Compositions and methods for treatment and mitigation of alcohol-induced skin flushing

Abstract

To provide pharmaceutical compositions for use in the prevention and treatment of alcohol-induced hypersensitivity reactions including alcohol-induced skin flushing.SOLUTION: A pharmaceutical composition comprises: a therapeutically effective amount of 4-methylpyrazole, or a pharmaceutically acceptable salt, hydrate, polymorph, or solvate thereof; Withania somifera / ashwaganda; and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition further comprises at least one pharmaceutically inert excipient in an amount effective for masking the taste of 4-methylpyrazole.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Application No. 62 / 852,000, filed on May 23, 2019, the entire content of which is incorporated herein by reference.

Summary of the Invention

[0002] This application relates to a pharmaceutical composition having a therapeutically effective amount of 4-methylpyrazole, or a pharmaceutically acceptable salt, hydrate, polymorph, or solvate thereof, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is configured for oral administration and further has at least one pharmaceutically inert coating useful for masking the odor of 4-methylpyrazole. In some embodiments, the at least one pharmaceutically inert coating is selected from the group consisting of hydroxyalkylcellulose, anti-adherent, plasticizer, sugar, methacrylate copolymer, hydroxyalkylcellulose, and water-soluble polymer. In some embodiments, the pharmaceutical composition is configured as an oral dosage form selected from powder, tablet, lozenge, chewing gum, pill, capsule, microcapsule, caplet, orally disintegrating tablet, osmotic controlled release oral delivery system, or any combination thereof. In some embodiments, it further has n-acetylcysteine, ampelopsin, Withania somifera / ashwagandha, L-cysteine, S-acetylglutathione, molybdenum, iron, zinc, iron chelating agent, L-ascorbic acid, L-threonine, or any combination thereof. In some embodiments, the iron chelating agent has curcumin, quercetin, inositol hexakisphosphate (IP6), or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier is water. In some embodiments, the pharmaceutical composition further has a taste masking agent, an odor masking agent, or a combination thereof. In some embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount between about 10 and about 20 mg / kg. In some embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount that results in a plasma concentration of about 0.1 μmol / L.

[0003] Some embodiments relate to a pharmaceutical composition having a therapeutically effective amount of 4-methylpyrazole or a pharmaceutically acceptable salt, hydrate, polymorph, or solvate thereof and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is configured for transdermal administration. In some embodiments, the pharmaceutical composition further comprises n-acetylcysteine, ampelopsin, Withania somifera / ashwagandha, L-cystine, S-acetylglutathione, molybdenum, iron, zinc, an iron chelating agent, L-ascorbic acid, L-threanine, or any combination thereof. In some embodiments, the iron chelating agent comprises curcumin, quercetin, inositol hexaphosphate, or any combination thereof.

[0004] Some embodiments relate to a method of treating and / or preventing an alcohol-induced allergic reaction in a subject, the method comprising administering the pharmaceutical composition disclosed herein. In some embodiments, the alcohol-induced allergic reaction is selected from flushing, increased heart rate, palpitations, low blood pressure, nausea, dizziness, headache, vomiting, diarrhea, stomach upset, ataxia, confusion, hives, systemic dermatitis, allergic rhinitis, bronchoconstriction, exacerbation of asthmatic bronchoconstriction, cardiovascular collapse, allergic conjunctivitis, atopic dermatitis, eosinophilic esophagitis, anaphylaxis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), and any combination thereof. In some embodiments, the alcohol flush is characterized by facial redness, increased skin temperature, increased heart rate, decreased diastolic blood pressure, or any combination thereof. In some embodiments, the pharmaceutical composition is administered before the subject consumes alcohol. In some embodiments, the pharmaceutical composition is administered from about 60 minutes to about 15 minutes before the subject consumes ethanol. In some embodiments, the pharmaceutical composition is administered simultaneously with or after the subject consumes ethanol.

[0005] Some embodiments relate to a method of removing acetaldehyde formed from ethanol in a subject, the method comprising administering the pharmaceutical composition described herein.

[0006] Some embodiments relate to a method of reducing and / or removing acetaldehyde formed from ethanol in the oral cavity, esophagus, stomach, large intestine, or combinations thereof of a subject, the method comprising administering the pharmaceutical composition described herein.

[0007] Some embodiments relate to a method of reducing and / or removing the blood acetaldehyde concentration of a subject, the method comprising administering the pharmaceutical composition described herein.

[0008] Some embodiments relate to a method of reducing and / or removing acetaldehyde from the gastrointestinal tract of a subject, the method comprising administering the pharmaceutical composition described herein.

[0009] Some embodiments relate to a method of inhibiting mitochondrial aldehyde dehydrogenase 2 (ALDH2) in a subject, the method comprising administering the pharmaceutical composition described herein.

[0010] Some embodiments relate to a method for reducing the risk of a disease or disorder caused by the consumption of ethanol in a subject, the method comprising administering the pharmaceutical composition described herein.

