Compositions and methods for reducing blood ethanol concentration through alcohol dehydrogenase and acetaldehyde scavengers

The present disclosure relates to compositions and methods for reducing blood ethanol concentration through a dual-action mechanism involving alcohol dehydrogenase (ADH) and acetaldehyde (AcH) scavengers. The present disclosure provides a formulation that accelerates ethanol (EtOH) metabolism via ADH and rapidly eliminates AcH using scavengers, thereby driving the reaction equilibrium forward to enhance EtOH clearance.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 69942-702_201_SL.xml, created Mar. 31, 2025, which is 1,909 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

BACKGROUND

Alcohol dehydrogenases (ADHes) are the primary enzymes for catalyzing the conversion of ethanol (EtOH) to acetaldehyde (AcH). Enhancing ADH activity can accelerate EtOH clearance, thereby reducing blood ethanol concentration more rapidly. However, the resulting increase in AcH concentration can exacerbate toxicity if not addressed concurrently.

AcH is a highly toxic metabolic byproduct of EtOH metabolism, primarily produced via the enzymatic action of alcohol dehydrogenase (ADH) in the liver. AcH is a major contributor to alcohol-induced tissue damage and adverse physiological effects.

The harmful effects of AcH include DNA damage, protein adduct formation, and increased oxidative stress, all of which contribute to mutagenesis and organ damage, particularly in the liver. AcH has been implicated in the pathogenesis of alcohol-induced liver diseases, facial flushing, and various cancers, particularly of the upper aero-digestive tract.

Genetic polymorphisms in aldehyde dehydrogenase (ALDH), particularly the ALDH2*2 variant common in East Asian populations, impair the metabolism of AcH, leading to its accumulation and associated symptoms such as “Asian flush.”

Elevated AcH levels contribute to alcohol hangover symptoms, including headache, nausea, and fatigue, which persist even after EtOH has been cleared from the bloodstream.

AcH has also been linked to alcohol dependence, as it produces reinforcing effects in the central nervous system (CNS) that contribute to addiction.

No existing solution effectively addresses both EtOH and AcH clearance simultaneously, particularly during the critical early phase when blood ethanol concentration is still high and AcH accumulates rapidly.

Accordingly, there is an unmet need for compositions and methods that simultaneously reduce blood ethanol concentration and mitigate AcH toxicity, providing a comprehensive solution for managing the adverse effects of alcohol consumption.

SUMMARY

In one aspect disclosed herein is a pharmaceutical composition comprising (1) an alcohol dehydrogenase (ADH) and (2) an acetaldehyde (AcH) scavenger.

In some embodiments, the ADH is a microbial alcohol dehydrogenase. In some embodiments, the ADH is a Saccharomyces cerevisiae alcohol dehydrogenase. In some embodiments, the Saccharomyces cerevisiae alcohol dehydrogenase comprises SEQ ID NO: 1.

In some embodiments, the ADH is a plant alcohol dehydrogenase. In some embodiments, the ADH is an animal alcohol dehydrogenase. In some embodiments, the ADH is a mammalian alcohol dehydrogenase. In some embodiments, the ADH is a human alcohol dehydrogenase.

In some embodiments, the ADH is produced recombinantly.

In some embodiments, the AcH scavenger comprises a thiol-containing compound or an agent that sequesters AcH. In some embodiments, the AcH scavenger comprises the thiol-containing compound. In some embodiments, the AcH scavenger comprises the agent that sequesters AcH. In some embodiments, the AcH scavenger comprises the thiol-containing compound and the agent that sequesters AcH. In some embodiments, the composition further comprises an antioxidant.

In some embodiments, the pharmaceutical composition is formulated for one or more of oral or transmucosal administration. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In another aspect disclosed herein is a method for reducing blood ethanol concentration and acetaldehyde (AcH) toxicity, the method comprising administering to a subject an effective amount of both (1) an alcohol dehydrogenase (ADH) and (2) an acetaldehyde (AcH) scavenger.

