as described above. The data shown in Figure 18 confirms the receptor expression data reported in the literature.

To study cytotoxicity, cells were seeded in 96-well flat-bottom culture plates in medium containing activated charcoal-stripped serum. After 24 hours, test compounds were added to the cells. DMSO was used as vehicle and negative control, and RU486 as positive control. After 72 hours of incubation, cells were harvested, lysed, and survival was determined using a long-term luminescence titration (glow) survival assay.

Table VI shows the IC ₅₀ values of CB-03-10 for the above breast cancer cell lines

MCF7
(ER ⁺ PR ⁺ GR ^+/- ) MDA-MB-231
(ER ^- PR ^- GR ⁺⁺ ) RU486 not active 435 CB-03-10 28 106 CB-03-05 50 not active

Table VI

CB-03-10 is active against both breast cancer cell lines, but appears to be more active against MCF7 cells than MDA-MB-231, possibly suggesting that GR is not the only target of this compound. RU486 (mifepristone, a GR/PR antagonist), as expected, had no effect on the survival of GR ^+/- MCF7 cells, but very slightly suppressed the survival of TNBC GR ⁺ MDA-MB-231 cells by a maximum of 25% at 100 μM. It is interesting to note that CB-03-05 is only active against MCF7 and not MDA-MB-231. It is unclear which receptor is responsible for the observed difference in results, since these cells differ in at least 4 receptors. If the cause is not the GR receptor, it could be ER (estrogen receptor), PR (progesterone receptor) or Her2, which are expressed in MCF7 cells but not in MDA-MB-231.

General conclusions

The examples discussed demonstrate that cortexolone 17α-valerate-21-propionate (CB-03-10), among other things, has excellent activity superior to that of other known cortexolone derivatives. The authors obtained improved results both in vitro and in vivo, for example in such aspects as:

I) general antitumor activity in vitro;

ii) in vitro antitumor activity not directly correlated with AR expression;

iii) in vitro antitumor activity directly correlated with GR expression;

IV) therapeutic index (TI); And

V) In vivo antitumor activity against pancreatic and prostate tumors;

VI) From the data shown in Table VII below, it is evident that cortexolone derivatives kill cancer cells at different concentrations and with different IC ₅₀ values. However, CB-03-06 and CB-03-10 demonstrate better IC ₅₀ values compared to other compounds in the cortexolone derivative series against cancer cell lines of various origins. Even the CB-03-10 metabolite, such as CB-03-05, shows a good IC ₅₀ value for LNCaP prostate cancer cells (IC ₅₀ 32 μM). Lower IC ₅₀ values indicate stronger antitumor activity in vitro.

Table VII: IC ₅₀ values of cortexolone derivatives when tested on prostate and pancreatic cancer cell lines

Naimenov. cellular lines CB-03-01 C17 prop. CB-03-03 C17.21 bottles. CB-03-04 9 dehydr 17 bottles. CB-03-05 C17 shaft. CB-03-06 C17 petrol. CB-03-10 C17, 21, shaft. Enza-lutamide CPA LNCaP 33 16 46 32 12 10 38 22 PC3 190 53 140 170 28 53 180 90 Panc1 490 70 340 74 28 60 110 46 MiaPaca2 110 thirty 160 59 20 37 65 39

II) AR expression was tested in cancer cell lines, see Table VIII

In prostate cancer cell lines, as expected, the growth inhibition demonstrated by potent antiandrogens such as CPA and enzalutamide correlates with AR expression in prostate cancer cells (the higher the AR expression, the greater the cytotoxic activity, resulting in lower _IC50 values ). Additionally, CB-03-04 exhibits an _IC50 value of 46 for LNCaP cells (a prostate cancer cell line that expresses high levels of the androgen receptor), but significantly higher _IC50 values (135) for PC3 cells, which express low or zero AR levels. Notably, the cytotoxic activity of CB-03-06 and CB-03-10 was not clearly influenced by androgen receptor expression on prostate cancer cells. CB-03-06 and CB-03-10 have very good IC ₅₀ values almost independent of AR expression.

In pancreatic cancer cell lines where there is little or almost no AR expression, CB-03-06 and CB-03-10 exhibit potent cytotoxic activity superior to that of CPA and enzalutamide. This higher activity could result from an additional mechanism of action involving binding of other receptors.

