RU2776343C2 - Compounds for reduction in lipotoxic damage - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a compound selected from a group consisting of

and

or to its pharmaceutically acceptable salt. The invention also relates to a pharmaceutical composition for the inhibition of lipase based on the specified compound.

EFFECT: new compounds and a pharmaceutical composition based on them are obtained, which can be used in medicine for the treatment of acute pancreatitis and acne.

12 cl, 39 dwg, 1 tbl, 7 ex

Description

Link to related applications

The present application claims priority under U.S. Provisional Application Serial No. 62/531454, filed July 12, 2017, the contents of which are incorporated herein by reference in their entirety.

The field of technology to which the present invention relates

The present disclosure relates to the use of lipase inhibitors for the treatment of severe pancreatitis and/or acne.

Background of the Invention

The pancreas produces enzymes that aid digestion and absorption of food; one such enzyme is lipase, which digests fat. Some people (eg, obese people) are at increased risk of developing multiple organ failure in acute inflammatory conditions such as severe burns, severe trauma, critical illness, and acute pancreatitis (AP). Pancreatitis is associated with the release of destructive digestive enzymes from the acinar cells of the pancreas into the pancreas itself. When AP starts, it can quickly become severe AP (SAP). This is of concern because SAP results in a mortality rate of 40 to 50% when complicated by acute renal failure, respiratory failure, hypocalcemia and other manifestations of multiple organ failure, or large areas of pancreatic necrosis. In the absence of effective treatments, the current standard of care is supportive care and management of complications when they occur.

Brief summary of the present invention

Provided herein are novel lipase inhibitors and methods for their use in the treatment of pancreatitis and/or organ failure and/or acne comprising administering to a subject in need of such treatment an effective amount of a lipase inhibitor as provided herein. The methods provided herein are based, at least in part, on the findings that lipotoxicity contributes to inflammation, multiple organ failure, necrotizing pancreatic acinar cell death, acute pancreatitis, sepsis (eg, culture-negative sepsis), burns, acne. and infections, and that inhibition of lipase activity can reduce the rates associated with these conditions. Accordingly, in some embodiments, provided herein are methods and compositions for limiting lipotoxicity and thereby reducing the likelihood of adverse outcomes associated with acute pancreatitis and other severe systemic conditions.

Provided herein is a compound selected from the group consisting of:

и

and

or their pharmaceutically acceptable salt. In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof. In some embodiments, the connection is:

or a pharmaceutically acceptable salt thereof.

Additionally provided herein is a pharmaceutical composition comprising one or more of the compounds provided herein and a pharmaceutically acceptable excipient.

The present disclosure also provides methods for treating acute pancreatitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof. In some embodiments, acute pancreatitis is severe. In some embodiments, the severity of acute pancreatitis is reduced from severe to mild after administration. In some embodiments, the subject is obese. In some embodiments, the risk of developing shock, renal failure, and/or lung failure is reduced.

Also provided herein is a method for treating acne in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.

Additionally provided herein is a method for treating sepsis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the sepsis is culture negative sepsis.

The present disclosure also relates to methods for treating burns in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof.

Also provided herein is a method for treating infections in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the infection is caused by one or more organisms selected from the group consisting of P. auregenosa , S. aureus , B. subtilis , and B. cepecia .

According to some of the above embodiments, the connection is:

or a pharmaceutically acceptable salt thereof. In some embodiments of the above embodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned in this document are incorporated by reference in their entirety. In the event of a conflict, the present description, including definitions, will control.

Other features and advantages of the present invention will be apparent from the following detailed description of the present invention and the figures, as well as from the claims.

Description of graphic materials

Fig. 1 is a diagram of the structure of orlistat, dividing the compound into three main regions.

Fig. 2 is a schematic diagram detailing the synthetic route for obtaining the amino ester modifications in the orlistat core.

In FIG. 3 shows the structures of the various non-formylated ester modified analog compounds obtained.

In FIG. 4 are line graphs showing the results of a lipase inhibition assay in which non-formylated ester modified analog compounds are tested. In FIG. 4A shows the results of freshly prepared compound solutions, and FIG. 4B shows the results of solutions stored overnight.

In FIG. 5 is a schematic diagram detailing the synthetic route for the preparation of formylated analogs with amino ester modification.

In FIG. 6 shows the structures of the various ester-modified formylated analog compounds obtained.

In FIG. 7 are line graphs showing the results of a lipase inhibition assay in which ester-modified formylated analog compounds were tested. In FIG. 7A shows the results of freshly prepared compound solutions, and FIG. 7B shows the results of solutions stored overnight.

In FIG. 8 are histograms detailing the efficacy of selected analog compounds in reducing lipotoxic damage to acinar cells when acinar cells and adipocytes are co-cultured (FIG. 8A) and assessing the toxicity of the compounds to these cells (FIG. 8B).

In FIG. 9 is a diagram detailing the chain extension reaction used to prepare β-chain analogues.