In some embodiments, the method comprises administering a composition as described above.

In some embodiments, the ADH and the AcH scavenger are administered before alcohol consumption. In some embodiments, the ADH and the AcH scavenger are administered concurrent with alcohol consumption. In some embodiments, the ADH and the AcH scavenger are administered after alcohol consumption.

In some embodiments, the administration of the ADH and the AcH scavenger reduces reactive oxygen species (ROS), increases cell viability, and protects the subject against alcohol-induced damages. In some embodiments, the administration of the ADH and the AcH scavenger decreases one or more symptoms associated with alcohol consumption. In some embodiments, the administration of ADH and the AcH scavenger results in reduced oxidative degradation. In some embodiments, the administration of the ADH and the AcH scavenger results in increased ADH stability, thereby preserving enzymatic activity of the ADH. In some embodiments, the administration of the ADH and the AcH scavenger both (1) accelerates EtOH metabolism due to action of the ADH and (2) removes AcH due to action of the AcH scavenger, thereby (1) shifting reaction equilibrium forward according to Le Chatelier's principle and (2) increasing the conversion rate of EtOH to AcH. In some embodiments, the presence of the AcH scavenger reduces the apparent Km and increases the apparent Vmax of the ADH, thereby increasing the rate of metabolism of EtOH.

In some embodiments, the AcH scavenger comprises a thiol-containing compound or an agent that sequesters AcH. In some embodiments, the AcH scavenger comprises the thiol-containing compound. In some embodiments, the AcH scavenger comprises the agent that sequesters AcH. In some embodiments, the AcH scavenger comprises the thiol-containing compound and the agent that sequesters AcH. In some embodiments, the method further comprises administering an antioxidant.

In another aspect disclosed herein is a kit comprising (1) an alcohol dehydrogenase (ADH) and (2) an acetaldehyde (AcH) scavenger.

In some embodiments, the alcohol dehydrogenase and the AcH scavenger are in a single composition. In some embodiments, the kit further comprises instructions for use.

INCORPORATION BY REFERENCE

DETAILED DESCRIPTION

As used herein, the term “scavenger” refers to a compound that interacts with AcH to decrease the concentration of free-AcH in solution. Scavengers include, but are not limited to, (1) thiol-containing compounds that form a hemiacetal or thioacetal adduct with AcH and (2) agents that sequester AcH to prevent it from interacting with ADH.

The present disclosure relates to compositions and methods for reducing blood ethanol concentration by combining alcohol dehydrogenase (ADH) with acetaldehyde (AcH) scavengers. The present disclosure provides novel approaches for managing elevated ethanol (EtOH) and AcH levels, particularly in the context of alcohol metabolism.

Combining ADH with AcH scavengers present a complementary approach: ADH accelerates EtOH breakdown, while the scavengers neutralize the resulting AcH, effectively mitigating both blood ethanol concentration and AcH toxicity.

This dual-action strategy can be particularly beneficial for individuals with ALDH deficiencies, chronic alcohol users, and patients experiencing acute alcohol intoxication.

Furthermore, this disclosure addresses a significant gap in current pharmacokinetic models, which often fail to account for AcH accumulation during EtOH metabolism.

The present disclosure provides a pharmaceutical composition combining ADH with one or more AcH scavengers. This combination achieves an effect by accelerating the conversion of ethanol (EtOH) to AcH (via ADH) and soon thereafter scavenging the AcH (via AcH scavengers), thereby reducing both blood ethanol concentration and AcH toxicity. The present disclosure is applicable for treating a range of alcohol-related conditions and for preventing alcohol-associated diseases. The composition may be administered through multiple delivery routes, ensuring effective bioavailability and therapeutic efficacy.

In some embodiments, the methods described herein may be used to treat humans or animals. In a preferred embodiment, the methods are used to treat humans.

Additional objects, advantages, and novel features of the present disclosure will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting.

