Methods and compositions for the treatment of alcoholism

Abstract

Provided herein is a method for treating acute alcoholism using agonists of fibroblast growth factor receptor (FGFR) and beta-klotho complex, such as fibroblast growth factor 21 (FGF21).Also provided are compositions and kits that contain these agonists (such as FGF21) for administration to subjects in need thereof.

Description

[Technical field]

[0001] [Acknowledgements for government support] This invention was made with Government support under Grant No. AA028473 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 319,073, filed March 11, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] [1. Field] The present disclosure is generally directed to compositions and methods for treating alcoholism. [Background technology]

[0004] [2. Description of Related Art] Acute alcohol poisoning is caused by ingesting toxic amounts of ethanol in a short period of time and can be life-threatening. Acute alcohol poisoning can lead to disorientation, loss of consciousness, impaired respiratory and cardiac function, and even death. Emergency treatment of alcohol poisoning includes keeping the intoxicated individual upright and awake, if possible. Therefore, drugs with sobering or "amethystic" (anti-intoxication) activity may be beneficial for treating this dangerous condition.

[0005] Norepinephrine (NE) is an important neuromodulator in the CNS. Most central NE is synthesized in the locus coeruleus (LC), a small nucleus located in the pons of the brainstem. LC neurons project widely throughout the brain and control diverse biological processes, including wakefulness and attention (Berridge et al., 2012; Poe et al., 2020). Fibroblast growth factor 21 (FGF21) is a hormone produced by the liver in response to various metabolic stresses, including starvation, protein deprivation, simple sugars, and ethanol. It acts on a cell surface receptor that is organized as a complex of the indigenous FGF receptor tyrosine kinase FGFR1c and the single-pass transmembrane protein β-Klotho (KLB). FGF21 directly binds to both FGFR1c and KLB, with FGFR1c acting as an effector of downstream signaling.

[0006] The present disclosure is based, at least in part, on the surprising discovery that FGF21 can activate noradrenergic pathways and exert sobering or "amethystic" effects on both wakefulness and motor coordination without altering ethanol catabolism.Therefore, the disclosure herein demonstrates that noradrenergic pathways can be pharmacologically targeted to treat acute alcohol intoxication. Summary of the Invention

[0007] According to one aspect of the present disclosure, provided herein is a method of treating acute alcoholism in a subject in need thereof, comprising administering to the subject an effective amount of an agonist of fibroblast growth factor receptor (FGFR) / β-Klotho complex. In various aspects, treating acute alcoholism may include reducing disorientation, increasing consciousness level, improving cardiac function, improving respiration, or any combination thereof.

[0008] Also provided herein is a method for preventing acute alcoholism in a subject in need thereof, comprising administering to the subject an effective amount of an agonist of fibroblast growth factor receptor (FGFR) / β-Klotho complex. In various embodiments, preventing acute alcoholism may include preventing disorientation, preventing or avoiding loss of consciousness, preventing cardiac or respiratory depression, or any combination thereof.

[0009] In any of the methods herein, the agonist of the FGFR / β Klotho complex may include FGF21 or an FGF21 homolog. In various embodiments, an effective amount of the agonist is administered as a pharmaceutical composition. In various embodiments, an effective amount of an FGFR / β Klotho agonist (e.g., FGF21 or an FGF21 homolog) may be about 0.5 mg / kg to about 2 mg / kg, but may vary depending on the identity of the FGF21 homolog, which may be more or less potent than native FGF21.

[0010] In any of the methods of treating acute alcohol intoxication described herein, the subject may have consumed alcohol less than 1 hour (e.g., within 10 minutes, within 20 minutes, within 30 minutes, within about 40 minutes, within about 50 minutes, or within about 60 minutes) prior to administration of the FGFR / β Klotho complex agonist (e.g., FGF21 or an FGF21 analog). In any of the methods of treating acute alcohol intoxication described herein, the subject may have consumed alcohol for about 1-24 hours, about 1-18 hours, about 1-12 hours, about 1-10 hours, about 1-8 hours, about 1-6 hours, about 1-5 hours, or about 1-4 hours prior to administration of the FGFR / β Klotho complex agonist (e.g., FGF21 or an FGF21 analog).

[0011] In any of the methods of preventing acute alcohol intoxication described herein, the subject may consume alcohol less than 1 hour (e.g., within 10 minutes, within 20 minutes, within 30 minutes, within about 40 minutes, within about 50 minutes, or within about 60 minutes) after administration of the FGFR / β Klotho complex agonist (e.g., FGF21 or an FGF21 analog). In any of the methods of preventing acute alcohol intoxication described herein, the subject may consume alcohol between about 1-24 hours, between about 1-18 hours, between about 1-12 hours, between about 1-10 hours, between about 1-8 hours, between about 1-6 hours, between about 1-5 hours, or between about 1-4 hours) after administration of FGF21.

[0012] In any of the methods provided herein, the subject can be a human.

[0013] Also provided is a composition comprising an agonist of fibroblast growth factor receptor (FGFR) beta Klotho complex and a pharma- ceutically acceptable carrier or excipient. In some aspects, the agonist may comprise FGF21 or its homologue. In various embodiments, the pharma-ceutically acceptable carrier or excipient may comprise a diluent, a binder, a filler, a buffer, a pH adjuster, a disintegrant, a dispersant, a preservative, a lubricant, or any combination thereof. In further aspects, the composition may be formulated for oral administration, nasal administration, or intravenous administration.

[0014] Further provided is a kit comprising an agonist of fibroblast growth factor receptor (FGFR) / β-Klotho complex, a pharma- ceutically acceptable carrier or excipient, and instructions for administering the activator to a subject suffering from acute alcoholism. In various embodiments, the agonist comprises FGF21 or its homologue. In some embodiments, the kit further comprises a container and / or an administration device. In various embodiments, the carrier or excipient included in the kit may comprise a diluent, a binder, a filler, a buffer, a pH adjuster, a disintegrant, a dispersant, a preservative, a lubricant, or any combination thereof. In various embodiments, the subject may be a human.