[0011] In some embodiments, the disease or disorder is selected from upper aerodigestive tract cancer, gastrointestinal cancer, or breast cancer. In some embodiments, the upper aerodigestive tract cancer comprises esophageal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, head or neck cancer. In some embodiments, the gastrointestinal cancer comprises gastric cancer or colorectal cancer. In some embodiments, the disease or disorder comprises late-onset Alzheimer's disease, hypertension, myocardial infarction, Parkinson's disease, amyotrophic lateral sclerosis, and cerebral ischemia.

[0012] Some embodiments relate to a method of blocking the histamine release effect of acetaldehyde in a subject, the method comprising administering the pharmaceutical composition described herein.

[0013] Some embodiments relate to a method of blocking alcohol-induced acetaldehyde production in a subject, the method comprising administering the pharmaceutical composition described herein. In some embodiments, the subject is a heterozygote or homozygote for the aldehyde dehydrogenase 2 (ALDH2) allele Glu487lys, ALDH2 * 2, ALDH2 * 487lys, Glu487lys, or rs671.

[0014] Some embodiments further comprise testing the subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele Glu487lys, ALDH2 * 2, ALDH2 * 487lys, Glu504lys, or rs671. In some embodiments, testing the subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele Glu487lys, ALDH2 * 2, ALDH2 * 487lys, Glu504lys, or rs671 comprises obtaining an exhaled breath sample from the subject, measuring the ethanol and acetaldehyde levels in the exhaled breath sample, and determining the ratio of the acetaldehyde level to the ethanol level in the exhaled breath sample, wherein a ratio of the acetaldehyde level to the ethanol level of about 23.3 or greater indicates the presence of Glu487lys, ALDH2 * 2, ALDH2 *Determining that indicates the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487Lys, Glu504Lys, or rs671, and administering to the subject the pharmaceutical composition disclosed herein if the subject has a ratio of acetaldehyde level to ethanol level of about 23.3 or greater. In some embodiments, the step of measuring the ethanol level and the acetaldehyde level in the breath sample is performed by semiconductor gas chromatography. In some embodiments, the breath sample is obtained after alcohol consumption. In some embodiments, Glu487Lys, ALDH2 * 2, ALDH2 * The step of testing a subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487Lys, Glu504Lys, or rs671 includes obtaining a biological sample from the subject, separating genomic DNA from the biological sample, Glu487Lys, ALDH2 * 2, ALDH2 * Identifying the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487Lys, Glu504Lys, or rs671, Glu487Lys, ALDH2 * 2, ALDH2 * And administering to the subject the medicament described herein if the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487Lys, Glu504Lys, or rs671 is present. In some embodiments, the biological sample is a swab sample. In some embodiments, the biological sample is a blood sample.

BRIEF DESCRIPTION OF THE INVENTION

[0015] The present application relates to a pharmaceutical composition for use in the prevention and treatment of alcohol-induced allergic reactions including alcohol-induced skin flushing.

[0016] Alcohol, also known by the chemical name ethanol, is consumed for various social, recreational, and pharmaceutical purposes. In this specification, the terms "alcohol" and "ethanol" are used interchangeably. Excessive alcohol intake causes damage to various tissues such as the liver, brain, skeletal muscle, and myocardium, and is a significant cause of morbidity and mortality in public health. Many of these effects of alcohol are caused by acetaldehyde produced during the process of alcohol metabolism. Acetaldehyde undergoes a two-step pathway in which alcohol is oxidized by alcohol dehydrogenase (ADH) to acetaldehyde and is rapidly metabolized to acetic acid by aldehyde dehydrogenase (ALDH), a mitochondrial liver enzyme.

[0017] There are polymorphisms in the ADH and ALDH genes that regulate individual differences in alcohol oxidation ability (Bosron et al., Hepatology 1986, 6, 502-510). People in East Asia have a mutant allele in which the amino acid of alcohol dehydrogenase subtype 2 (ADH2 * 2) encoded by the ADH enzyme is substituted with R47H (Matsuo et al., Carcinogenesis 2006, 27(5), 1018-1023; Tamakoshi et al., Alcohol 2003, 38, 407-410). The H47 ADH enzyme is "superactive" and exhibits a Vmax approximately 40 times higher than that of the low-activity R47 ADH enzyme encoded by the "typical" allele (ADH2 * 1) (Bosron et al., Hepatology 1986, 6, 502-510; Yoshida ct al., Prog. Nucleic Acid Res. Mol. Biol. 1991, 40, 255-287). The ADH2 * 2 allele is associated with the accumulation of acetaldehyde (Crabb et al., Proc.Nutr.Soc.2004, 63(1), 49-63).

[0018] In addition, East Asians have an aldehyde dehydrogenase subtype 2 (ALDH2 * 2, Glu487lys, ALDH2 * 487lys, Glu504lys, or also known as rs671) mutant allele (Chen et al., Am. J. Hum. Genet. 1999, 65(3), 795 - 807). The K487 ALDH enzyme shows a decrease in activity such that the acetaldehyde removal rate is reduced by 40 - 90% compared to the more active E487 ALDH2 enzyme encoded by the typical allele (ALDH2 * 1). Therefore, individuals expressing the mutant allele will have reduced or absent ALDH2 activity.