Various embodiments of the present disclosure are directed to formulations comprising one or more ADHs and one or more AcH scavenger(s).

The present disclosure includes embodiments addressing the metabolic pathway of EtOH by first converting EtOH to AcH using ADH, followed by the rapid elimination of AcH using one or more scavengers.

The present disclosure recognizes the interplay between ADH and AcH scavengers, emphasizing their combined effect. This dual-action mechanism harnesses the catalytic activity of ADH in converting EtOH to AcH and the subsequent rapid elimination of AcH through the action of AcH scavengers. This cooperation results in a significant increase of EtOH metabolism (e.g., an increase in the rate of metabolism), and a reduction of both blood ethanol concentration and AcH toxicity.

The mechanism of the dynamic equilibrium between ADH and AcH scavengers is grounded in Le Chatelier's principle, which states that a system at equilibrium will shift to counteract a change in concentration of a reactant or product. In the context of the present disclosure, the rapid removal of AcH by scavengers shifts the equilibrium of the enzymatic reaction catalyzed by ADH. As ACH is continually removed from the system, the reaction converting EtOH to AcH is driven forward, resulting in faster EtOH clearance from the body.

The reaction sequence may proceed as follows: a) EtOH oxidation—the enzyme ADH catalyzes the oxidation of EtOH to AcH according to the reaction; b) AcH scavenger(s) chemically react with AcH, in many cases forming non-toxic adducts or derivatives, thereby reducing free AcH concentration; and c) Equilibrium shift (Le Chatelier's Principle) as AcH is continuously removed from the system by scavenger(s), the equilibrium of the ADH-catalyzed reaction is shifted to the right, favoring further conversion of EtOH to AcH to restore equilibrium. The continuous elimination of AcH by scavenger(s) sustains a low AcH concentration, which accelerates the forward reaction of EtOH oxidation by ADH. This results in a self-reinforcing cycle where more EtOH is converted to AcH and subsequently removed from the system.

This dual mechanism demonstrates the biochemical interplay between ADH and AcH scavenger(s) and their influence on the EtOH metabolism pathway, driven by principles of enzymatic kinetics and equilibrium dynamics. This dual action lowers blood ethanol concentration faster than ADH or scavenger(s) alone and simultaneously prevents the accumulation of toxic AcH.

The present disclosure incorporates ADH, an enzyme responsible for converting EtOH to AcH. The ADH used may be of human origin (recombinant or purified), animal origin, plant origin, or microbial origin (e.g., yeast, bacterial, or fungal ADH).

In some embodiments, multiple variants of ADH are combined to enhance the enzymatic efficiency under various physiological conditions.

One of more scavengers may be used in combination with one or more ADHs. In some embodiments, the one or more scavengers include a thiol-containing compound that forms a hemiacetal or thioacetal adduct with AcH. For example, in some embodiments, the one or more AcH scavengers can comprise D-penicillamine, N-acetylcysteine (NAC), glutathione (GSH), L-cysteine, or cysteinylglycine (Cys-Gly), a salt thereof, or a combination thereof. In some embodiments, the one or more scavengers include an agent that sequesters AcH to prevent it from interacting with ADH. In some embodiments, AcH is sequestered via encapsulation, chelation, or adsorption. For instance, in some embodiments, the one of more scavengers that sequester, encapsulate, chelate to, or adsorb AcH comprise cyclodextrin (e.g., β-Cyclodextrin, α-Cyclodextrin, γ-Cyclodextrin), MXDA (meta-xylenediamine), anthranilamide, titanium dioxide (TiO2), zeolites, activated carbon, zinc acetate, copper (II) salts, a salt thereof, or a combination thereof.

In some embodiments, the composition can comprise one or more antioxidants. In some embodiments, the one or more antioxidants can reduce oxidative stress caused by AcH. In some embodiments, the one or more antioxidants can scavenge free radicals. In some embodiments, the one or more antioxidants can comprise vitamin C, lipoic acid (e.g., α-lipoic acid), grapeseed extract, ginger root extract, curcumin, milk thistle extract, prickly pear leaf, Picrorhiza kurroa extract, tectoridin, a salt thereof, or a combination thereof.