In FIG. 10 is a schematic diagram detailing the synthesis route for β-chain analogs.

In FIG. 11 is a schematic diagram detailing the synthesis route for β-chain analogs.

In FIG. 12 is a schematic diagram detailing the synthesis route for β-chain analogs.

In FIG. 13 is a schematic diagram detailing the synthesis route for β-chain analogues.

In FIG. 14 shows the structures of various β-chain analogues prepared.

In FIG. 15 is a line chart showing the results of a lipase inhibition assay testing β-chain analogs in fresh solutions.

In FIG. 16 is a schematic diagram detailing the synthesis route for α-chain analogs.

In FIG. 17 are line graphs showing the results of a lipase inhibition assay in which α-chain analogs are tested. In FIG. 17A shows the results of freshly prepared compound solutions, and FIG. 17B shows the results of solutions stored overnight.

In FIG. 18 are line graphs showing the results of an assay that examines % free fatty acid (FFA) production. In FIG. 18A shows the results of freshly prepared compound solutions, while FIG. 18B shows the results of solutions stored overnight.

In FIG. 19 shows tables of values derived from toxicity studies in mouse pancreatic cells.

In FIG. 20 are line graphs showing the results of an analysis that examines FFA formation in %. In FIG. 20A shows results with hPLRP2 lipase, while FIG. 20B shows results with hCEL lipase.

In FIG. 21 is a summary table of the in vitro studies described herein.

In FIG. 22 provides a summary of the use of orlistat to reduce severe pancreatitis.

In FIG. 23 shows the results of an in vivo test of compound 767. In FIG. 23A shows the chronology of the study; in fig. 23B is a histogram of serum lipase concentration; in fig. 23C is a graph showing serum urea nitrogen levels at various time points; in fig. 23D is a graph showing serum calcium levels at various time points; and in FIG. 23E is a graph showing protection against systemic inflammatory response syndrome (SIRS); in fig. 23F is a graph showing shock protection, and FIG. 23G is a graph showing improved survival.

In FIG. 24 shows photographs of treated mice.

In FIG. 25 is a summary table of in vivo studies with compound 767 and orlistat.

In FIG. 26 is a line graph showing % FFA production in P. acnes medium.

In FIG. 27 shows images of alcohol blue agar inoculated with P. acnes and exposed to both orlistat and compound 767.

In FIG. 28 shows the effectiveness of 10 mM agents in reducing LDH leakage from acinar cells incubated with 600 mM GTL.

Detailed disclosure of the present invention

Currently, severe pancreatitis is often treated with the FDA-approved lipase inhibitor orlistat. Although lipase inhibition reduces the severity of pancreatitis, the use of orlistat is complicated by the need for repeated dosing (eg, 50 mg/kg twice daily for two days) during pancreatitis and the hypertriglyceridemia associated with the use of this compound. Provided herein are compounds that provide therapeutic effects in pancreatitis without the need for repeated doses or the complications associated with orlistat.

Compounds provided herein include those described in Table 1.

Table 1.

Connection number Structure 728

731

729

727

725

716

718

717

726

724

732

733

734

738

736

737

741

739

740

743

742

762

763

760

767

768

or a pharmaceutically acceptable salt thereof. In some embodiments, the connection is

or a pharmaceutically acceptable salt thereof. In some embodiments, the connection is

or a pharmaceutically acceptable salt thereof.

In some embodiments, a compound provided herein is stable in aqueous solution. For example, the compound provided herein may be more stable than orlistat in aqueous solution. In some embodiments, the connection is

or a pharmaceutically acceptable salt thereof. In some embodiments, the connection is

or a pharmaceutically acceptable salt thereof.

Synthesis

The compounds described herein can be obtained, for example, in accordance with the procedures described in the examples and in the corresponding figures.

One skilled in the art will appreciate that the methods described are not the exclusive means by which the compounds provided herein can be synthesized, and that a wide range of synthetic organic reactions are available for potential use in the synthesis of the compounds provided herein. One skilled in the art will know how to select and implement suitable synthetic routes. Suitable methods for synthesizing starting materials, intermediates, and products can be determined by referring to the literature, including reference sources such as: Advances in Heterocyclic Chemistry , Vols. 1-107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al . (Ed.) Science of Synthesis , Vols. 1-48 (2001-2010) and Knowledge Updates KU2010/1-4; 2011/1-4; 2012/1-2 (Thieme, 2001-2012); Katritzky, et al . (Ed.) Comprehensive Organic Functional Group Transformations , (Pergamon Press, 1996); Katritzky et al . (Ed.); Comprehensive Organic Functional Group Transformations II (Elsevier, ^{2nd ed} ., 2004); Katritzky et al . (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984); Katritzky et al ., Comprehensive Heterocyclic Chemistry II , (Pergamon Press, 1996); Smith et al ., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure , 6th ^ed . (Wiley, 2007); Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

The reactions for preparing the compounds described herein can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents may be substantially unreactive with the starting materials (reagents), intermediates, or products at the temperatures at which the reactions are carried out (eg, temperatures that may range from the solvent's freezing point to the solvent's boiling point). This reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the particular reaction step, one skilled in the art can select suitable solvents for the particular reaction step.