[0019] Acetaldehyde is said to increase long - term health risks such as upper gastrointestinal cancer, breast cancer, liver disease, Alzheimer's disease, hypertension, and myocardial infarction, in addition to acute symptoms such as facial flushing, tachycardia, shortness of breath, dizziness, nausea, vomiting, and headache (Visapaa, Gut 2004, 53, 871 - 876; Yokoyama et al., Jpn. J. Clin. Oncol. 2003, 33(3), 111 - 121; Ohsawa et al., J. Hum. Genet. 2003, 48, 404 - 409; and references cited therein). Individuals expressing the ALDH2*2 allele with reduced or absent ALDH2 activity show alcohol - related hypersensitivity such as facial flushing and tachycardia when consuming a small amount of ethanol (Goedde et al., Hum. Genet. 1992, 88, 344 - 346; Xiao et al., J. Clin. Invest. 1995, 96, 2180 - 2186). Therefore, reducing acetaldehyde levels in individuals with reduced or absent ALDH2 activity is thought to help reduce the acute and long - term health risks these individuals experience when consuming ethanol.

[0020] 4-Methylpyrazole (also known as homopyrazole or 4-MP) inhibits alcohol dehydrogenase (ADH), an enzyme that oxidizes alcohol. It is an enzyme that oxidizes alcohols as part of a two-step metabolic elimination pathway in which ethanol is oxidized to acetaldehyde by ADH and then to acetic acid by aldehyde dehydrogenase (ALDH).

[0021] In some embodiments, the compounds for use in the methods described herein may be formulated as pharmaceutical compositions. The pharmaceutical compositions of the invention may comprise a compound described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Such compositions may optionally have at least one additional therapeutic agent useful for the prevention and treatment of alcohol-induced allergic reactions, including alcohol-induced skin flushing.

[0022] The compounds of the invention can be employed in conventional ways to control, treat, or reduce the progression and severity of the diseases described herein, including but not limited to alcohol-induced allergic reactions, including alcohol-induced skin flushing. Such treatment methods, their dosage levels, and requirements can be selected from the methods and techniques available to those skilled in the art. For example, the compounds of the invention can be administered in an amount effective to reduce the occurrence and severity of symptoms in a patient having or susceptible to an alcohol-induced allergic reaction, including alcohol-induced skin flushing, in combination with a pharmaceutically acceptable adjuvant.

[0023] In each embodiment disclosed herein, the compounds and methods can be utilized with or on a subject in need of such treatment, which may also be referred to as "in need thereof". As used herein, the phrase "in need thereof" means that a subject has been identified as having a need for a particular method or treatment and that the treatment has been administered to the subject for that particular purpose.

[0024] The terms "patient" and "subject" are interchangeable and are considered to mean any organism that may be treated with the compounds of the present invention. Thus, the terms "patient" and "subject" include, but are not limited to, any non-human mammal, primate or human. In some embodiments, the "patient" or "subject" is a mammal such as a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, primate, or human. In some embodiments, the patient or subject is an adult, child, or infant. In some embodiments, the patient or subject is a human.

[0025] As used herein, the terms "combination", "combined", and related terms mean the simultaneous or sequential administration of therapeutic agents in accordance with the present invention. For example, the described compounds may be administered separately in unit dosage forms simultaneously with or sequentially to another therapeutic agent, or may be administered together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form having the described compound, additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Two or more agents are typically considered to be administered "in combination" when a patient or individual is simultaneously exposed to both agents. In many embodiments, two or more agents are considered to be administered "in combination" when a patient or individual simultaneously exhibits therapeutically appropriate levels of the agents in a particular target tissue or sample (e.g., in the brain, in serum, etc.).

[0026] The term "pharmaceutically acceptable excipient" means a non-toxic excipient that, even when administered to a patient together with the compound of the present invention, does not destroy its pharmacological activity. Pharmaceutically acceptable excipients that can be used in these compositions include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, lanolin, but are not limited thereto. Pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present invention include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, lanolin, α-tocopherol, self-emulsifying drug delivery systems (SEDDS) such as polyethylene glycol 1000 succinate, or other similar polymeric delivery matrices, but are not limited thereto.

[0027] As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent that elicits a biological or pharmacological response in a tissue, system, animal, individual, or human that is sought by a clinician such as a researcher, veterinarian, medical doctor, etc., and includes one or more of the following: (1) prevention of a disease; for example, preventing a disease, condition, or disorder in an individual who may have a predisposition to the disease, condition, or disorder but has not yet experienced the pathology or symptoms of the disease, (2) suppression of a disease; for example, suppressing a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., preventing further development of the pathology and / or symptoms), (3) amelioration of a disease; for example, ameliorating a disease, condition, or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms). In some embodiments, the therapeutically effective amount of a compound represents the daily dosage of a particular compound. In some embodiments, the daily dosage of a particular compound may be administered as a single daily dose or divided into two or more doses of equal or unequal amounts administered throughout the day.