Additionally, any salts, enantiomers, diastereomers, or derivatives of the listed scavengers are included within the scope of the present disclosure.

The present disclosure encompasses the use of one or more of these AcH scavengers, either alone or in combination. In an embodiment of the present disclosure, the selected AcH scavengers can lead to higher and/or faster AcH degradation.

The compositions may be administered via any suitable mode of administration. Modes of administration: the compositions may be formulated for various routes of administration, including but not limited to: (a) oral administration, (b) an intravenous administration or (c) transmucosal administration.

In some embodiments, the formulation includes liposomal encapsulation or other drug delivery technologies, such as micelles, nanoparticles, or hydrogels, to improve the bioavailability of ADH and AcH scavengers.

Pharmaceutical formulations: Pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers, excipients, or diluents, including but not limited to: a) inert diluents such as calcium carbonate, mannitol, or lactose; b) binders such as starch or gelatin; c) disintegrants such as sodium starch glycolate; d) lubricants such as magnesium stearate or talc; e) solubilizers or stabilizers such as polyethylene glycol (PEG) or Tween 80; f) preservatives such as methylparaben and propylparaben; or g) sweeteners or flavoring agents.

The compositions of the present disclosure may include additional ingredients that are not physiologically active but serve to enhance the properties of the final composition. Further techniques for formulation and administration of active ingredients may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 23rd edition, 2018, which is incorporated herein by reference as if fully set forth herein.

Compositions of the present disclosure may, as desired, be presented in a pack, injector or dispenser device, such as an FDA (the U.S. Food and Drug Administration)-approved kit.

While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present disclosure be limited by the specific examples provided within the specification. While the present disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. Furthermore, it shall be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present disclosure. It is therefore contemplated that the present disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

Example 1: In Vitro Study on the Combined Effect of ADH and AcH Scavengers on EtOH and AcH Concentration Reduction

An in vitro study was conducted to evaluate the effect of a combined formulation of ADH and AcH scavengers on the reduction of EtOH and AcH concentrations. The study was designed to assess the enzymatic conversion of EtOH to AcH by ADH and the subsequent removal of AcH by scavengers. The experiment was performed using enzyme preparation from yeast (Saccharomyces cerevisiae).

SEQ ID

The experiment was conducted in triplicate with appropriate controls to ensure reproducibility.

A total reaction volume of 1 mL was prepared for each sample, consisting of 100 μL of EtOH (final concentration: 100 mM); 200U of ADH enzyme; AcH scavenger mixture (total 455 mg, with components as listed above); and 100 mM sodium phosphate buffer (pH 7.4).

All samples were incubated at 37° C. for 30 minutes with gentle shaking (150 rpm) to ensure uniform mixing and optimal enzyme activity. The reaction was stopped by adding 100 μL of 0.1 M hydrochloric acid (HCl) to each tube to denature the enzyme and halt the conversion of EtOH to AcH.

EtOH concentration was measured using a NAD/NADH-based enzymatic assay with a spectrophotometer at 340 nm. AcH concentration was measured using a modified Hantzsch reaction assay, which detects AcH at 412 nm.

The combined formulation of ADH and AcH scavengers demonstrated a significant reduction in both EtOH and AcH concentrations compared to the control groups.

The following results were obtained:

EtOH Reduction
EtOH Final
AcH Final

The highest reduction in both EtOH and AcH was achieved due to the effect of ADH converting EtOH to AcH and scavengers rapidly eliminating AcH. The results demonstrate the dual mechanism of the ADH and AcH scavenger combination. ADH facilitated the rapid conversion of EtOH to AcH, while the scavengers efficiently eliminated AcH, thereby driving the reaction forward (according to Le Chatelier's principle). This action led to a substantial reduction in both EtOH and AcH concentrations.

