Novel modified tetracyclines for treatment of alcohol use disorder, pain and other disorders involving potential inflammatory processes

Abstract

To provide a novel tetracycline derivative having reduced antimicrobial activity for use in treating disorders of the central nervous system.SOLUTION: Provided are novel molecules comprising a modified tetracycline molecule including Deamino Diacetyl Minocycline, Methyl Ether Minocycline, Ethyl Ether Minocycline, Propyl Ether Minocycline, Butyl Ether Minocycline, Butyl Ether Monoacetyl Minocycline, Butyl Ether Diacetyl Minocycline, Butyl Ether Triacetyl Minocycline, or Butyl Ether Tetra Acetyl Minocycline, pharmaceutically acceptable salts, pro-drugs, biologically active metabolites, and tautomers thereof, and methods for using the same to treat Alcohol Use Disorder (AUD), Substance Use Disorder (SUD), tobacco use, pain, or proinflammatory disorders.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 987,700, filed March 10, 2020, the entire contents of which are incorporated herein by reference.

[0002] Statement of Federally Funded Research This invention was made with government support under grants R21AA021142, R41AA027447, and U01AA027401 awarded by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health. The federal government has certain rights in this invention.

[0003] The present invention relates generally to the field of novel tetracycline derivatives with reduced antimicrobial activity for use in the treatment of disorders of the central nervous system. [Background technology]

[0004] Without limiting the scope of the invention, its background will be described in connection with modified tetracyclines.

[0005] Alcohol use disorder (AUD), or alcoholism, affects approximately 5% of people worldwide. AUD is characterized by a physical dependence on alcohol, leading to an increased tolerance and difficulty for individuals in controlling their consumption. Some long-term effects of ethanol consumption include cognitive and psychological changes, liver cirrhosis, gastritis, cardiomyopathy, anemia, and certain types of cancer. Alcoholism is caused by a complex interaction of genetic and environmental factors. Several genes are known to be involved in how humans metabolize alcohol and in the development of AUD. The increasing availability of alcohol has also contributed to the increasing prevalence of AUD. Alcohol is the most accessible and widely abused recreational drug, with beer being the third most popular beverage after water and tea. Currently, there are few treatments for alcoholism other than rehabilitation, which can be expensive and highly visible. There is an unmet need for medications to address the debilitating effects of AUD.

[0006] One such modified tetracycline derivative was filed by Lorenz et al. and is entitled "Method for the synthesis of A-ring aromatized acetyl minocyclines" in the U.S. Briefly, these applicants disclose a compound of formula:

[0007] [ka]

[0008] The application appears to teach a less complicated method for the preparation of A-ring aromatized acetyl minocycline of formula (wherein R1-R5 are acetyl and / or H), which involves reacting minocycline hydrochloride with acetic anhydride in the presence of a proton catcher and subjecting the reaction product to chromatographic filtration using a carrier material and an eluent. The eluent is removed by distillation, followed by purification of the product by recrystallization. However, the application does not disclose the treatment of addictive disorders, and only teaches the treatment of neurodegeneration.

[0009] Other similar modified tetracycline derivatives have also been described by Lorenz et al., in "Method for the synthesis of A-ring aromatized acetyl minocyclines This is taught in International Publication No. 2009012741, which is incorporated herein by reference. This application is believed to teach a less complicated method for preparing A-ring aromatized acetyl minocycline. The preparation of A-ring aromatized acetyl minocycline of formula (I), where R1-R5 are acetyl and / or H, is accomplished by reacting minocycline hydrochloride with acetic anhydride in the presence of a proton catcher, followed by chromatographic filtration of the reaction product using a carrier material and an eluent. The eluent is then distilled off, and the product is then washed by recrystallization. Like the above application, this application does not disclose the treatment of addictive disorders, and only teaches the treatment of neurodegeneration.

[0010] However, there remains a need for novel molecules for the treatment of alcohol use, including but not limited to high alcohol consumption, withdrawal symptoms, increased pain sensitization and duration, altered innate immune responses, reduced tobacco consumption, and all aspects of the addiction process associated with other drugs of abuse, such as opioids. (Mark R. Hutchinson, Alexis L. Northcutt, Lindsey W. Chao, Jeffrey J. Kearney, Yingning Zhang, Debra L. Berkelhammer, Lisa C. Loram, Robert R. Rozeske, Sondra T. Bland, Steven F. Maier, Todd T. Gleeson, and Linda R. Watkins, Minocycline suppresses morphine-induced respiratory depression, suppresses morphine-induced reward, and enhances systemic morphine-induced analgesia, Brain, Behavior and Immunity 22(8): 1248-1256, 2009) [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent Application Publication No. 20100173991 [Patent Document 2] International Publication No. 2009012741 [Non-patent literature]