[0028] The compounds described in this specification can form their acid addition salts. It will be understood that the salts of the compounds described herein should be pharmaceutically acceptable for use as pharmaceuticals. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art and include those described in J. Pharm. Sci., 1977, 66, 1-19, for example, acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and organic acids such as succinic acid, maleic acid, acetic acid, fumaric acid, citric acid, tartaric acid, benzoic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid. The present invention includes within its scope all possible stoichiometric and non-stoichiometric forms. Pharmaceutically acceptable salts of the compounds described herein include, for example, conventional non-toxic salts or quaternary ammonium salts of the compounds from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isothionic acid. In other cases, the compounds described may contain one or more acidic functional groups and can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can likewise be prepared in situ in the administration vehicle or dosage form manufacturing process, or by separately reacting the purified compound in the free acid form with a suitable base, for example, a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali salts and alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, aluminum salts, etc.Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.

[0029] The compounds described herein can be prepared in crystalline or amorphous forms, and in the case of crystalline forms, they may optionally be dissolved in a solvent, for example, as a hydrate. The present invention includes not only stoichiometric solvates (e.g., hydrates) but also compounds having a variable amount of solvent (e.g., water) within its scope. The specific compounds described herein can exist as stereoisomers (e.g., diastereomers and enantiomers), and the present invention extends to each of these stereoisomers and mixtures thereof including racemates. Different stereoisomers may be separated from each other by conventional methods or any isomer may be obtained by stereospecific or asymmetric synthesis. Further, the present invention extends to any tautomers and mixtures thereof.

[0030] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but preferred methods, apparatus, and materials are described herein.

[0031] Also, the compounds of the present invention can be used in compositions and methods for treating or protecting an individual over a long period of time against the conditions described herein including, but not limited to, neurodegenerative diseases. This compound may be employed in such compositions alone or together with other compounds of this invention in a manner consistent with the conventional use of such compounds in pharmaceutical compositions. For example, by combining the compounds of the present invention with a pharmaceutically acceptable adjuvant conventionally employed in vaccines and administering in a prophylactically effective amount, an individual can be protected over a long period of time against the diseases described herein including, but not limited to, neurodegenerative diseases.

[0032] This application relates to a pharmaceutical composition having a therapeutically effective amount of 4-methylpyrazole, or a pharmaceutically acceptable salt, hydrate, polymorph, or solvate thereof, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for oral administration.

[0033] In some embodiments, the pharmaceutical composition further has at least one pharmaceutically inert coating useful for masking the odor of 4-methylpyrazole. In still other embodiments, the pharmaceutical compositions described herein can be encapsulated. In some embodiments, the encapsulation medium includes, but is not limited to, capsules, soft gel caps, gel caps, coatings, or any combination thereof. In some embodiments, the coating can include, but is not limited to, films, waxes, varnishes, glazes, polymer coatings, sugar coatings, polysaccharide-based coatings, enteric coatings, or combinations thereof.

[0034] In some embodiments, at least one pharmaceutically inert coating is selected from the group consisting of hydroxyalkylcellulose, hydroxypropylcellulose, anti-adhesive agents, plasticizers, saccharides, methacrylic acid copolymers, hydroxyalkylcellulose, and water-soluble polymers. Examples of anti-adhesive agents include, but are not limited to, talc (Alphafil 500), silicon dioxide, silica hydrogel, microcrystalline cellulose, alkali stearates, starch, and combinations thereof. Examples of plasticizers include, but are not limited to, propylene glycol, glycerin, trimethylolpropane, polyethylene glycol, dibutyl sebacate, acetylated monoglyceride, diethyl phthalate, triacetin, glyceryl triacetate, acetyltriethyl citrate, triethyl citrate, and combinations thereof. Examples of water-soluble polymers include, but are not limited to, hydroxypropylcellulose, hydroxypropylmethylcellulose, acacia, sodium carboxymethylcellulose, dextrin, alginic acid, ethylcellulose resin, gelatin, guar gum, liquid glucose, methylcellulose, pregelatinized starch, sodium alginate, starch, zein, polyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate copolymer, vinyl acetate-crotonic acid copolymer, ethyl acrylate-methacrylic acid copolymer, and combinations thereof.

[0035] In some embodiments, the pharmaceutical composition is configured as an oral dosage form selected from powders, tablets, lozenges, chewing gums, pills, capsules, microcapsules, caplets, orally disintegrating tablets, osmotic pressure-controlled release oral delivery systems, or combinations thereof.

[0036] The formulations described herein suitable for oral administration are in the form of capsules, cachets, tablets, lozenges (usually using a flavor base such as sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids. Further, troches (using inert bases such as gelatin and glycerin, sucrose and acacia, etc.) and / or mouthwashes containing a predetermined amount of the compound of the present invention as an active ingredient can also be used. Further, the compounds described herein may be administered as a bolus, a gargle or a paste.

[0037] For solid agents for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is one or more pharmaceutically acceptable carriers such as sodium citrate and dicalcium phosphate, and / or any of the following: fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, silicic acid, binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia, wetting agents such as glycerol, disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, solution retardants such as paraffin, absorption promoters such as quaternary ammonium compounds, wetting agents such as cetyl alcohol, glyceryl monostearate, nonionic surfactants, adsorbents such as kaolin, bentonite clay, lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof, and is mixed with a coloring agent. In the case of capsules, tablets, and pills, the pharmaceutical composition may have a buffering agent. Further, solid compositions of the same type can also be used as fillers for soft-shell and hard-shell gelatin capsules using excipients such as lactose and lactulose, high molecular weight polyethylene glycol, etc.