[0015] Embodiments of the present disclosure are described below by way of example, where like reference numbers indicate similar elements. [Brief description of the drawings]

[0016] [Fig. 1A-1E] Fgf21- / - mice showed delayed recovery of righting reflex after excessive ethanol administration. (A) Plasma FGF21 concentrations were measured 1 h before and at the indicated times after ethanol administration (5 g / kg, oral gavage) in wild-type (WT) mice (n=6 / group). (B-E) WT and Fgf21- / - mice were administered ethanol (5 g / kg, oral gavage) and the time to loss of righting reflex (LORR) (B), LORR duration (C), plasma ethanol concentration (D), and brain ethanol concentration (E) were measured (n=5-6 / group). All data represent mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001 compared to -1 h (A) or WT (C). [Fig. 2A-2F] Hepatocyte-specific Fgf21- / - and neuron-specific Klb- / - mice showed delayed recovery of righting reflex after excessive ethanol administration. Control mice (Fgf21fl / fl) and hepatocyte-specific Fgf21- / - mice (Fgf21Alb) (A-C), or control mice (Klbfl / fl) and neuron-specific Klb- / - mice (KlbCamk2a) (D-F) were administered ethanol (5 g / kg, oral gavage), and the time to loss of righting reflex (LORR) (A, D), LORR duration (B, E), and plasma ethanol concentration (C, F) were measured (n=5-7 / group). All data are mean ± SEM. [Figure 3A]Pharmacological FGF21 attenuates alcohol-induced sedation. Wild-type (WT) male mice were administered ethanol (5 g / kg, oral gavage) and 1 h later received ip injections of vehicle or FGF21 at the indicated doses. Loss of righting reflex (LORR) duration was measured after administration of FGF21 or vehicle (n=5–6 / group). Data represent mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001 compared to vehicle. [Figure 3B] Pharmacological FGF21 attenuates alcohol-induced sedation. Wild-type (WT) female mice were administered ethanol (5 g / kg, oral gavage) and 1 h later received ip injections of vehicle or FGF21 at the indicated doses. Loss of righting reflex (LORR) duration was measured after administration of FGF21 or vehicle (n=5–6 / group). Data represent mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001 compared to vehicle. [Figure 3C] Pharmacological FGF21 attenuates alcohol-induced sedation. WT and Fgf21- / - mice (n=13-15 / group) were administered ethanol (5 g / kg, oral gavage) and 1 h later received an ip injection of FGF21 (1 mg / kg) or vehicle. LORR duration was measured after administration of FGF21 or vehicle. Data represent the mean ± SEM. Different lowercase letters indicate statistical significance (P<0.05). [Figure 3D] Pharmacological FGF21 attenuates alcohol-induced sedation. Control (Klbfl / fl) and neuron-specific Klb- / - mice (KlbCamk2a) (n=12–16 / group) were administered ethanol (5 g / kg, oral gavage) and 1 h later received an ip injection of FGF21 (1 mg / kg) or vehicle. LORR duration was measured after administration of FGF21 or vehicle. Data represent the mean ± SEM. Different lowercase letters indicate statistical significance (P<0.05). [Figure 3E]Pharmacological FGF21 attenuates alcohol-induced sedation. WT mice were administered ethanol (2 g / kg, ip) and 30 min later received an ip injection of FGF21 (1 mg / kg, indicated by arrow) or vehicle. The time that mice could remain on the rotarod was measured up to 60 s (n=8 / group). Data represent the mean ± SEM. [Figure 3F] Pharmacological FGF21 attenuates alcohol-induced sedation. WT and Fgf21- / - mice were administered ethanol (2 g / kg, ip) and time on the rotarod was measured as in (E). Data represent the mean ± SEM. [Figure 4A-4D] Pharmacological FGF21 does not attenuate other sedatives. Wild-type mice were administered ethanol (4.3 g / kg) (A) (n = 9–12 / group), ketamine (200 mg / kg) (B) (n = 6 / group), diazepam (30 mg / kg) (C) (n = 5–6 / group), or pentobarbital (55 mg / kg) (D) (n = 7–8 / group) by ip injection. Thirty minutes later, mice were injected ip with either FGF21 (1 mg / kg) or vehicle. Loss of righting reflex (LORR) duration was measured after administration of FGF21 or vehicle. All data represent the mean ± SEM. *, P < 0.05 compared with vehicle-treated mice. [Figure 5A] FGF21 is a physiological regulator of noradrenergic neurons. Representative confocal images of c-Fos and norepinephrine transporter (NET) immunostaining in locus coeruleus sections prepared from wild-type (WT) and Fgf21- / - mice 2.5 hours after oral gavage with either water or ethanol (5 mg / kg). Scale bar represents 50 μM. The drawings do not limit the disclosure to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, with emphasis instead being placed on clearly illustrating the principles of certain embodiments of the disclosure. [Figure 5B]FGF21 is a physiological regulator of noradrenergic neurons. Quantification of c-Fos / NET co-expression (upper panel) and total NET-positive cell numbers (lower panel) (n=3 sections / mouse, 4 mice / group). Data represent mean±SEM. Different lowercase letters indicate statistical significance (P<0.05). The figures do not limit the disclosure to the specific embodiments disclosed and described herein. [Figure 6A] Pharmacological FGF21 activates norepinephrine neurons in the locus coeruleus. Immunostaining was performed on locus coeruleus (LC) sections prepared from mice expressing TdTomato fused to the C-terminus of KLB. Antibodies against TdTomato and the norepinephrine transporter (NET) were used. The top panel is a confocal image. The bottom panel is a composite Z-stack image. Scale bar represents 50 μM. [Figure 6B] Pharmacological FGF21 activates norepinephrine neurons in the locus coeruleus. Immunostaining for c-Fos and NETs in LC sections prepared from wild-type mice treated with vehicle or FGF21 (1 mg / kg, ip) for 2 h. Representative confocal images are shown in (B). Scale bar represents 50 μM. [Figure 6C] Pharmacological FGF21 activates norepinephrine neurons in the locus coeruleus. Immunostaining for c-Fos and NETs in LC sections prepared from wild-type mice treated with vehicle or FGF21 (1 mg / kg, ip) for 2 h. Quantification of c-Fos / NET co-expression (upper panel) and total NET-positive cell numbers (lower panel) is shown in (C) (n=4 sections / mouse, 3 mice / group). Data represent mean ± SEM. ****, P<0.0001. [Figure 6D]Pharmacological FGF21 activates norepinephrine neurons in the locus coeruleus. Immunostaining for c-Fos and NETs in LC sections from groups of control mice (Klbfl / fl) and neuron-specific Klb- / - mice (KlbCamk2a) treated for 2 h with vehicle or FGF21 as in (B) (n=3 mice / group). Quantification of c-Fos / NET co-expression (left panel) and total NET-positive cell numbers (right panel) is shown in (E) (n=3 sections / mouse, 3 mice / group). Data represent mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 7A] FGF21 exerts its amethystic activity through the noradrenergic nervous system. Wild-type (WT) mice were injected with DSP-4 (50 mg / kg, ip) or vehicle. Two days later, mice were administered ethanol (5 g / kg, oral gavage) and 1 h later received an ip injection of FGF21 (1 mg / kg) or vehicle (n=16 / group). Loss of righting reflex (LORR) duration was measured after administration of FGF21 or vehicle. Data represent mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 7B] FGF21 exerts its amethystic activity through the noradrenergic nervous system. WT mice were administered ethanol (5 g / kg, oral gavage) and 1 h later received ip injections of vehicle, FGF21 (1 mg / kg), prazosin (0.8 mg / kg) or FGF21 + prazosin (n = 8–10 / group). LORR duration was measured after administration of vehicle or FGF21. Data represent the mean ± SEM. Different lowercase letters indicate statistical significance (p < 0.05). [Figure 7C]FGF21 exerts its amethystic activity through the noradrenergic nervous system. Experiments were performed as in (B), except that mice were injected ip with vehicle, FGF21 (1 mg / kg), propranolol (10 mg / kg), or FGF21 + propranolol (n=15-17 / group). LORR duration was measured after administration of vehicle or FGF21. Data represent the mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 7D] FGF21 exerts its amethystic activity through the noradrenergic nervous system. Control (Klbfl / fl) and KlbDbh mice were administered ethanol (5 g / kg, gavage) and 1 h later received ip injections of vehicle or FGF21 (1 mg / kg). LORR duration was measured after administration of FGF21 or vehicle (n=4-6 / group). Data represent the mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 7E] FGF21 exerts its amethystic activity through the noradrenergic nervous system. Immunostaining for c-Fos and norepinephrine transporter (NET) was performed in locus coeruleus (LC) sections from groups of control (Klbfl / fl) and KlbDbh mice treated with vehicle or FGF21 (1 mg / kg) for 2 h (n=3-4 mice / group). Quantification of c-Fos / NET co-expression (left panel) and total NET-positive cell numbers (right panel) are shown (n=3 sections / mouse, 3-4 mice / group). Data represent mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 7F]FGF21 exerts its amethystic activity through the noradrenergic nervous system. Klbfl / fl mice bilaterally injected in the LC region with adeno-associated virus (AAV) expressing either control (GFP) or Cre were administered ethanol (5 g / kg, gavage) and 1 h later ip injection of vehicle or FGF21 at the indicated doses. LORR duration was measured after administration of FGF21 or vehicle (n=8-11 / group). Data represent mean ± SEM. Different lowercase letters indicate statistical significance (p<0.05). [Figure 8] In situ hybridization of Klb mRNA (top panel) and Fgfr1c mRNA (middle panel) and their two synthesis (bottom panel). Two different magnifications are shown, with the boxed area in the left panel expanded in the right panel. The chromogenic signals of Klb and Fgfr1c are highlighted in green and red, respectively. The positions of the locus coeruleus (LC), dorsolateral tegmental nucleus (LDTg) and ependymal cells (ep) in the vicinity of the fourth ventricle are indicated. The arrow indicates the strong Klb signal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure is directed to compositions and methods for treating and / or preventing acute alcoholism in a subject in need thereof. The methods described herein are based on the surprising discovery that noradrenergic pathways can be modulated to produce an amythestic effect. As shown below, FGF21 is a hormone induced in the liver by starvation, protein deprivation, simple sugars and ethanol, and can act on the FGF receptor / β-kotho complex to stimulate the noradrenergic system and eliminate or alleviate alcoholism. Thus, administration of an agonist of the FGF receptor / β-kotho complex (such as FGF21 or its homologues) may be particularly useful for treating or preventing severe alcoholism.