This in vitro study confirms that the combination of ADH and AcH scavengers effectively increases EtOH metabolism and eliminates its toxic intermediate, AcH. The results support the potential therapeutic use of this combination for reducing blood ethanol concentration and mitigating AcH-related toxicity in vivo. Further studies, including in vivo animal models, are recommended to confirm these findings and assess clinical applicability.

Example 2: In Vivo Study of Combined ADH and AcH Scavengers in Reducing Blood EtOH and AcH Concentrations in Rodents

The study was performed to evaluate the efficacy of a combined formulation of ADH and AcH scavengers in reducing blood EtOH and AcH concentrations in an in vivo rodent model.

Blood samples were collected from the tail vein at 0, 15, 30, 60, and 120 minutes post-EtOH administration. EtOH concentration was measured using a NAD/NADH-based enzymatic assay.

Results

Group 
Group 2
Group 3
Group 4

The combined group showed the greatest reduction in both EtOH concentrations due to ADH-facilitated conversion and subsequent scavenging of AcH. This effect was consistent across all time points (FIG. 1).

Example 3: Enzyme Kinetics Analysis of ADH with AcH Scavengers Using Michaelis-Menten Parameters (Vmax and KM)

Control 
With

The presence of AcH scavengers increased Vmax because removing AcH allows the enzyme to operate at full capacity. KM decreases (apparent) because the reaction is pulled forward, making it appear as though ADH has higher substrate affinity. kcat (Catalytic Rate Constant) increases, indicating that each enzyme molecule processes more substrate per unit time, confirming that the enzyme is working more efficiently.

Example 4: Stability and Degradation Profile of ADH in the Presence of AcH Scavengers Over Time

Conditions: 37° C. for up to 60 min with gentle shaking (100 rpm), ADH activity assessed via NADH production (340 nm).

Time 
ADH activity (% of t = 0)

The AcH scavenger-containing composition protected ADH from oxidative degradation, likely due to free radical scavenging (GSH, NAC, α-lipoic acid), thiol protection (NAC) and metal ion chelation (GSH).

Class
Mechanism
Examples

Activated Carbon

Example 5: In Vitro Cellular Study on HepG2 Liver Cells Treated with ADH and AcH Scavengers to Evaluate the Protective Effect of ADH and AcH Scavengers Against EtOH and AcH-Induced Cytotoxicity

Cell viability was detected by MTT assay (absorbance at 570 nm). ROS (Reactive Oxygen Species) was detected by DCFH-DA assay (fluorescence at 488 nm).

Results

Cell viability
ROS (% of

Results Interpretation

ROS 
Overall Impact on
Viability

Group
EtOH Effect
AcH Effect
Impact
Viability
(%)

Group 1
High EtOH toxicity
Low AcH (since
High ROS
Lowest viability
52.30%

(EtOH only,
results in membrane
ADH is not
from EtOH
due to prolonged

dysfunction

Group 2
Lower EtOH due to
the higher AcH is
Moderate
Viability
67.10%

(EtOH +
ADH convertion fo
toxic, but
ROS from
increases since

ADH)
EtOH to AcH,
metabolized by
AcH
EtOH toxicity is

reducing direct EtOH
endogenous
accumulation
reduced, though

AcH still causes

stress

Group 3
High EtOH toxicity
Low AcH
Lower ROS
Viability
71.40%

(EtOH +
(same as Group 1)
(scavengers
due to
improves due to

AcH
since ADH is not
prevent
antioxidant
antioxidant

Scavengers,
added
accumulation)
effects of
protection but

No ADH)

remains

Group 4
Lowest EtOH toxicity
Lowest AcH
Lowest ROS
Highest viability
92.80%

(EtOH +
due to ADH
toxicity since the
from both
due to clearance

ADH + AcH
conversion of EtOH to
scavengers
EtOH
of EtOH and AcH

Scavengers)
AcH rapidly
remove AcH
reduction and

efficiently
antioxidant

protection