[0038] Tablets are made by compression or molding, and one or more accessory components (excipients) may optionally be used. Compressed tablets can be prepared using a binder (e.g., gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, or cross-linked carboxymethylcellulose), surfactant, or dispersant, etc. Molded tablets can be made with a suitable machine that wets a mixture of the powdered compound with an inert liquid diluent. When a solid carrier is used, the preparation can be in the form of tablets, in hard gelatin capsules in the form of powder or pellets, or in the form of troches or lozenges. The amount of the solid carrier varies, for example, from about 0.01 to 800 mg, preferably from about 0.01 mg to 400 mg, or about 3 mg to about 400 mg. When a liquid carrier is used, the preparation can be, for example, in the form of a syrup, emulsion, sterile injection such as soft gelatin capsules, ampoules, or a non-aqueous liquid suspension. When the composition is in the form of capsules, for example, any conventional encapsulation using the aforementioned carriers within a hard gelatin capsule shell is appropriate.

[0039] Tablets and other solid dosage forms, such as sugar-coated tablets, capsules, pills, and granules, can optionally be coated or prepared with coatings or shells such as enteric coatings or other coatings well known in pharmaceutical formulation technology. In addition, the active ingredient can be formulated for sustained release using hydroxypropyl methylcellulose with varying ratios, other polymer matrices, liposomes, and / or microparticles to obtain a desired release profile. Formulations for rapid release, such as lyophilization, are also possible. Sterilization can be carried out, for example, by filtration through a bacteria-proof filter or by incorporating in the form of a sterile solid composition that can be dissolved in sterile water or other sterile injectable immediately before use of a bactericide. Also, these compositions can optionally contain an opacifier and be in a composition that releases the active ingredient only or preferentially at a specific part of the digestive tract, optionally with a delay. Examples of implantable compositions that can be used include polymeric substances and waxes. The active ingredient can, if appropriate, be in the form of being microencapsulated using one or more of the excipients described above.

[0040] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0041] In addition to the inert diluent, oral compositions can contain adjuvants such as wetting agents, emulsifiers, suspending agents, sweeteners, flavors, colorants, flavoring agents, preservatives, and the like.

[0042] The suspension can contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

[0043] The amount of the active ingredient, or its active salt or derivative, required for treatment varies not only depending on the specific salt selected, but also on the route of administration, the nature of the condition being treated, the age and condition of the patient, and ultimately is left to the judgment of the attending physician or clinician. Generally, those skilled in the art understand how to extrapolate in vivo data obtained in a model system, typically an animal model, to another model such as a human. Depending on the circumstances, these extrapolations may be based solely on the weight of the animal model compared to other animals such as mammals, preferably humans, but more often these extrapolations incorporate various factors rather than being based simply on weight. Representative factors include the type, age, weight, sex, diet, medical condition, route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics, toxicity profile of the particular compound employed, whether a drug delivery system is utilized, whether an acute or chronic medical condition is being treated, whether prophylaxis is being carried out, or whether, in addition to the compounds of the present invention, further active compounds are being administered as part of a drug combination. The dosing regimen for treating a medical condition using the compounds and / or compositions of the present invention is selected according to the various factors as cited above. Accordingly, the dosing regimen actually employed can vary widely and thus may deviate from the preferred dosing regimen, and those skilled in the art will recognize that they can test these dosing amounts and regimens outside of these typical ranges and, where appropriate, use them in the methods of the present invention.

[0044] The desired dosage is preferably presented as a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-doses themselves can be further divided, for example, for administration at several discrete loose intervals. The daily dosage can be divided into several, for example two, three or four partial administrations, especially when a relatively large amount of administration is considered particularly appropriate. Depending on the individual's behavior, it may be necessary to deviate above or below the indicated daily dosage as required.

[0045] In some embodiments, the pharmaceutical compositions disclosed herein may further comprise N-acetylcysteine, ampelopsin, Withania somifera / ashwagandha, L-cysteine, S-acetylglutathione, molybdenum, iron, zinc, iron chelating agents (curcumin / quercetin / IP6), L-ascorbic acid, L-threonine, or any combination thereof. In some embodiments, the iron chelating agent has curcumin, quercetin, inositol hexaphosphate (IP6), or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier is water. In some embodiments, the pharmaceutical composition further comprises a taste masking agent, an odor masking agent, or a combination thereof.

[0046] In some embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount between about 0.1 and about 200 mg / kg. In some embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount between about 10 and about 20 mg / kg. In still other embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount that results in a plasma concentration between about 0.01 μmol / L and about 10 μmol / L. In still other embodiments, the therapeutically effective amount of 4-methylpyrazole is an amount that results in a plasma concentration of about 0.1 μmol / L.