[0018] The following detailed description refers to the accompanying drawings, which illustrate various embodiments of the present disclosure. The drawings and description describe aspects and embodiments of the present disclosure in sufficient detail to enable those skilled in the art to practice the disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. Thus, the following description should not be taken in a limiting sense.

[0019] [1.Terminology] The phrases and terms used herein are for the purpose of description and should not be considered limiting. For example, the use of singular terms, such as "a," is not intended to limit the number of items. Also, the use of relationship terms, such as, but not limited to, "top," "bottom," "left," "right," "upper," "lower," "down," "up," and "side," are used in the description for clarity with particular reference to the drawings, and are not intended to limit the scope of the disclosure or the appended claims.

[0020] Furthermore, the present disclosure is intended to be considered as an example of the principles of the present disclosure, since embodiments in many different forms are possible, and the present disclosure is not intended to be limited to the specific embodiments shown and described. Any one of the features of the present disclosure may be used individually or in combination with any other feature. When "an embodiment", "embodiments" and / or similar terms are used herein, it means that the feature and / or features referred to are included in at least one aspect of the present disclosure. When "an embodiment", "embodiments" and / or similar terms are used herein individually, they do not necessarily refer to the same embodiment, nor are they mutually exclusive, unless otherwise specified and / or readily understood by one of ordinary skill in the art from the present description. For example, a feature, structure, process, step, or act, etc. described in one embodiment may be included in other embodiments, but are not necessarily included. Thus, the present disclosure may include various combinations and / or integrations of the embodiments described herein. In addition, not all aspects of the present disclosure described herein are necessary for the practice of the present disclosure. Similarly, other systems, methods, features, and advantages of the present disclosure will be or become apparent to one of ordinary skill in the art upon examination of the figures and descriptions thereof, and it is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be covered by the claims.

[0021] Any term expressing a degree, for example but not limited to, "substantially" as used herein and in the appended claims, should be understood to include an exact configuration or a similar but not exact configuration. For example, a "substantially flat surface" means an exactly flat surface or a similar but not exactly flat surface. Similarly, the term "about" or "approximately" as used herein and in the appended claims should be understood to include the stated value or a value three times or one third of the stated value. For example, about 3 mm includes all values ​​between 1 mm and 9 mm, and approximately 50 degrees includes all values ​​between 16.6 degrees and 150 degrees. For example, they can be ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0022] The terms "comprising," "including," and "having" are used interchangeably in this disclosure. The terms "comprising," "including," and "having" are meant to include, but not necessarily be limited to, what is so set forth.

[0023] Finally, the terms "or" and "and / or," as used herein, should be construed as being inclusive or meaning any one or any combination. Thus, "A, B or C" or "A, B and / or C" means any of "A," "B," or "C"; "A and B"; "A and C"; "B and C"; "A, B and C." Exceptions to this definition occur only when combinations of elements, features, steps or acts are in some way inherently mutually exclusive.

[0024] In various embodiments, the subject is a mammal, for example, in various embodiments, the subject is a human.

[0025] Although several embodiments have been described, it will be understood by those skilled in the art that various modifications, alternative configurations, and equivalents may be used without departing from the spirit of the present disclosure. In addition, several well-known processes and elements have not been described to avoid unnecessarily obscuring the present disclosure. Thus, this description should not be interpreted as limiting the scope of the present disclosure.