The preparation of the compounds described herein may involve the attachment and deprotection of various chemical groups. The need for attaching and deprotecting groups, as well as the choice of suitable protecting groups can be easily determined by a person skilled in the art. The chemistry of protecting groups can be found, for example, in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis , ^{3rd ed} ., Wiley & Sons, Inc., New York (1999).

Reactions can be monitored in accordance with any suitable method known in the present technical field. For example, product formation can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (for example, ¹ H or ¹³ C), infrared spectroscopy, spectrophotometry (for example, in the UV and visible spectrum), mass spectrometry, or chromatographic methods. such as high performance liquid chromatography (HPLC), liquid chromatography/mass spectroscopy (LC/MS), or thin layer chromatography (TLC). Compounds can be purified by those skilled in the art using a variety of methods, including high performance liquid chromatography (HPLC) and silica gel normal phase chromatography.

As used herein, the term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structures. Compounds identified herein by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise indicated.

Compounds provided herein also include tautomeric forms. The tautomeric forms result from the replacement of a single bond by an adjacent double bond, along with concomitant proton migration. Tautomeric forms include prototropic tautomers, which are isomeric protonation states characterized by the same empirical formula and overall charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and ring forms where the proton can occupy two or more positions of the heterocyclic system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. The tautomeric forms may be in equilibrium or be sterically locked into one form by appropriate substitution.

In some embodiments, the compounds described herein may contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, enantiomerically enriched mixtures, single enantiomers, single diastereomers, and diastereomeric mixtures (e.g., including ( R )- and ( S )-enantiomers, diastereomers, ( D )-isomers, ( L )-isomers, (+) (right- handed ) forms, (-) ( left -handed) forms, their racemic mixtures and other mixtures thereof). Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomeric forms, as well as their mixtures of these compounds, are specifically included in the present description. The compounds described herein may also or additionally contain bonds in which the rotation of the bond is restricted relative to that particular bond, e.g., restricted as a result of the presence of a ring or a double bond (e.g., carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds) . Accordingly, all cis/trans and E/Z isomers and rotational isomers are expressly included in this specification. Unless otherwise mentioned or indicated, the chemical designation of a compound includes the mixture of all possible stereochemically isomeric forms of that compound.

Optical isomers can be obtained in pure form using standard procedures known to those skilled in the art and include, but are not limited to, diastereomeric salt formation, kinetic optical resolution, and asymmetric synthesis. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972), each of which is incorporated herein by reference in its entirety. In addition, it should be understood that the compounds described herein include all possible regioisomers and mixtures thereof, which can be obtained in pure form using standard separation procedures known to specialists in this field of technology, they include, without limitation, column chromatography, thin layer chromatography and high performance liquid chromatography.

Unless otherwise stated, the compounds provided herein may also include all isotopes of atoms present in the intermediate or final compounds. Isotopes include those atoms that have the same atomic number but different mass numbers. Unless otherwise noted, when an atom is designated as an isotope or radioisotope (e.g., deuterium, [ ¹¹ C], [ ¹⁸ F]), the atom is considered to contain an isotope or radioisotope in an amount at least greater than the natural abundance of the isotope or radioisotope . For example, when an atom is designated "D" or "deuterium", it is understood that a deuterium content is present at a position that is at least 3,000 times the natural abundance of deuterium, which is 0.015% (i.e., at least 45 % inclusion of deuterium).

All compounds and their pharmaceutically acceptable salts can be found together with other substances such as water and solvents (eg hydrates and solvates), or they can be isolated.

In some embodiments, the preparation of compounds may include the addition of acids or bases to affect, for example, the catalysis of the desired reaction or the formation of salt forms such as acid addition salts.

Exemplary acids may be inorganic or organic acids and include, without limitation, strong and weak acids. Some examples of acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include, without limitation, acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid.

Illustrative bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some examples of strong bases include, without limitation, hydroxide, alkoxides, metal amides, metal hydrides, metal dialkylamides, and arylamines, the alkoxides including lithium, sodium, and potassium salts of methyl, ethyl, and tert-butyl oxides; metal amides include sodium amide, potassium amide and lithium amide; metal hydrides include sodium hydride, potassium hydride and lithium hydride; and metal dialkylamides include the lithium, sodium, and potassium salts of methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, trimethylsilyl, and cyclohexyl substituted amides.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms that, based on careful clinical evaluation, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems. or complications, commensurate with a reasonable risk/benefit ratio.