Table VIII: AR expression by prostate and pancreatic cancer cell lines and IC ₅₀ of cortexolone derivatives

Naimenov. cellular lines CB-03-01 C17 prop. CB-03-03 C17.21 bottles. CB-03-04 9 dehydr 17 bottles. CB-03-05 C17 shaft. CB-03-06 C17 petrol. CB-03-10 C17, 21, shaft. Enza-lutamide CPA Expression
AR LNCaP 33 16 46 32 12 10 38 22 9 PC3 190 53 140 170 28 53 180 90 1 Panc1 490 70 340 74 28 60 110 46 4 MiaPaca2 110 thirty 160 59 20 37 65 39 1

III) The therapeutic index (TI) (also called therapeutic window, safety window or therapeutic ratio) is the ratio of the amount of a therapeutic agent that causes a therapeutic effect to the amount that causes a toxic effect. The IC ₅₀ of the compounds was determined on fresh cells isolated from human blood (PBMC). The toxicity of compounds was determined using the following equation:

Therapeutic Index=Safety/Efficacy=IC ₅₀ for activated PBMC/IC ₅₀ for cancer cells;

The results are shown in Table IX. All cortexolone derivatives demonstrate a consistently safe toxicity profile. However, CB-03-06 and CB-03-10 demonstrated the highest therapeutic index when studied in all 7 cancer cell lines tested in vitro.

Table IX: Therapeutic Index of Cortexolone Derivatives for a Range of Cancer Cell Lines

Therapeutic index=IC ₅₀ for activated PBMC/IC ₅₀ for cancer cells Fabric type Name cellular lines CB-03-01 C17 prop. CB-03-03 C17, C21 bottle. CB-03-04 9 dehydr. 17 bottles CB-03-05 C17 shaft. CB-03-06 C17 bin. CB-03-10 C17.21 shaft. Enza-lut-amide CPA Prostate cancer LNCaP 0 9 8 4 8 9 2 3 PC3 0 3 3 1 3 2 1 1 The cancer has grown. glands Panc1 0 2 1 2 3 2 1 1 MiaPaca2 0 5 2 2 5 3 1 2 BxPC3 3 3 1 Breast cancer MCF7 0 4 4 3 4 3 1 1 Cancer rim. guts HT29 7 4 6 3 Average 0 4 4 3 5 4 1 1

IV) Triple negative breast cancer (TNBC) as shown in Example 25. The cytotoxic activity of CB-03-10 is particularly impressive because conventionally used therapeutic agents do not act on triple negative breast cancer (TNBC) cell lines. TNBC is defined as the absence of estrogen and progesterone receptor expression and ERBB2 amplification. Such cells do not respond to endocrine or anti-ERBB2 therapy. Current research has identified some potential therapeutic targets for TNBC. However, the disease still leads to poor outcomes. Considering the cytotoxic activity and excellent safety profile of CB-03-10, this substance is a new and improved candidate for the clinical treatment of these cancers.

Below, the present invention will be described using the following numbered embodiments.

1. In one embodiment, the invention relates to a compound of formula (I)

(I)

where R is hydrogen or a C(O)-R ₁ fragment, where R ₁ is a linear alkyl chain containing from 2 to 5 carbon atoms, and where R' is a linear alkyl chain containing from 3 to 6 carbon atoms, or optionally a substituted aryl group, or an optionally substituted heteroaryl group.

2. In another embodiment, the invention relates to a compound of formula (I) according to claim 1, wherein the optionally substituted aryl group is phenyl.

3. In another embodiment, the invention relates to a compound of formula (I) according to claim 1, wherein R ₁ represents hydrogen or a CH ₂ CH ₃ fragment, and R' represents a -(CH ₂ ) ₃ -CH ₃ fragment or phenyl .

4. In another embodiment, the invention relates to a compound according to claim 1, having the formula:

5. In another embodiment, the invention relates to a compound according to claim 1, having the formula:

6. In another embodiment, the invention relates to a compound according to any one of claims 1 to 5 for use as a medicine.

7. In another embodiment, the invention relates to a compound according to any one of claims 1 to 5 for use in the treatment of precancerous diseases, dysplasias, metaplasias and tumor diseases.

8. In another embodiment, the invention relates to a compound for use according to claim 7, characterized in that said tumor disease includes malignancies and metastases.