[0026] Those skilled in the art will understand that the presently disclosed embodiments are taught by way of example and not by way of limitation. Therefore, the matter contained in this specification or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all of the general and specific features described herein, as well as all of the descriptions of the methods and ranges of aggregates that may be linguistically said to fall therebetween.

[0027] [2. Method] Various aspects of the present disclosure provide a method for preventing or treating acute alcoholism. The method generally comprises administering to a subject in need thereof an effective amount of an agonist of FGFR / β-kotho complex. In various embodiments, the agonist may comprise FGF21 or an FGF21 homologue.

[0028] FGF21 functions through a receptor complex consisting of FGF receptor (FGFR) and co-receptor β-Klotho. Briefly, β-Klotho is a transmembrane protein that interacts with FGF receptor and is required to promote FGF21 binding and subsequent activation of the complex. As further described herein, the interaction between FGF21 and FGF receptor / β-Klotho complex results in a surprising amythestic effect that can be utilized to treat or prevent alcohol poisoning / intoxication.

[0029] As used herein, an FGF21 congener is a substance, compound, or moiety that acts as an agonist of the FGFR / β-Klotho complex, and thus activates the noradrenergic system in a similar or equivalent manner to FGF21. In some embodiments, an FGF21 congener may be a modified form of FGF21. For example, an FGF21 congener may be a native FGF21 that has been modified to extend or improve its pharmacokinetics (e.g., half-life). In some examples, an FGF21 congener may be an antibody or antibody-like moiety that binds with high affinity to and activates the FGFR / β-Klotho complex (see, e.g., Min et al., "Agonistic β-Klotho antibody mimics fibroblast growth factor 21 (FGF21) functions" J Biol Chem. 2018 Sep 21; 293(38): 14678-14688, which is incorporated herein by reference in its entirety). In some examples, the FGF21 homologue may be a small compound that can bind to or regulate the FGFR / β-Klotho complex. In other examples, the FGF21 homologue may be a protein or peptide that has affinity for the FGFR / β-Klotho complex.

[0030] In various embodiments, FGF21 homologs may be described in Taludar et al. ("FGF19 and FGF21: In NASH we trust" Molecular Mechanism Vol. 46, 2021, 101152), which is incorporated by reference in its entirety. For example, suitable FGF21 homologs to be used in the methods of the present invention may be selected from the group consisting of LY2405319 (Eli Lilly), 05231023 (Pfizer), PF-05231023 (Pfizer), AKR-001 (Akero (AMG-876)), BMS-986036 (BMS), BKB8488A (Genentech), and NGM313 (MSD MK-3655).

[0031] Thus, in one aspect of the disclosure, a method of treating acute alcoholism in a subject in need thereof is provided. In various aspects, the method comprises activating the noradrenergic system in the subject. For example, the method may comprise activating the FGF21-NE pathway in the subject. In some embodiments, the method comprises administering an effective amount of a compound that acts on and activates the FGF21-NE pathway. In some embodiments, the method comprises administering an effective amount of an agonist of the FGFR / β-kotho complex. In some embodiments, the method comprises administering FGF21 or an FGF21 homolog to the subject. In some embodiments, the method comprises administering an effective amount of FGF21 or an FGF21 homolog to the subject. In some embodiments, the effective amount of FGF21 or an FGF21 homolog is administered after the subject consumes alcohol. For example, in some embodiments, the subject consumes alcohol between 1 and 24 hours, between 1 and 18 hours, between 1 and 12 hours, between 1 and 10 hours, between 1 and 8 hours, between 1 and 6 hours, between 1 and 5 hours, between 1 and 4 hours, between 1 and 3 hours, or between 1 and 2 hours prior to administration of FGF21.

[0032] Also provided are methods of preventing acute alcohol intoxication and / or teaching the effects of acute alcohol intoxication in a subject in need thereof, comprising administering to the subject an effective amount of an agonist of the FGF21 receptor / β-Klotho complex (e.g., FGF21 or an FGF21 analog) prior to ingestion of alcohol by the subject. In some embodiments, the effective amount of FGF21 is administered prior to ingestion of alcohol. For example, in some embodiments, the subject consumes alcohol between 1-4 hours, 1-3 hours, or 1-2 hours after administration of the agonist of the FGF21 receptor / β-Klotho complex (e.g., FGF21).

[0033] In various embodiments, methods of treating and / or preventing acute alcohol intoxication may include reducing disorientation, increasing the level of consciousness, improving cardiac function, improving breathing, or any combination thereof.

[0034] In any of these embodiments, the effective amount of the agonist (e.g., FGF21 or a homolog thereof) may be about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 9 mg / kg, about 0.5 mg / kg to about 8 mg / kg, about 0.5 mg / kg to about 7 mg / kg, about 0.5 mg / kg to about 6 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 4 mg / kg, about 0.5 mg / kg to about 3 mg / kg, or about 0.5 mg / kg to about 2 mg / kg. For example, in some embodiments, the effective amount of the agonist (e.g., FGF21 or a homolog thereof) is about 0.5 mg / kg to about 2 mg / kg.

[0035] In any of these embodiments, the effective amount of the agonist may be administered to the subject in various doses. The dose may be delivered once or over several days. In some aspects, the effective amount of the agonist comprises a dose of about 1 to 100 mg of FGF21 or FGF21 congener. In some aspects, the effective dose may be about 1 to 10 mg, about 3 to 10 mg, or about 5 to 10 mg. For example, the effective dose may be about 3 mg, about 5 mg, or about 10 mg. In various aspects, the effective dose may be about 10 to 50 mg, about 10 to 40 mg, about 10 to 30 mg, about 10 to about 20 mg, about 20 to about 50 mg, about 20 to about 40 mg, about 20 to about 30 mg, about 30 to about 50 mg, about 30 to about 40 mg, or about 40 to about 50 mg. For example, the effective dose may be about 20 mg, about 25 mg, about 28 mg, or about 50 mg. In further embodiments, the effective dose may be about 50 mg to about 200 mg, about 60 mg to about 200 mg, about 70 mg to about 200 mg, about 80 mg to about 200 mg, about 90 mg to about 200 mg, about 100 mg to about 200 mg, about 110 mg to about 200 mg, about 120 mg to about 200 mg, about 130 mg to about 200 mg, about 140 mg to about 200 mg, about 150 mg to about 200 mg, about 160 mg to about 200 mg, about 170 mg to about 200 mg, about 180 mg to about 200 mg, or about 190 mg to about 200 mg. In some embodiments, a suitable dosage is about 50 mg to about 150 mg, about 60 mg to about 150 mg, about 70 mg to about 150 mg, about 80 mg to about 150 mg, about 90 mg to about 150 mg, about 100 mg to about 150 mg, about 110 mg to about 150 mg, about 120 mg to about 150 mg, about 130 mg to about 150 mg, or about 140 mg to about 150 mg. For example, in some embodiments, a suitable dosage may be about 50 mg, about 70 mg, about 100 mg, about 140 mg, or about 150 mg.