The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, the term "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, without limitation, salts of inorganic or organic acids and basic residues such as amines; alkali or organic salts of acid residues such as carboxylic acids; etc. Pharmaceutically acceptable salts according to the present application include conventional non-toxic salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts according to the present application can be synthesized from the parent compound, which contains a basic or acidic fragment, using conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or mixtures thereof; in general, non-aqueous media such as ether, ethyl acetate, alcohols (eg methanol, ethanol, isopropanol or butanol) or acetonitrile (MeCN) are preferred. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences , 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science , 66, 2 (1977). Conventional methods for preparing salt forms are described, for example, in Handbook of Pharmaceutical Salts : Properties, Selection and Use , Wiley-VCH, 2002.

Pharmaceutical compositions

When used as pharmaceuticals, the compounds provided herein and their pharmaceutically acceptable salts can be administered in the form of pharmaceutical compositions. These compositions can be prepared as described herein or elsewhere and can be administered in a variety of ways depending on whether topical or systemic therapy is required and the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic, and mucosal administration, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal administration), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular administration or injection or infusion; or intracranial (eg, intrathecal or intraventricular administration). Parenteral administration can be in the form of a single bolus dose or can be done, for example, using a continuous infusion pump. In some embodiments, the compounds provided herein, or pharmaceutically acceptable salts thereof, are suitable for parenteral administration. In some embodiments, the compounds provided herein are suitable for intravenous administration. In some embodiments, the compounds provided herein are suitable for oral administration. In some embodiments, the compounds provided herein are suitable for topical administration.

Pharmaceutical compositions and topical formulations may include, without limitation, transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily bases, thickeners and the like may be necessary or desirable. In some embodiments, the pharmaceutical compositions provided herein are suitable for parenteral administration. In some embodiments, the compounds provided herein can be formulated for topical delivery (eg, for the treatment of acne). The illustrative composition includes the following:

about 10-about 50 mM of the compound provided herein (for example, compound 767)

approximately 50% (v/v) 99.5 - 100% ethanol or isopropanol

about 0.25 - about 2% (w/v) carbomer (e.g. carbomer 934 or carbomer 940)

about 0.5 - about 1% (w/v) trolamine

water (up to 100%).

In some embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for oral administration. In some embodiments, the pharmaceutical compositions provided herein are suitable for topical administration.

In addition, pharmaceutical compositions are contemplated which contain, as an active ingredient, a compound provided herein, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers (eg excipients). In preparing the pharmaceutical compositions provided herein, the active ingredient is typically mixed with an excipient, diluted with an excipient, or enclosed in such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it may be a solid, semi-solid or liquid material which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions may, for example, be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations may further include, without limitation, lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; flavors or combinations thereof.

The active compound may be effective over a wide dosage range and is generally administered in an effective amount. However, it should be understood that the amount of the compound actually administered will generally be determined by the physician in accordance with the relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound administered, the age, body weight, and response of the individual subject, the severity of the subject's symptoms; and the like.

The compositions provided herein can be administered from one or more times a day to one or more times a week; including once every two days. One skilled in the art will appreciate that certain factors may influence the dosage and administration intervals necessary to effectively treat a subject, including, without limitation, the severity of the disease or disorder, prior treatment, the subject's general health and/or age, and other diseases present. In addition, treatment of a subject with a therapeutically effective amount of a compound described herein may include a single treatment or a series of administrations. In some embodiments, the compositions provided herein are administered as a single dose.

The dosage, toxicity and therapeutic efficacy of the compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD ₅₀ (dose that is lethal in 50% of the population) and ED ₅₀ (the dose that is therapeutically effective in 50% of the population ). The dose ratio between toxic and therapeutic effects is a therapeutic index, and can be expressed as the ratio LD ₅₀ /ED ₅₀ . Compounds exhibiting high therapeutic performance are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of diseased tissue in order to minimize potential damage to non-infected cells and thus reduce side effects. In some embodiments, the compounds provided herein exhibit lower toxicity compared to similar dosages of orlistat. A toxicity score can be determined, for example, using methods such as those described herein.

Methods of treatment

The present disclosure further provides methods for treating disorders associated with elevated lipid levels (eg, lipotoxicity). A number of such violations are known in the present technical field and can be easily identified by a person skilled in the present technical field. In some embodiments, the methods provide for a method of inhibiting serum lipases in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the compounds provided herein reduce serum lipase concentrations. In some embodiments, the lipase is a pancreatic lipase, such as human pancreatic triacylglycerolipase (hPNLIP), human pancreatic lipase-associated protein 2 (hPLRP2), and human colipase (hCEL). In some embodiments, the compounds provided herein may reduce or inhibit the formation of free fatty acids. For example, the compounds provided herein can decrease serum free fatty acid concentrations.

As used herein, the term "subject" refers to any animal, including mammals. For example, the term "subject" includes, without limitation, mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject is obese. As used herein, the term "obese" refers to a subject having a body mass index greater than 23, 25, and in particular greater than or equal to 30. Alternatively, it may represent an increase in abdominal fat or girth greater than normal. for that race, ethnic group, gender, or nationality of the individual.