[0047] Embodiments of the present specification also relate to a pharmaceutical composition having a therapeutically effective amount of 4-methylpyrazole or a pharmaceutically acceptable salt, hydrate, polymorph, or solvate thereof and a pharmaceutically acceptable carrier, and this pharmaceutical composition is configured for transdermal administration.

[0048] In some embodiments, the pharmaceutical composition may further have n-acetylcysteine, ampelopsin, Withania somifera / ashwagandha, L-cystine, S-acetylglutathione, molybdenum, iron, zinc, an iron chelating agent (curcumin / quercetin / IP6), L-ascorbic acid, L-threanine, or any combination thereof. In some embodiments, the iron chelating agent has curcumin, quercetin, inositol hexaphosphate, or any combination thereof.

[0049] Also described herein is a method for treating and / or preventing alcohol-induced allergic reactions in a subject, the method comprising administering the pharmaceutical composition described herein. In some embodiments, the alcohol-induced allergic reaction is selected from flushing, increased heart rate, palpitations, low blood pressure, nausea, dizziness, headache, vomiting, diarrhea, stomach upset, ataxia, confusion, urticaria, systemic dermatitis, allergic rhinitis, bronchoconstriction, exacerbation of asthmatic bronchoconstriction, cardiovascular collapse, allergic conjunctivitis, atopic dermatitis, eosinophilic esophagitis, anaphylaxis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), and any combination thereof. In some embodiments, alcohol-induced flushing is characterized by facial redness, increased skin temperature, increased heart rate, decreased diastolic blood pressure, or any combination thereof. In some embodiments, the pharmaceutical composition is administered before the subject consumes alcohol.

[0050] In some embodiments, the pharmaceutical composition is administered about 60 minutes to about 15 minutes before the subject consumes alcohol. In some embodiments, the pharmaceutical composition is administered simultaneously with or after the subject consumes alcohol.

[0051] Also disclosed is a method for removing acetaldehyde generated from alcohol in a subject, which comprises the step of administering the pharmaceutical composition described herein.

[0052] Also described herein is a method for reducing and / or removing acetaldehyde formed from alcohol in the oral cavity, esophagus, stomach, large intestine, or combinations thereof of a subject, which comprises the step of administering the pharmaceutical composition described herein. According to research, acetaldehyde has been shown to be most abundant in these parts rather than in the bloodstream immediately after consumption (Salaspuro M P, Acetaldehyde, microbes and cancer of the digestive tract. Crit Rev Clin Lab Sci 2003; 40:183-208; Salaspuro M. Acetaldehyde as a common denominator and cumulative carcinogen in digestive tract cancers. Scand J Gastroenterol 2009; 44:912-25; Salaspuro M. Acetaldehyde and gastric cancer. J Dig Dis 2011; 12:51-9).

[0053] Also described herein is a method for reducing and / or removing the blood acetaldehyde concentration in a subject, which comprises the step of administering the pharmaceutical composition described herein.

[0054] Also described herein is a method for reducing and / or removing acetaldehyde from the digestive tract of a subject, which comprises the step of administering the pharmaceutical composition described herein.

[0055] Also described herein is a method for inhibiting mitochondrial aldehyde dehydrogenase 2 (ALDH2) in a subject, which comprises the step of administering the pharmaceutical composition described herein.

[0056] Also described herein is a method for reducing the risk in a subject of a disease or disorder caused by alcohol consumption, the method comprising the step of administering the pharmaceutical composition described herein. In some embodiments, the disease or disorder is selected from upper aerodigestive tract cancer, gastrointestinal cancer or breast cancer. In some embodiments, the upper aerodigestive tract cancer comprises esophageal cancer, pharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, cancer of the head or neck. In some embodiments, the gastrointestinal cancer comprises gastric cancer or colorectal cancer. In some embodiments, the disease or disorder has late-onset Alzheimer's disease, hypertension, myocardial infarction, Parkinson's disease, amyotrophic lateral sclerosis, and cerebral ischemia.

[0057] Some embodiments relate to a method of blocking the histamine-releasing effect of acetaldehyde in a subject, the method comprising the step of administering the pharmaceutical composition disclosed herein.

[0058] Some embodiments relate to a method of blocking alcohol-induced acetaldehyde production in a subject, the method comprising the step of administering the pharmaceutical composition disclosed herein.

[0059] In some embodiments, the subject is heterozygous or homozygous for the aldehyde dehydrogenase 2 (ALDH2) allele Glu487lys, ALDH2 * 2, ALDH2 * 487lys, Glu504lys, or rs671.

[0060] In some embodiments, Glu487lys, ALDH2 * 2, ALDH2 * further comprises the step of testing the subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele Glu487lys, ALDH2

[0061] In some embodiments, Glu487lys, ALDH2 * 2, ALDH2 *The step of testing a subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487lys, Glu504lys, or rs671 comprises obtaining a breath sample from the subject, measuring the alcohol level and acetaldehyde level in the breath sample, and determining the ratio of the acetaldehyde level to the alcohol level in the breath sample, wherein a ratio of the acetaldehyde level to the alcohol level of about 23.3 or more indicates the presence of Glu487lys, ALDH2 * 2, ALDH2 * determining that indicates the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487lys, Glu504lys, or rs671; and administering the pharmaceutical composition according to claim 1 or claim 12 to the subject if the subject has a ratio of acetaldehyde level to alcohol level of about 23.3 or more.