[0036] In various embodiments, the effective amount of the agonist may depend on what the agonist is (e.g., whether FGF21 or a congener is used). As noted above, suitable FGF21 congeners may be selected from LY2405319 (Eli Lilly), 05231023 (Pfizer), PF-05231023 (Pfizer), AKR-001 (Akero (AMG-876)), BMS-986036 (BMS), BKB8488A (Genentech), and NGM313 (MSD MK-3655).

[0037] In any of the methods provided herein, the subject in need thereof can be a human.

[0038] 3. Pharmaceutical Compositions In any of the methods herein, a suitable FGF21 / β-Klotho complex agonist (eg, FGF21 or a homolog thereof) may be administered as a pharmaceutical composition.

[0039] FGF21 is a protein expressed by the FGF21 gene (NCBI gene ID: 26291). It can therefore be prepared by standard techniques in the art for any of the pharmaceutical compositions provided herein or for any of its uses. For example, recombinant FGF21 can be prepared from transfected nucleic acid molecules using molecular biology methods known in the art. In such methods, a polynucleotide sequence encoding FGF21 is inserted into a vector that can express FGF21 when introduced into a suitable host cell. Suitable host cells are known to those skilled in the art and can include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. FGF21 can be expressed and obtained from cells using common purification methods. For example, a secretion signal can be added to FGF21, which allows it to be secreted and isolated from cell culture supernatant. Alternatively, FGF21 lacking a secretion signal can be purified from inclusion bodies and / or cell extracts. FGF21 can be isolated from culture supernatant, inclusion bodies or cell extracts using any method known to those skilled in the art, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for a specific antigen after protein A, and size exclusion column chromatography), centrifugation, differential solubility, e.g., ammonium sulfate precipitation, or any other standard technique for purifying proteins. See, for example, Scopes, "Protein Purification", Springer Verlag, NY (1982). Isolation of a polypeptide is greatly facilitated when the polypeptide contains a purification moiety.

[0040] In various embodiments, the pharmaceutical compositions provided herein comprise FGF21 analogs (e.g., compounds, peptides, proteins, or moieties that activate FGF / β-Klotho complex). In some embodiments, the analogs may be modified FGF21 to improve its pharmacokinetics or pharmaceutical usefulness. For example, FGF21 may be modified to improve its half-life. Modifications known in the art that are suitable for this purpose include PEGylation, glycoPEGylation, and modification of certain amino acid residues. Chimeric molecules that include FGF21 or FGF21 analogs fused to Fc domains are also envisioned. Bispecific agonist antibodies that bind to the complex of FGFR1 and KLB can also be used.

[0041] Accordingly, various aspects of the present disclosure are directed to pharmaceutical compositions, eg, pharmaceutical compositions that include FGF21 and a pharma- ceutically suitable carrier or excipient.

[0042] The composition can include at least one additive.Suitable additives include pharmaceutically acceptable additives, such as diluents, binders, fillers, buffers, pH adjusters, disintegrants, dispersants, preservatives, lubricants, flavorings, flavorings, colorings, or combinations thereof.The amount and type of additives used to form pharmaceutical compositions can be selected according to known pharmaceutical principles.

[0043] In one embodiment, the additive may be a diluent. The diluent may be compressible (i.e., plastically deformable) and may be susceptible to abrasion. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., mixed esters of acetate and butyrate), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, corn starch, phosphorylated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable low-wear diluents include calcium hydrogen phosphate (anhydrous or dihydrate), tricalcium phosphate, calcium carbonate, and magnesium carbonate.

[0044] In another embodiment, the additive may be a binder. Suitable binders include, but are not limited to, starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, or sugar.

[0045] In another embodiment, the additive may be a filler.Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone.As non-limiting examples, the filler may be calcium sulfate, both dibasic and tribasic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, calcium hydrogen phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starch, lactose, sucrose, mannitol, or sorbitol.

[0046] In yet another embodiment, the additive may be a buffer. Representative examples of suitable buffers include, but are not limited to, phosphate buffers, carbonate buffers, citrate buffers, Tris buffers, and buffered saline (e.g., Tris buffered saline or phosphate buffered saline).

[0047] In various embodiments, the additive may be a pH adjuster. By way of non-limiting example, the pH adjuster may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0048] In further embodiments, the additive may be a disintegrant.The disintegrant may be non-effervescent or effervescent.Suitable examples of non-effervescent disintegrants include, but are not limited to, starches, such as corn starch, potato starch, pregelatinized starch and modified starch; sweeteners; clays, such as bentonite; microcrystalline cellulose; alginates; sodium starch glycolate; gums, such as agar, guar, locust bean, karaya, pectin and tragacanth.Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0049] In yet another embodiment, the additive may be a dispersant or dispersion enhancer. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0050] In another alternative embodiment, the additive may be a preservative.Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelating agents, such as EDTA or EGTA; and antimicrobial agents, such as parabens, chlorobutanol, or phenol.

[0051] The compositions disclosed herein may be formulated into various dosage forms and administered by a number of different means that deliver a therapeutically effective amount of the active ingredient. The additives included in the compositions containing FGF21 may be based on the form in which such compositions are administered. Such compositions may be administered orally, parenterally, or topically in dosage unit formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles, as desired. Topical administration may also include the use of transdermal administration, such as transdermal patches or iontophoresis devices. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques. In some embodiments, the pharmaceutical compositions containing FGF21 are formulated for nasal, intravenous, or oral administration. Drug formulation is discussed, for example, in Gennaro, A.R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, NY (1980).

[0052] [3. Kit] Various aspects of the present disclosure are directed to a kit for treating acute alcoholism.In some embodiments, the kit comprises FGF21, a pharma- ceutically acceptable carrier or additive, and instructions for administering FGF21 to the subject suffering from acute alcoholism.

[0053] In various embodiments, the kit comprises a pharmaceutical composition (e.g., any of the pharmaceutical compositions provided herein) comprising FGF21 and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the kit further comprises a container. In some embodiments, the kit further comprises an administration device.