In some embodiments, the methods described herein may include in vitro methods , such as contacting a sample (eg, cell or tissue) with a compound provided herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the disorder is acute pancreatitis. For example, acute pancreatitis may be severe. In some embodiments, the methods provided herein may be useful for reducing the severity of acute pancreatitis from a severe case to a mild case of pancreatitis. The present disclosure provides methods for treating one or more symptoms of pancreatitis in a subject in need thereof by administering a therapeutically effective amount of a compound provided herein, or a pharmaceutically acceptable salt thereof, to treat one or more symptoms of acute pancreatitis. Exemplary symptoms of acute pancreatitis include abdominal pain, back pain, bloating, nausea, vomiting, fever, rapid pulse, shortness of breath, low body temperature, and the like.

Non-limiting embodiments of the present invention provide a method for reducing the risk of organ failure in a subject suffering from acute pancreatitis, comprising administering to the subject an effective amount of a pancreatic lipase inhibitor. Organs for which the risk of organ failure may be reduced include the kidney (where the failure is called kidney failure), the lung (where the failure is called pulmonary failure), shock with low blood pressure or an increase in heart rate or the appearance of pulmonary edema ( noted as crackles on auscultation or chest x-ray or CT scan), as well as multiple organ failure (eg, multiple organ failure syndrome affecting at least these two organs). The condition of these organs can be determined using clinical methods well known in the present technical field. For example, without limitation, kidney function (and the development of kidney failure) can be assessed by elevated blood urea nitrogen, elevated creatinine, decreased urine output, and/or histological findings; lung function and progression of pulmonary insufficiency can be assessed by examining respiratory function, blood gases (oxygen and carbon dioxide), oxygen support needs (e.g. nasal cannula or face mask or ventilator, with various percentages and oxygen consumption) and/or histological data; (For indicators of organ failure see J. Wallach, 1978, Interpretation of Diagnostic Tests, Third Edition, Little, Brown and Co., Boston, and/or J. Wallach, 2006, Interpretation of Diagnostic Tests, Eighth Edition, Lippincott Williams & Wilkins, both documents are hereby incorporated by reference in their entirety). Similarly, a measure of systemic inflammation is an increase in one or more inflammatory mediators, including, but not limited to, CRP or parts of the systemic inflammatory response syndrome (SIRS) criteria, tumor necrosis factor alpha, monocyte chemotactic protein 1, and/or interleukin 6. These embodiments are supported, at least in part, by the following working examples, which show the effectiveness of pancreatic lipase inhibitors in reducing the risk of multiple organ failure.

In some embodiments, the compounds provided herein are useful for reducing the risk of secondary effects of acute pancreatitis in a subject in need thereof. For example, the risk of developing shock may be reduced. In some embodiments, the risk of developing kidney failure is reduced. In some embodiments, the risk of developing lung failure is reduced.

In some embodiments, the disorder is acne. In other embodiments, the disorder is trauma, hemorrhage, critical illness, or sepsis. For example, sepsis is culture-negative sepsis. In some embodiments, the violation is a burn. In other embodiments, the disorder is an infection. For example, the infection may be caused by one or more organisms selected from the group consisting of P. auregenosa , S. aureus , B. subtilis , and B. cepecia . See, for example, Ryan CM, Sheridan RL, Schoenfeld DA, Warshaw AL, Tompkins RG: Postburn pancreatitis. Ann Surg 1995, 222(2):163-170; Subramanian A, Albert V, Mishra B, Sanoria S, Pandey RM: Association Between the Pancreatic Enzyme Level and Organ Failure in Trauma Patients. Trauma Mon 2016, 21(2):e20773; Malinoski DJ, Hadjizacharia P, Salim A, Kim H, Dolich MO, Cinat M, Barrios C, Lekawa ME, Hoyt DB: Elevated serum pancreatic enzyme levels after hemorrhagic shock predict organ failure and death. J Trauma 2009, 67(3):445-449; Lee CC, Chung WY, Shih YH: Elevated amylase and lipase levels in the neurosurgery intensive care unit. J Chin Med Assoc 2010, 73(1):8-14; and Manjuck J, Zein J, Carpati C, Astiz M: Clinical significance of increased lipase levels on admission to the ICU. Chest 2005, 127(1):246-250.

EXAMPLES

Example 1 Preparation and Testing of Unformylated Amino Acid Analogs

A number of unformylated amino acid analogs were prepared in which the L-leucine amino ester was replaced by substituents. The synthesis steps involved in this process are described in FIG. 2. As shown in FIG. 3, the resulting compounds included compounds where the amino ester of L-leucine (No. 723, which is also orlistat) was replaced with substituents such as N-methylleucine (No. 724) or more polar or shorter substituents, including L-alanine (No. 716), N-Acetyl-L-Cystine (No. 717), L-Aspartate (No. 718), L-Histidine (No. 725), L-Methionine Sulfone (No. 726), L-Arginine (No. 727), L -diaminopimelic acid (No. 728) L-serine (No. 729), L-ornithine (No. 731) or N-acetyleucine (No. 732).