[0062] In some embodiments, the measurement of the alcohol and acetaldehyde levels in the breath sample is performed by semiconductor gas chromatography.

[0063] In some embodiments, the breath sample is obtained after alcohol consumption.

[0064] In some embodiments, Glu487lys, ALDH2 * 2, ALDH2 * The step of testing a subject for the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487lys, Glu504lys, or rs671 comprises obtaining a biological sample from the subject, separating genomic DNA from the biological sample, Glu487lys, ALDH2 * 2, ALDH2 * identifying the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called Glu487lys, Glu504lys, or rs671, Glu487lys, ALDH2 * 2, ALDH2 *administering the pharmaceutical composition according to claim 1 or claim 12 when there is an aldehyde dehydrogenase 2 (ALDH2) allele called 487lys, Glu504lys, or rs671. In some embodiments, the biological sample is a swab sample. In some embodiments, the biological sample is a blood sample.

[0065] For the purpose of clarity of understanding, the foregoing invention has been described in some detail by way of illustration and example. However, it will be apparent to those skilled in the art that certain changes and modifications can be made. Therefore, the description and examples should not be construed as limiting the scope of the invention as defined by the appended claims. Although the present disclosure has been described in considerable detail with reference to its particular preferred version, other versions are possible. Therefore, the spirit and scope of the present invention should not be limited to the description of the preferred version described herein.

[0066] Compositions, materials, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, but suitable preparations, methods, and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the specific embodiments described below are illustrative only and not intended to be limiting.

[0067] All features disclosed in this specification, including the abstract and drawings, and all steps of any method or process disclosed, can be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification, including the abstract and drawings, can be replaced by an alternative feature that serves the same purpose, an equivalent purpose, or a similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each disclosed feature is only an example of a general series of equivalent or similar features. In addition to what is described in this specification, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be included within the scope of the appended claims.

[0068] Unless otherwise specified, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, etc., used in this specification and the claims are to be understood as being modified in all instances by the term "about". As used herein, the term "about" means plus or minus 10% of a given value. For example, "about 50%" means a range of 45% to 55%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, each numerical value inherently contains certain errors resulting from the standard deviation found in its respective testing measurements.

[0069] The recitation of a range of values herein is merely intended to serve as a shorthand reference to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and is not intended to limit the scope of the invention otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0070] The grouping of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Members of each group can be referred to and claimed individually, or in any combination with other members of the group or other elements described herein. For convenience and / or for reasons of patentability, one or more members of a group are expected to be included in or deleted from the group. When such inclusion or deletion occurs, the specification is to be regarded as including the modified group and will satisfy the written description of all Markush groups used in the appended claims.

[0071] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the present invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to adopt such variations as appropriate, and the inventors intend for the present invention to be practiced in ways not specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. Further, unless otherwise stated herein or clearly inconsistent from the context, all possible combinations of the above-described elements are included in the present invention.

[0072] The specific embodiments disclosed herein can be further limited in the claims using the recitation "consisting of" or "consisting essentially of" rather than "having". When used in the claims, whether at the time of filing or added by amendment, the transitional term "consisting of" excludes elements, steps, or components not specified in the claims. The transitional term "consisting essentially of" limits the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are those essentially or explicitly described herein and are operative herein.

[0073] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the present invention. Accordingly, the alternative configurations of the present invention can be utilized in accordance with the teachings herein as examples and not limitations. Therefore, the present invention is not limited to what has been precisely shown and described.

Claims

1. a therapeutically effective amount of 4-methylpyrazole, or a pharma- ceutically acceptable salt, hydrate, polymorph, or solvate thereof; Withania somifera / Ashwagandha, and A pharma- ceutically acceptable carrier; 1. A pharmaceutical composition comprising: the pharmaceutical composition is formulated for oral administration to an adult subject in an amount of 10 mg of 4-methylpyrazole per kg of the subject's body weight to 20 mg of 4-methylpyrazole per kg of the subject's body weight; The pharmaceutical composition further comprises at least one pharma- ceutical inactive excipient in an amount effective to mask the taste of 4-methylpyrazole. Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the at least one pharma- ceutical inert carrier is present in a taste masking coating further comprising one or more of an anti-adherent agent, a plasticizer, a hydroxyalkyl cellulose, and a water-soluble polymer.

3. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is configured as an oral dosage form selected from a powder, a tablet, a lozenge, a chewing gum, a pill, a capsule, a microcapsule, a caplet, an orally disintegrating tablet, an osmotic controlled release oral delivery system, or any combination thereof.

4. 10. The pharmaceutical composition of claim 1, further comprising n-acetylcysteine, ampelopsin, L-cystine, S-acetylglutathione, molybdenum, iron, zinc, an iron chelator, L-ascorbic acid, L-threonine, or any combination thereof.

5. 5. The pharmaceutical composition of claim 4, wherein the iron chelator comprises curcumin, quercetin, inositol hexakisphosphate (IP6), or any combination thereof.