[0054] In various embodiments, instructions are included in a kit for administering FGF21, or a pharmaceutical composition comprising FGF21, to an individual suffering from acute alcohol intoxication. EXAMPLES

[0055] The following examples are included to illustrate preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples are techniques that the inventors have discovered to work well in implementing the present disclosure, and therefore may be considered to constitute preferred modes of its implementation. However, those skilled in the art should understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0056] Example 1: General Method <Mouse test> The use of mice and all related procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. Experiments were performed in male mice unless otherwise specified. Fgf21- / - (Potthoff et al., 2009), KlbCamk2a (Bookout et al., 2013), Fgf21Alb (Song et al., 2018) and Klb-TdTomato reporter mice (KLB-T) (Coate et al., 2017) have been described. KlbDbh mice were generated by crossing Dbh-Cre mice (Jackson Laboratory, stock number: 033951) with Klbfl / fl mice (Bookout et al., 2013). Fgf21- / -, Fgf21Alb, and KLB-T mice were of C57BL / 6J strain, and KlbCamk2a and KlbDbh mice were of mixed C57BL / 6J;129 / Sv strain. C57BL / 6J mice were generated at the University of Texas Southwestern Medical Center Animal Breeding Core or purchased from Jackson Laboratory. Mice were housed in a temperature-controlled environment with a 12-h light-dark cycle and fed standard rodent chow ad libitum. All experiments were performed on age- and sex-matched mice. Recombinant human FGF21 protein was obtained from Novo Nordisk and administered at a dose of 1 mg / kg by ip injection unless otherwise stated in the figure legends.

[0057] <Virus injection> Klbfl / fl mice were anesthetized using isoflurane anesthesia (3%-4% for induction, 1.5%-2% for maintenance), placed in a stereotaxic apparatus (David Kopf Instruments), and body temperature was maintained using a thermoregulator. The skull was exposed, the bregma identified, and two small holes were drilled for bilateral injections into the LC (coordinates from lambda: mediolateral ±0.9 mm, anteroposterior -0.9 mm, and dorsoventral -3.82 mm (Paxinos and Franklin, 2019)). Dorsal-ventral coordinates are referenced to the pia mater. A volume of 300 nl (1 × 10 12 AAV8-GFP or AAV8-GFP-Cre viruses (UNC Vector Core) were injected bilaterally into the LC at 100 ng / μl (genomic particles / μl). Virus was infused at 50 nl / min using a microinjection syringe pump and Micro2T controller system with a 34G Nanofil needle (World Precision Instrument, UMP3T-1). After each injection, the needle was kept in place for 10 min to prevent backflow and then slowly removed over 5 min. The skin was closed by sutures. Mice were allowed to recover for 2 weeks before being used for experiments.

[0058] <Loss of righting reflex (LORR) test> Time to LORR was defined as the time between ethanol administration and LORR. Ataxic mice were placed in a supine position in a V-shaped plastic trough, and the time it took for them to rise three times within 30 seconds was measured, which was defined as the duration of LORR. To test the effect of FGF21 on ethanol-induced sedation, mice were gavaged with ethanol (5 g / kg) and 1 h later received an ip injection of either FGF21 or vehicle. For anesthetic agent testing, mice were given an ip injection of ketamine (200 mg / kg), diazepam (30 mg / kg), pentobarbital (55 mg / kg) or ethanol (4.3 g / kg), followed 30 min later by an ip injection of FGF21 or vehicle. Diazepam and ketamine were diluted in 0.9% saline, and pentobarbital was diluted in 0.9% saline containing 10% ethanol.

[0059] <DSP-4, Prazosin, and Propranolol Tests> For the DSP-4 test, C57BL / 6J mice were injected i.p. with either DSP-4 (50 mg / kg) or vehicle. Two days later, the mice were force-fed ethanol (5 g / kg), and 1 hour later, either FGF21 or vehicle was injected i.p. For the prazosin and propranolol tests, the mice were force-fed ethanol, and 1 hour later, FGF21 or vehicle was injected i.p. in the presence or absence of prazosin (0.8 mg / kg) or propranolol (10 mg / kg). DSP-4, prazosin, and propranolol were all diluted in 0.9% saline immediately before use.

[0060] <Rotarod Test> Mice were placed on a Rotamex-5 rotarod (Columbus Instruments) at 5 rpm for training, and training was considered complete when the mice were able to stay on the rotarod for 60 seconds. To evaluate the effect of FGF21 on ethanol-impaired motor coordination, the mice were injected i.p. with ethanol (2 g / kg), and 1 hour later, either FGF21 or vehicle was injected i.p. The time on the rotarod was measured at regular intervals with a maximum of 60 seconds.

[0061] <c-Fos Induction and Immunohistochemistry Tests> For the c-Fos induction test, mice were habituated to either i.p. injection of 0.9% saline or forced oral administration of water for 4 days. On day 5, mice were injected i.p. with vehicle or FGF21 (2-hour treatment), or were given forced oral administration of water or ethanol (5 g / kg, 2.5-hour treatment). Mice were anesthetized with isoflurane and perfused transcardially first with PBS and then with 10% neutral buffered formalin (NBF). Brains were fixed in 10% NBF at 4 °C for 24 hours, and sections were prepared at a thickness of 50 μm using a Leica VT1000S vibratome. Brains of untreated KLB-T mice were processed similarly. Sections were incubated in blocking buffer (1% bovine serum albumin, 5% normal goat serum, 0.3% Triton X-100 in PBS) for 1 hour with shaking at room temperature, and then incubated in primary antibodies containing each antibody against NET (Mab Technologies, 1:1000 dilution), cFos (Cell Signaling Technology, 1:1000 dilution), red fluorescent protein (Rockland, 1:500 dilution), and GFP (Aves Labs, 1:2000 dilution) at 4 °C for 48 hours. Free-floating sections were washed 3 times with PBS for 10 minutes each, and then incubated at room temperature for 1 hour in secondary antibodies conjugated with AlexaFluor containing goat anti-mouse IgG, goat anti-chicken IgG, goat anti-rabbit IgG (Invitrogen, 1:500 dilution), and DAPI (Fisher Scientific, 1:5000 dilution). Sections were washed 3 times with PBS for 10 minutes each and mounted using Aqua-Poly / Mount (Polysciences). Images were acquired using a Zeiss LSM780 confocal microscope and processed using ImageJ software. Counting of c-Fos was performed blindly.

[0062] <in situ hybridization analysis> C57BL / 6J mice were anesthetized with isoflurane and first perfused transcardially with PBS and then with 10% neutral buffered formalin. The brains were fixed in 10% NBF at 4 °C for 24 h. Brain sections were prepared at a cross-sectional thickness of 25 μm using a Leica cryostat microtome. In situ hybridization was performed using the RNAscope multiplex fluorescence kit. The Klb probe and the Fgfr1c probe were purchased from Advanced Cell Diagnostics. The hybridized sections were counterstained with hematoxylin, dehydrated, cleared, and mounted using EcoMount (BioCare Medical). Images were acquired using a Zeiss LSM780 confocal microscope.