To test the efficacy of these compounds, the agents were dissolved as a 20 mM stock solution in ethanol and further diluted to PBS as a 600 µmol working stock solution (3.3% ethanol). It was used either immediately or left at room temperature for 16-20 hours overnight before retesting efficacy. Agents were incubated with pancreatic lysate in serial 1/3 dilutions ranging from 200 to 0.3 µmol for 30 minutes. Then 5 μl of these mixtures were used to measure lipase activity using a commercial lipase assay (Pointe Scientific) containing monoacylglycerolipase in the reagent. Lipase assay was performed using reagents in proportion to the recommended protocol. Values are presented as % maximum lipase activity in the well without any inhibitor. As can be seen, there was no increase in efficacy of the unformylated orlistat analogs compared to that of orlistat (FIG. 4).

Example 2 Preparation and Testing of Formylated Amino Acid Analogs

A number of formylated amino acid analogs were prepared in which the L-leucine amino ester was replaced by substituents. The synthesis steps involved in this process are described in FIG. 2 and FIG. 5.

These agents (Nos. 733, 734, 738, 739, 740, 742, 743, as shown in Fig. 6) were initially tested as described in Example 1. It was noted that compounds 741, 736, 743 were characterized by an approximately 10-fold improvement in stability and efficacy compared to orlistat after overnight storage (FIG. 7). The compounds were further tested for efficacy in reducing lipotoxic damage to acinar cells in co-culture of acinar cells and adipocytes as described in Navina S, Acharya C, DeLany JP, Orlichenko LS, Baty CJ, Shiva SS, Durgampudi C, Karlsson JM, Lee K, Bae KT et al: Lipotoxicity causes multisystem organ failure and exacerbates acute pancreatitis in obesity. Sci Transl Med 2011, 3(107):107ra110 (FIG. 8A), and while all agents were effective at 66 µmol (the closest concentration to the IC _{50 of} orlistat after overnight incubation), compound 743 was noted to be toxic for acinar cells (FIG. 8B) and compound 741 was the least toxic at this concentration while maintaining its efficacy. Compound 736 also retained its efficacy with low toxicity.

Example 3 Preparation and Testing of β-Chain Analogs

To further explore options for improving the performance of analogs, the 741 beta chain was modified to be more hydrophilic. For this, compounds 760, 762, 763 were prepared as described in FIG. 9-14. Modifications at this position were associated with no improvement in lipase inhibition efficiency and, in fact, a decrease in efficiency was observed. See fig. fifteen.

Example 4 Preparation and Testing of α-Chain Analogs

A series of analogs was generated based on alpha chain modifications as shown in FIG. 16, creating compounds 767 and 768. Although these agents had equivalent performance in the fresh orlistat lipase activity assay, agent 767 retained its potency significantly when stored overnight. See fig. 17.

Example 5 Inhibition of Free Fatty Acid (FFA) Formation, Toxicity, and Efficacy in Inhibiting Recombinant Human Pancreatic Triacylglycerolipase [hPNLIP], Human Pancreatic Lipase-Associated Protein 2 (hPLRP2), and Human Carboxyether Lipase (hCEL)

The selection efficiency of the obtained analogs was investigated to determine their ability to inhibit the formation of free fatty acids (FFA) from recombinant human lipases while maintaining stability. To test these characteristics, analogs were dissolved in ethanol (200 mM stock solution) and further diluted to 600 µmol (0.3% ethanol) working stock solution in PBS. Agents were incubated with recombinant enzymes at serial 1/3 dilutions ranging from 200 to 0.3 µmol for 30 minutes and then added to the substrate with 1 mM glyceryltrilinoleate (GTL) in PBS, pH 7.4 along with cofactor colipase (CLPS; 0.5 µg/ml). As seen in FIG. 18, Agents 767, 733, 734, 740, and 743 had an IC ₅₀ against recombinant human pancreatic triacylglycerolipase [PNLIP; hPTL, in the presence of colipase (CLPS)], which were 50 times lower than orlistat, or 741, maintaining an IC ₅₀ of <2 μmol when stored overnight. In particular, compounds 767 and 740 showed better stability.

Toxicity studies of the respective compounds were also performed using a dilution of 200 mM stock stock solution in ethanol in acinar cell incubation medium (HEPES buffer, pH 7.4, as described by Navina et al.) to 600 µmol stock solutions of the compounds in 0.3% ethanol. at PBS. They were added to acinar cells in 1/3 dilutions starting from 200 µmol. Mouse pancreatic ATP levels and LDH leakage were measured over 4 hours and compared with controls. Any number positive compared to control indicates % damage greater than control acinar cells. Agent 743 (see FIG. 8B) and 733, however, were toxic to acinar cells, causing an ATP drop and LDH leakage (see FIG. 19) at concentrations in the range of 20-200 µmol, concentrations relevant to use in vivo.