9. In another embodiment, the invention relates to a compound for use according to claim 8, characterized in that said tumor diseases are solid tumors, preferably epithelial tumors such as prostate carcinoma; breast carcinoma; pancreatic carcinoma; lung carcinoma; gastrointestinal carcinoma, such as colon carcinoma; kidney cancer; thyroid carcinoma; uterine carcinoma; adrenal carcinoma.

10. In another embodiment, the invention relates to a compound for use according to claim 9, characterized in that said epithelial tumors are prostate carcinoma or pancreatic carcinoma, preferably exocrine pancreatic carcinoma.

11. In another embodiment, the invention relates to a pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 5, in combination with at least one physiologically acceptable excipient.

12. In another embodiment, the invention relates to a pharmaceutical composition according to claim 11, characterized in that it is in solid or liquid form.

13. In another embodiment, the invention relates to a pharmaceutical composition in solid form according to claim 12, characterized in that it is in the form of a powder, freeze-dried powder, granules, dragees, tablets or capsules.

14. In another embodiment, the invention relates to a pharmaceutical composition in liquid form according to claim 12, characterized in that it is in the form of a solution, emulsion, suspension or syrup.

15. In another embodiment, the invention relates to a pharmaceutical composition according to any one of claims 11 to 14, characterized by the presence of at least one other active ingredient, preferably a chemotherapeutic active ingredient, as a combination for simultaneous, separate or sequential administration.

16. In another embodiment, the invention relates to a pharmaceutical composition according to claims 11-15 for use in the treatment of precancerous diseases, dysplasias, metaplasias and tumor diseases.

17. In another embodiment, the invention relates to a pharmaceutical composition for use according to claim 16, characterized in that said tumor diseases include malignancies and metastases.

18. In another embodiment, the invention relates to a pharmaceutical composition for use according to claim 17, characterized in that said tumor disease is a solid tumor, preferably an epithelial tumor such as prostate carcinoma, breast carcinoma; pancreatic carcinoma; lung carcinoma; gastrointestinal carcinoma, such as colon carcinoma; kidney cancer; thyroid carcinoma; uterine carcinoma; adrenal carcinoma.

19. In another embodiment, the invention relates to a pharmaceutical composition for use according to claim 18, characterized in that said epithelial tumor is prostate carcinoma or pancreatic carcinoma, preferably exocrine pancreatic carcinoma.

Claims (23)

1. Use of a compound having the formula: Cortexolone 17α-valerate to obtain a pharmaceutical composition for the treatment of tumor diseases, wherein said tumor diseases are epithelial tumors selected from prostate carcinoma, breast carcinoma; pancreatic carcinoma and colon carcinoma. 2. Use according to claim 1, wherein prostate carcinoma has become refractory to antiandrogen therapy. 3. Use according to claim 2, wherein the antiandrogen is enzalutamide. 4. Use according to claim 1, wherein the pancreatic carcinoma is exocrine pancreatic carcinoma. 5. Use according to claim 1, wherein the breast carcinoma is triple negative breast cancer (TNBC). 6. Use according to claim 1, wherein the breast carcinoma is in a subject who is suffering from relapses or is refractory to standard therapy. 7. A pharmaceutical composition comprising an effective amount of a compound having the formula: Cortexolone 17α-valerate for use in the treatment of tumor diseases, wherein the tumor disease is an epithelial tumor selected from prostate carcinoma, breast carcinoma, pancreatic carcinoma, and colon carcinoma. 8. The pharmaceutical composition according to claim 7, wherein prostate carcinoma has become refractory to antiandrogen therapy. 9. Pharmaceutical composition according to claim 8, where the antiandrogen is enzalutamide. 10. The pharmaceutical composition according to claim 7, wherein the pancreatic carcinoma is an exocrine pancreatic carcinoma. 11. The pharmaceutical composition according to claim 7, wherein the breast carcinoma is triple negative breast cancer (TNBC). 12. The pharmaceutical composition according to claim 7, wherein the breast carcinoma is in a subject who is suffering from relapses or is refractory to standard therapy. 13. Pharmaceutical composition for use according to any one of claims 7 to 12, further comprising at least one additional active ingredient. 14. The pharmaceutical composition according to claim 13, wherein the additional active ingredient is a chemotherapeutic active ingredient, for simultaneous, separate or sequential administration. 15. Pharmaceutical composition for use according to any one of claims 7 to 14, for use with a second composition comprising a chemotherapeutic active ingredient.