[0063] <Measurement of FGF21 and Ethanol> To measure the concentrations of FGF21 and ethanol in the plasma of mice, the blood was centrifuged at 3,000 rpm for 15 min immediately after collection, and the plasma was stored at -80 °C until analysis. The FGF21 concentration in the plasma was measured using an FGF21 mouse / rat ELISA kit (BioVendor) according to the manufacturer's instructions. The ethanol concentration in the plasma was measured using an EnzyChrom ethanol assay kit (BioAssay Systems) according to the manufacturer's instructions. To measure the ethanol concentration in the brain, the brain was removed, immediately frozen in liquid nitrogen, and stored at -80 °C. The frozen whole brain was homogenized in 0.1 N HCl and centrifuged at 13,000 rpm at 4 °C for 30 min. The ethanol concentration in the supernatant was measured using an EnzyChrom ethanol assay kit.

[0064] <Statistical Analysis> All data are expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism software, version 9.0. For two-group analyses, unpaired two-tailed Student's t-tests were used. Multiple groups were tested by one-way or two-way ANOVA with Tukey's multiple comparison test. For rotarod analyses, linear mixed-effects models were fitted using the R package lme4, and P values ​​for between-group effects were derived using likelihood ratio tests. In all analyses, P values ​​<0.05 were considered significant.

[0065] [Example 2: Deficiency of GF21 exacerbates ethanol-induced intoxication] The effects of a single excessive dose of ethanol (5 g / kg, by oral gavage) were compared between wild-type (WT) and global Fgf21- / - mice. As expected, plasma FGF21 was induced by ethanol in WT mice, peaking at 2 h (Figure 1A). In rodents, impaired righting reflex is a standard marker of intoxication. WT and Fgf21- / - mice lost righting reflex at similar times after ethanol gavage (Figure 1B). Notably, however, Fgf21- / - mice took significantly longer to regain righting reflex (Figure 1C). WT and Fgf21- / - mice cleared ethanol from plasma at similar rates (Figure 1D), and brain ethanol concentrations were similar between genotypes (Figure 1E). Thus, FGF21 protects against ethanol-induced sedation without affecting ethanol catabolism.

[0066] Similar results were obtained in hepatocyte-specific Fgf21 knockout (Fgf21Alb) mice (Fig. 2A and 2B) and neuron-specific Klb knockout (KlbCamk2a) mice (Fig. 2D and 2E) for both the time to and duration of loss of righting reflex. There was no difference in plasma ethanol clearance between the knockout and control mice (Fig. 2C and 2F). These results indicate that liver-derived FGF21 attenuates ethanol-induced sedation by acting on the nervous system.

[0067] Example 3: Pharmacological FGF21 is amethystic Pharmacological FGF21 treatment was tested to shorten the time to recovery of the righting reflex after ethanol administration. WT mice were gavaged with an excessive dose of ethanol and 1 h later received an ip injection of FGF21. Notably, administration of FGF21 shortened the time to recovery of the righting reflex by about 90 min for both male and female mice, representing a reduction of approximately 50% (Figures 3A and 3B). This effect was dose-dependent and was maximally effective at 1 mg / kg FGF21 in male mice (Figure 3A).

[0068] The same pharmacological FGF21 rescue experiment was performed in Fgf21- / - and KlbCamk2a mice. In Fgf21- / - mice, administration of FGF21 shortened the righting reflex recovery time to that observed in WT mice (Figure 3C). In contrast, in KlbCamk2a mice, FGF21 had no effect on the righting reflex recovery time (Figure 3D). This indicates that pharmacological FGF21 exerts its amethyst effect by acting on the nervous system.

[0069] We also investigated whether FGF21 inhibits ethanol-induced impairment of motor coordination. Pharmacological FGF21 treatment shortened the time it took WT mice to recover coordination on the rotarod after administration of a moderate dose of ethanol (2 g / kg, ip) (Figure 3E). Conversely, the recovery time was significantly prolonged in Fgf21- / - compared to WT mice (Figure 3F). Thus, FGF21 is amethystic for ethanol-induced impairment of both righting reflex and motor coordination.

[0070] [Example 4: Amethystic activity of FGF21 is selective for ethanol] We tested whether FGF21 inhibits the effects of other sedatives that act by different mechanisms, such as the glutamatergic receptor antagonist ketamine, and the GABA receptor agonists diazepam and pentobarbital. These sedatives act more rapidly and have a shorter duration than ethanol, so we shortened the experimental time schedule. Under these modified conditions, FGF21 retained its ability to shorten the righting reflex recovery time in ethanol-treated mice (Figure 4A). In contrast, FGF21 administration did not shorten the righting reflex recovery time for ketamine, diazepam, and pentobarbital (Figures 4B-4D). Thus, the amethystic activity of FGF21 is selective for ethanol.

[0071] [Example 5: FGF21 is a physiological regulator of noradrenergic neurons] To determine whether FGF21 contributes to ethanol-induced activation of noradrenergic neurons in the LC, WT and Fgf21- / - mice were gavaged with ethanol and euthanized 2.5 hours later. Immunostaining of LC sections was performed for c-Fos and NE transporter (NET), a marker of noradrenergic neurons. As reported, ethanol induced c-Fos expression in NET+LC neurons in WT mice (Figure 5A and Figure 5B) (Chang et al., 1995; Kolodziejska-Akiyama et al., 2005; Thiele et al., 1997). Notably, this induced response was completely absent in Fgf21- / -, although there was a trend toward increased basal c-Fos in knockout mice (Figure 5A and Figure 5B). These data indicate that FGF21 is a crucial physiological signal by which ethanol activates noradrenergic neurons in the LC.