These compounds were further tested for efficacy in reducing recombinant human pancreatic lipase-associated protein 2 (hPLRP2) and human carboxyether lipase (hCEL), as shown in FIG. 20. These lipases make up 15-30% of pancreatic lipases. Agents 740, 734 and 767 again showed IC ₅₀ of 2 µmol or less. Relative potency and toxicity are summarized in the table in FIG. 21. Noting this, along with the strengths of 767, i.e. a) higher potency against mouse pancreatic lysates (FIGS. 7 and 17), b) higher potency against the most abundant lipase (hPTL) (FIGS. 18), c) better stability in aquatic environments (FIGS. 17 and 18) and d) lower toxicity (FIG. 19), compound 767 was selected for further in vivo studies. In addition, agents 740 and 767 at a concentration of 10 μmol were less toxic and more effective than orlistat in reducing GTL-induced damage to acinar cells (FIG. 28).

Example 6 In Vivo Studies of Compound 767

The aim of this in vivo study was to see if the severity of lethal pancreatitis in mice can be reduced when compound 767 is administered as a single therapeutic dose and if this compound can avoid the limitations of orlistat, such as repeated high doses and hypertriglyceridemia. See fig. 22.

Compound 767 or orlistat was dissolved as 1 mg/10 μl ethanol, which was diluted to 400 μl with saline (final concentration 2.5% ethanol). Genetically obese male ob/ob mice (50-60 g) were used and blood was taken from the tail vein to determine baseline serum levels of lipase, triglycerides and BUN (blood urea nitrogen). The timeline is shown in Fig. 23A. Severe pancreatitis was induced by subcutaneous implantation of an Alzet pump containing 2.5 mg/ml cerulein administered at a dose of 50 μg/kg/hour. Six hours later, a blood sample from the tail vein (50 μl) was analyzed for serum lipase to confirm an increase of more than 3 times baseline. As seen in FIG. 23B, this was achieved (P = 0.003).

The agents were then administered as an intraperitoneal dose (20 mg/kg single dose [n=8 for 767 or orlistat n=7]. Animals were administered saline 1 ml intraperitoneally twice a day, vital organs were monitored daily, paying attention to the severity (temperature with fever or hypothermia and pulse with shock.) They are respectively part of the SIRS and the revised Atlanta classification of severity criteria (Classification of acute pancreatitis - 2012: revision of the Atlanta classification and definitions by international consensus. (Classification of acute pancreatitis--2012: revision of the Atlanta classification and definitions by international consensus) Gut 2013, 62(1):102-111)). Tail vein blood samples were collected for severity parameters (serum BUN, serum calcium, and total white blood cells) which are part of the Ranson and Glasgow criteria) and triglycerides. Animals were killed if they experienced agony.

As seen in FIG. 23, all untreated mice developed renal failure in vivo (BUN 85 ± 18 mg/dL) FIG. 23C, hypocalcemia (serum calcium 4.9 ± 0.2 mg/dL) FIG. 23D, SIRS of FIG. 23E, the shock of FIG. 23F and 100% mortality of FIG. 23g. Agent 767 was more effective than orlistat in normalizing BUN (peak 18.5 ± 0.9 vs. orlistat 50 ± 4.4 mg/dL, p<0.05) FIG. 23C, serum calcium levels (8.8 ± 1.0 mg/dL vs. 4.2 ± 0.3 mg/dL, p<0.05) FIG. 23D, UFA (2.4±0.4 mM vs. 5.4±0.9 mM), shock prevention (PD: 290±20 µm vs. 171±21 µm, p<0.05) FIG. 23F, leukopenia (in 0/8 mice vs. 4/7 mice, p<0.03, see Fig. 25), hypothermia (94.1 ± 0.8 vs. 86 ± 0.9°F, p <0.01; Fig. 23E) and improved 5-day survival (8/8 vs. 2/7, p < 0.01) of Figs. 23g. In the vehicle group, mortality was 8/8 (mean time 44 hours), in the orlistat group, 5/7. The macroscopic picture for the two agents was compared in FIG. 24. As can be seen, the orlistat-treated animal showed much more extensive fat necrosis. This protection with agent 767 was achieved without the hypertriglyceridemia associated with the dosage required for protection with orlistat (FIG. 25). The results are summarized in the table in FIG. 25.

Example 7 Creation of FFA in P. acnes Medium

P. acnes-induced lipolysis was inhibited by 767 (FIG. 26 and FIG. 27).

Broth culture of P. acnes:

For medium: 2.0 g Casein Enzymatic Hydrolyzate (Cat. No. 12855, Affymetrix Inc, Ohio, USA), 1.0 g Yeast Extract (Cat. No. 61180-5000, Acros Organics, NJ, USA) and 0.03 g of Victoria Blue B (Cat. No. sc-216055, Santa Crutz Biotechnology Inc, CA, USA) was completely dissolved in 200 ml of double distilled water with gentle heating. The mixed solution was sterilized in an autoclave at a pressure of 15 pounds (121°C) for 15 minutes. The autoclaved solution was allowed to cool to 50° C., then 6 ml of emulsified lipase substrate was slowly added with continuous stirring to obtain uniform distribution and stored at 4° C. for P. acnes culture.