[0012] [Non-Patent Document 1] Mark R. Hutchinson, Alexis L. Northcutt, Lindsey W. Chao, Jeffrey J. Kearney, Yingning Zhang, Debra L. Berkelhammer, Lisa C. Loram, Robert R. Rozeske, Sondra T. Bland, Steven F. Maier, Todd T. Gleeson, and Linda R. Watkins, Minocycline suppresses morphine-induced respiratory depression, suppresses morphine-induced reward, and enhances systemic Morphine-induced analgesia, Brain, Behavior and Immunity 22(8): 1248-1256, 2009 Summary of the Invention [Means for solving the problem]

[0013] In another embodiment, the present invention provides modified tetracycline molecules, including deaminodiacetylminocycline, methyl ether minocycline, ethyl ether minocycline, propyl ether minocycline, butyl ether minocycline, butyl ether monoacetylminocycline, butyl ether diacetylminocycline, butyl ether triacetylminocycline, or butyl ether tetraacetylminocycline, and their pharmaceutical uses. In one aspect, the modified tetracycline molecules, their pharmaceutically acceptable salts, prodrugs, biologically active metabolites, and tautomers include those of Formulas 1-48.

[0014] [ka] JPEG2025169433000003.jpg237121JPEG2025169433000004.jpg240137JPEG2025169433 000005.jpg208148JPEG2025169433000006.jpg211159JPEG2025169433000007.jpg22615 0JPEG2025169433000008.jpg234154JPEG2025169433000009.jpg237149JPEG2025169433 000010.jpg224163JPEG2025169433000011.jpg230156JPEG2025169433000012.jpg68170 [ka]

[0015] In one embodiment, the molecule has reduced or substantially no antibacterial activity. In another embodiment, the molecule has reduced or substantially no antifungal activity. In another embodiment, the molecule is provided in an amount that inhibits alcohol use disorder (AUD), substance use disorder (SUD), smoking, pain, and inflammatory response. In another embodiment, the inflammatory response is rheumatoid arthritis. In another embodiment, the molecule does not inhibit ribosomes, e.g., bacterial ribosomes. In another embodiment, the modification is at least one of creating steric hindrance, blocking hydrogen bonds, or altering coordination bonds with divalent cations. In another embodiment, the molecule is disposed in a pharmaceutically acceptable buffer, excipient, filler, or carrier. In another embodiment, the molecule is formulated into a composition for oral, enteral, intramuscular, parenteral, intravenous, or intraperitoneal administration.

[0016] In another embodiment, the invention includes pharmaceutical compositions comprising compounds of Formulas 1-48, pharmaceutically acceptable salts, prodrugs, biologically active metabolites, and tautomers thereof.

[0017] In another embodiment, the invention includes a method of treating an alcohol use disorder (AUD), a substance use disorder (SUD), smoking, pain, or an inflammation-induced disorder, optionally comprising identifying a subject in need of treatment for an alcohol use disorder (AUD), a substance use disorder (SUD), smoking, pain, or an inflammation-induced disorder, and providing to the subject one or more modified tetracyclines of Formulas 1-48, wherein the modified tetracyclines have reduced or no antimicrobial activity, in an amount effective to ameliorate or eliminate the AUD, SUD, pain, or inflammation-induced disorder. In one aspect, the molecule has reduced or substantially no antibacterial activity. In another aspect, the inflammation-induced disorder is rheumatoid arthritis. In another aspect, the molecule has reduced or substantially no antifungal activity. In another aspect, the inflammation-induced disorder is rheumatoid arthritis. In another aspect, the molecule has reduced or substantially no antifungal activity. In embodiments, the molecule inhibits alcohol use disorder (AUD), substance use disorder (SUD), pain, and disorders involving an underlying inflammatory process. In another embodiment, the modified molecule is doxycycline, minocycline, or tigecycline, or a tautomeric structure thereof. In another embodiment, the modification is at least one of creating steric hindrance, blocking hydrogen bonds, or altering coordination bonds with divalent cations. In another embodiment, the method further comprises providing the molecule in a pharmaceutically acceptable buffer, excipient, filler, or carrier. In another embodiment, the method further comprises formulating into a composition for oral, enteral, intramuscular, parenteral, intravenous, or intraperitoneal administration.