[0072] [Example 6: FGF21 acts directly on noradrenergic neurons] To determine whether FGF21 acts directly on noradrenergic neurons, initial experiments tested whether KLB, an obligate coreceptor for FGF21 that is selectively expressed in a few brain regions (Bookout et al., 2013), is present within LCs. In the absence of a reliable KLB antibody, we measured KLB expression using established reporter mice in which TdTomato was knocked into the endogenous Klb gene to generate a KLB-TdTomato fusion protein (Coate et al., 2017). The fusion protein was detected by immunostaining within LCs. It colocalized with NETs in most, but not all, cells (Figure 6A). The fusion protein was also detected in areas adjacent to LCs where there was little or no staining for NETs (Figure 6A). Thus, KLB is expressed in noradrenergic neurons and possibly other cell types in and around the LC. In the absence of an antibody selective for FGFR1c, we tested whether Fgfr1c.mRNA was present in LCs by in situ hybridization. Fgfr1c mRNA was detected throughout the LC region and colocalized with Klb mRNA in some cells (Figure 8). Consistent with the Fgfr1c / Klb expression data, pharmacological FGF21 administration induced c-Fos immunoreactivity in NET+LC neurons of WT mice (Figures 6B and 6C). When this same pharmacological experiment was performed in neuron-specific KlbCamk2a mice, no trend toward increased FGF21-induced and basal c-Fos expression was observed (Figure 6D).

[0073] To test whether noradrenergic neurons are required for the amethystic activity of FGF21, we first used DSP-4 (Ross and Stenfors, 2015), a neurotoxin that readily crosses the blood-brain barrier and selectively and irreversibly inhibits NETs, ​​thereby disrupting NE signaling. Mice pretreated with DSP-4 were completely refractory to the amethystic effect of FGF21 on the righting reflex (Figure 7A). Upon release from neurons, NE acts on α1- and β-adrenergic receptors in multiple subcortical regions to stimulate wakefulness (Berridge et al., 2012). Accordingly, the amethystic effect of FGF21 was also blocked by prazosin and propranolol, which are selective α1- and β-adrenergic receptor antagonists, respectively (Figure 7B and Figure 7C). As previously reported, prazosin alone prolonged ethanol-induced sedation (Figure 7B) (Malinowska et al., 1999). These pharmacological data indicate that FGF21 exerts its amethystic activity via the noradrenergic nervous system.

[0074] Finally, to test whether FGF21 acts directly on (i) neurons that are noradrenergic and (ii) neurons located within the LC region, we performed a complementary gene Klb knockout study. Klbfl / fl mice were crossed with Dbh-Cre mice to selectively inhibit FGF21 activity in noradrenergic neurons or were injected bilaterally into the LC region with adenovirus-associated viruses (AAV) expressing either Cre recombinase fused to green fluorescent protein (GFP) or GFP alone. Consistent with the pharmacological studies, the amethystic effect of FGF21 on the righting reflex was abolished in KlbDbh mice (Figure 7D) and Klbfl / fl mice injected with AAV-Cre, but not in control AAV-GFP (Figure 7F). Interestingly, unlike Fgf21- / - and KlbCamk2a mice, no prolongation of the time it took for these knockout mice to regain the righting reflex after ethanol exposure was observed. In Klbfl / fl mice injected with AAV-Cre, the recovery time was even shortened (Figure 7G). The reason for this difference is unclear, but the data raise the possibility of a compensatory response to the loss of KLB in noradrenergic neurons. In contrast to control mice, in KlbDbh mice, FGF21 did not induce c-Fos expression in NET+LC neurons (Figure 7E). Taken together, these experiments suggest that FGF21 exerts its amethystic activity by acting directly on neurons present in the noradrenergic and LC regions.

Claims

1. 1. A method of treating acute alcohol intoxication in a subject in need of such treatment, comprising administering to the subject an effective amount of an agonist of the fibroblast growth factor receptor (FGFR) / β-Klotho complex.

2. 10. The method of claim 1, wherein treating acute alcohol intoxication comprises reducing disorientation, increasing level of consciousness, improving cardiac function, improving respiration, or any combination thereof.

3. The method of claim 1 or claim 2, wherein the agonist comprises FGF21 or an FGF21 homolog.

4. The method of any one of claims 1 to 3, wherein the effective amount of the agonist is administered as a pharmaceutical composition.

5. The method of any one of claims 1 to 4, wherein the effective amount of the agonist is from about 0.5 mg / kg to about 2 mg / kg.

6. 6. The method of any one of claims 1 to 5, wherein the subject consumed alcohol for about 1 to 24 hours, about 1 to 18 hours, about 1 to 12 hours, about 1 to 10 hours, about 1 to 8 hours, about 1 to 6 hours, about 1 to 5 hours, or about 1 to 4 hours prior to administration of the agonist.

7. The method of any one of claims 1 to 6, wherein the subject is a human.

8. A pharmaceutical composition comprising an agonist of the fibroblast growth factor receptor (FGFR) / β-Klotho complex and a pharma- ceutically acceptable carrier or excipient.

9. The pharmaceutical composition of claim 8 , wherein the agonist comprises FGF21 or a homolog thereof.

10. 10. The pharmaceutical composition of claim 8 or 9, wherein the carrier or excipient comprises a diluent, binder, filler, buffer, pH adjuster, disintegrant, dispersant, preservative, lubricant, or any combination thereof.

11. The pharmaceutical composition according to any one of claims 8 to 10, which is formulated for oral, nasal or intravenous administration.

12. A kit comprising an agonist of the fibroblast growth factor receptor (FGFR) / β-Klotho complex, a pharma- ceutically acceptable carrier or excipient, and instructions for administering said agonist to a subject suffering from acute alcohol intoxication.

13. The kit of claim 12 , wherein the agonist comprises FGF21 or a homolog thereof.

14. 14. The kit of claim 12 or 13, further comprising a container.

15. The kit of any one of claims 12 to 14, further comprising an administration device.

16. The kit of any one of claims 12 to 15, wherein the carrier or additive comprises a diluent, binder, filler, buffer, pH adjuster, disintegrant, dispersant, preservative, lubricant, or any combination thereof.

17. The kit according to any one of claims 12 to 16, wherein the subject is a human.

18. A method for preventing acute alcoholism and / or informing a subject of the effects of acute alcoholism in a subject in need of such prevention and / or informing the subject, comprising administering to the subject an effective amount of an agonist of the fibroblast growth factor receptor (FGFR) / β-Klotho complex prior to the ingestion of alcohol by the subject.

19. The method of claim 18 , wherein the agonist comprises FGF21 or an FGF21 homolog.

20. 20. The method of claim 18 or 19, wherein the effective amount of the agonist is administered as a pharmaceutical composition comprising a pharma- ceutically suitable carrier or excipient.

21. The method of any one of claims 18 to 20, wherein the effective amount of the agonist is from about 0.5 mg / kg to about 2 mg / kg.

22. The method of any one of claims 18 to 21, wherein the subject consumes alcohol between about 1 to 24 hours, about 1 to 18 hours, about 1 to 12 hours, about 1 to 10 hours, about 1 to 8 hours, about 1 to 6 hours, about 1 to 5 hours, or about 1 to 4 hours after administration of the FGF21.

23. The method of any one of claims 18 to 22, wherein the subject is a human.

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