Preparation of Lipase Substrate: Lipase substrate was prepared by mixing 200 μl Tween 80 (Cat. No. P4780, Sigma, St. Louis, USA) in 100 ml of warm double-distilled water and 25 ml of cottonseed oil (Cat. No. C0145, Spectrum chemical , California, USA). The mixture was vigorously stirred, followed by sonication (pulse 10 sec x 5 times) to obtain an emulsion.

P. acnes culture-propagation medium, storage conditions, inoculum size used in the medium.

A small portion of lyophilized Propionibacterium acnes (strain VPI0389, ATCC) was added to the medium and allowed to grow at 37°C for 48 hours in a standard BD GasPak EZ incubation container (Cat. No. 260671, Beckton Dickinson, MD, USA) with two anaerobic sachets (Cat. No. 260683, Beckton Dickinson, MD, USA) that maintained anaerobic conditions in the container. After 48 h of cultivation, the number of bacteria was measured by the turbidimetric method (OD 600 nm). The amount of secreted lipase and produced FFA was measured in the culture supernatant.

For storage of P. acnes , cultured bacteria in a 1.5 ml autoclaved Eppendorf microcentrifuge (1 ml/microcentrifuge) were centrifuged at 5 rpm for 5 minutes, the spent medium was removed, the precipitated bacteria were resuspended in P. acnes culture medium containing 15% glycerol ( 1 ml/microcentrifuge) without substrate and stored at -80°C for later use.

For further cultivation of P acnes , one aliquot of P acnes concentrate was removed from -80° C. and thawed on ice. Nearly 30 µl of P. acnes concentrate (1 x 10°/30 µl) was added to 1 ml of lipase substrate culture medium in each 2 ml Eppendorf microcentrifuge and incubated at 37° C. for 48 hours under anaerobic conditions.

Efficacy trial or lipase inhibitors against P. acnes:

Orlistat or agent 767 (stock solution 200 mM in ethanol) was added at the indicated concentrations at the beginning of the incubation. At the end of the incubation period, a portion of the well-mixed medium was removed and free fatty acid formation was analyzed using the HR Series NEFA-HR(2) kit from Wako Diagnostics (Richmond Virginia).

Agar culture of P. acnes

Alcohol Blue Agar: Supplier, Cat. No. Receipt, storage. Culture inoculum size, culture conditions.

To prepare a plate, 3.215 g of alcohol blue agar (Cat. No. M445, HiMedia Laboratories Pvt Ltd, Mumbai, India) was dissolved in 100 ml of double distilled water, autoclaved, left to cool to 55°C, 3 ml of warm lipase substrate was added and well mixed. While warm, the mixture was poured into tissue culture plates (6-well, 12-well plates) under aseptic conditions. The solidified agar plates were wrapped in parafilm and kept at 37° C. overnight to confirm the absence of contamination. Additional plates were stored at 4° C. in a freezer for later use. Thawed P. acnes concentrates recovered from -80° C. were either dispensed (30 μl/well) into 6-well/12-well plates or inoculated throughout the well with inoculation loops. The plates were then stored at 37° C. in a BD GasPak EZ chamber with one anaerobic sachet and waited 5-7 days to see bacterial colonies. Then they were visualized and photographed on a trans-illuminator.

Other embodiments

It should be understood that while the present invention has been described in conjunction with its detailed disclosure, the foregoing description is intended to be illustrative and not to limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims (26)

1. A compound selected from the group consisting of

and

or their pharmaceutically acceptable salt. 2. The compound according to claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof. 3. The compound according to claim 1, wherein the compound is

or a pharmaceutically acceptable salt thereof. 4. Pharmaceutical composition for lipase inhibition, containing a therapeutically effective amount of a compound according to any one of paragraphs. 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. 5. Application of a compound selected from the group consisting of

and

or a pharmaceutically acceptable salt thereof for the treatment of acute pancreatitis in a subject in need thereof. 6. Use according to claim 5, in which acute pancreatitis is severe. 7. Use according to claim 5 wherein the severity of acute pancreatitis is reduced from severe to mild after administration. 8. Use according to claim 5 wherein the subject is obese. 9. Use according to claim 5, in which the risk of developing shock is reduced. 10. Application of the compound of the formula

or a pharmaceutically acceptable salt thereof for the treatment of acne in a subject in need thereof. 11. Use according to claim 5, wherein the compound is

or a pharmaceutically acceptable salt thereof. 12. Use according to claim 5, wherein the compound is

or a pharmaceutically acceptable salt thereof.

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