6. 10. The pharmaceutical composition of claim 1, wherein the pharma- ceutically acceptable carrier is water.

7. 2. The pharmaceutical composition of claim 1, wherein the therapeutically effective amount of 4-methylpyrazole is an amount that results in a plasma concentration of about 0.1 μmol / L.

8. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating and / or preventing alcohol-induced hypersensitivity reactions in a subject.

9. 9. The use of claim 8, wherein the alcohol-induced hypersensitivity reaction is selected from flushing, elevated heart rate, palpitations, hypotension, nausea, dizziness, headache, vomiting, diarrhea, upset stomach, ataxia, confusion, urticaria, generalized dermatitis, allergic rhinitis, bronchoconstriction, exacerbation of asthmatic bronchoconstriction, cardiovascular collapse, allergic conjunctivitis, atopic dermatitis, eosinophilic esophagitis, anaphylaxis, chronic bronchitis, and chronic obstructive pulmonary disease (COPD), and any combination thereof.

10. 10. The use of claim 9, wherein the alcohol flush is characterized by facial redness, increased skin temperature, increased heart rate, decreased diastolic blood pressure, or a combination thereof.

11. 9. The use of claim 8, wherein the pharmaceutical composition is administered before the subject consumes alcohol.

12. 9. The method of claim 8, wherein the pharmaceutical composition is administered about 60 minutes to about 15 minutes before the subject consumes alcohol.

13. 9. The use of claim 8, wherein the pharmaceutical composition is administered simultaneously with or after the subject consumes alcohol.

14. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for removing acetaldehyde produced from alcohol in a subject.

15. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for reducing and / or removing acetaldehyde produced from alcohol in the oral cavity, esophagus, stomach, large intestine, or a combination thereof, of a subject.

16. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for reducing and / or removing acetaldehyde blood levels in a subject.

17. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for reducing and / or removing acetaldehyde from the gastrointestinal tract of a subject.

18. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for inhibiting mitochondrial aldehyde dehydrogenase 2 (ALDH2) in a subject.

19. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for reducing the risk of a disease or disorder resulting from alcohol consumption in a subject.

20. 20. The use of claim 19, wherein the disease or disorder is selected from upper aerodigestive tract cancer, gastrointestinal cancer, or breast cancer.

21. 20. The use of claim 19, wherein the upper aerodigestive tract cancer comprises esophageal cancer, oropharyngeal cancer, hypopharyngeal cancer, laryngeal cancer, or head or neck cancer.

22. 20. The use of claim 19, wherein the gastrointestinal cancer comprises gastric cancer or colon cancer.

23. 20. The use of claim 19, wherein the disease or disorder comprises late-onset Alzheimer's disease, hypertension, myocardial infarction, Parkinson's disease, amyotrophic lateral sclerosis, or cerebral ischemia.

24. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for inhibiting the histamine releasing effect of acetaldehyde in a subject.

25. 13. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for blocking alcohol-induced acetaldehyde production in a subject.

26. In the use according to any one of claims 8 to 25, the subject is ＊ 2 or ALDH2 ＊ Use, who is heterozygous or homozygous for the aldehyde dehydrogenase 2 (ALDH2) allele called 487lys.

27. The method according to any one of claims 8 to 25, comprising: ＊ 2. ALDH2 ＊ The use of claim 1, further comprising the step of testing a biological sample obtained from said subject for the presence of an aldehyde dehydrogenase 2 (ALDH2) allele called rs671, rs704, or rs487lys.

28. 28. The use according to claim 27, wherein the Glu487lys, ALDH2 ＊ 2. ALDH2 ＊ testing a biological sample obtained from said subject for the presence of an aldehyde dehydrogenase 2 (ALDH2) allele designated rs671, rs671, (a) measuring alcohol and acetaldehyde levels in said biological sample; and (b) calculating a ratio of acetaldehyde to alcohol levels in the biological sample, The ratio of the acetaldehyde level to the alcohol level is about 23.3 or more, ＊ 2. ALDH2 ＊ indicating the presence of the aldehyde dehydrogenase 2 (ALDH2) allele called rs671, rs671, A process of calculating; To have, to use.

29. 30. The use of claim 28, wherein the step of measuring alcohol and acetaldehyde levels in the biological sample is performed by semiconductor gas chromatography.

30. 30. The use of claim 28, wherein the biological sample is obtained after consumption of alcohol.

31. 28. The use according to claim 27, wherein the Glu487lys, ALDH2 ＊ 2. ALDH2 ＊ testing a biological sample obtained from said subject for the presence of an aldehyde dehydrogenase 2 (ALDH2) allele designated rs671, rs671, (a) isolating genomic DNA from the biological sample; (b) Glu487lys, ALDH2 ＊ 2. ALDH2 ＊ determining the presence of an aldehyde dehydrogenase 2 (ALDH2) allele designated rs671, rs487lys, Glu504lys, or rs671; To have, to use.

32. 30. The use of claim 28, wherein the biological sample is a swab sample.

33. 30. The use of claim 28, wherein the biological sample is a blood sample.