[0018] In another embodiment, the invention includes a method of treating alcohol use disorder (AUD), substance use disorder (SUD), smoking, pain, or an inflammation-induced disorder, comprising identifying a subject in need of treatment for at least one of AUD, SUD, pain, or an inflammation-induced disorder, and providing to the subject one or more modified tetracyclines of Formulas 1-48, wherein the modified tetracyclines have reduced or no antimicrobial activity, in an amount effective to ameliorate or eliminate the AUD, SUD, pain, or inflammation-induced disorder. In one aspect, the molecule has reduced or substantially no antibacterial activity. In another aspect, the inflammation-induced disorder is rheumatoid arthritis. In another aspect, the molecule has reduced or substantially no antifungal activity. In another aspect, the molecule inhibits alcohol use disorder (AUD), substance use disorder (SUD), pain, and disorders involving underlying inflammatory processes. In another embodiment, the modified molecule is doxycycline, minocycline, or tigecycline, or a tautomeric structure thereof. In another embodiment, the molecule does not inhibit ribosomes, e.g., bacterial ribosomes. In another embodiment, the modification is at least one of creating steric hindrance, blocking hydrogen bonds, or changing coordination bonds with divalent cations. In another embodiment, the method further comprises providing the molecule in a pharmaceutically acceptable buffer, excipient, filler, or carrier. In another embodiment, the method further comprises formulating the molecule into a composition for oral, enteral, intramuscular, parenteral, intravenous, or intraperitoneal administration.

[0019] In another embodiment, the present invention provides a method for evaluating candidate agents potentially useful in the treatment of alcohol use disorder (AUD), substance use disorder (SUD), including opioid use disorder, smoking, pain, or an inflammation-induced disorder, comprising the steps of: a) measuring the level of alcohol use disorder (AUD), substance use disorder (SUD), smoking, pain, or an inflammation-induced disorder in a series of patients; b) administering a candidate agent to a first subset of the patients and a placebo to a second subset of the patients, wherein the candidate agent is a modified doxyl group having different combinations of halogens, acetyl esters, methyl esters, and diacetals having formulas 1-48. and d) determining whether the candidate agent results in a statistically significant reduction in alcohol use disorder (AUD), substance use disorder (SUD), pain, or an inflammatory disorder relative to any reduction occurring in a second subset of patients, wherein a statistically significant reduction indicates that the candidate agent is useful for treating alcohol use disorder (AUD), substance use disorder (SUD), smoking, pain, or an inflammatory disorder. In one aspect, the molecule has no more than moderate antibacterial activity or no antibacterial activity. In another aspect, the molecule has no more than moderate antifungal activity or no antifungal activity. In another aspect, the molecule is provided in an amount sufficient to inhibit alcohol use disorder (AUD), substance use disorder (SUD), pain, and disorders involving underlying inflammatory processes. In another embodiment, the modified molecule is doxycycline, minocycline, or tigecycline, or a tautomeric structure thereof. In another embodiment, the modification is at least one of creating steric hindrance, blocking hydrogen bonds, or changing coordination bonds with divalent cations. [Brief explanation of the drawings]

[0020] For a more complete understanding of the features and advantages of the present invention, reference should now be made to the detailed description of the invention taken in conjunction with the accompanying drawings, in which: [Figure 1]Figure 1 shows the proposed mechanism by which ethanol consumption is reduced. [Figure 2-1] ~ [Figure 2-2] FIG. 2 shows a basic strategy for the generation of novel compounds for the treatment of alcohol use disorder (AUD) that have low or no antimicrobial activity. [Figure 3] FIG. 3 shows that minocycline inhibits bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome. [Figure 4A] ~ [Figure 4C] Figure 4A) Zone of inhibition assay shows that CMM lost its antibacterial activity against Escherichia coli (E. coli) after 24 hours of cell culture. Figure 4B) Colony-forming unit assay shows that CMM lost its antibacterial activity against E. coli after 24 hours of cell culture. Figure 4C) Colony-forming unit assay shows that CMM lost its antifungal activity against Candida albicans (C. albicans) after 48 hours of cell culture. MINO = minocycline, BeMAc = butyl ether monoacetate minocycline, DeDiAc = deaminodiacetyl minocycline, TRI = triacetyl minocycline. [Figure 5A] ~ [Figure 5C] Figure 5A) CMM rapidly reduced ethanol consumption in the drinking-in-the-dark (DID) model of AUD. MINO, DeDiAc, and TRI reduced ethanol consumption by more than 50% via intraperitoneal injection (left). MINO, DeDiAc, TRI, and BeMAc slightly reduced ethanol consumption via gavage (right). Figure 5B) Gavage dose-response curve for DeDIAc in female mice. Figure 5C) Gavage dose-response curve for DeDIAc in male mice. MINO = minocycline, BeMAc = butyl ether monoacetate minocycline, DeDiAc = deaminodiacetyl minocycline, TRI = triacetyl minocycline. *p<0.05 compared to control. [Figure 6]Figure 6 shows the screening method for modified minocycline. The purity and structure of the CMM analogs were confirmed using HPLC, NMR, and LC-MS. The loss of antibiotic activity was confirmed by ZOI and CFU assays, and alcohol consumption was tested in C67BL / 6J mice. [Figure 7] Figure 7 shows the chemical modification site of minocycline and, for reference, the structure of ribosome dissociation by minocycline. Schedlbauer, A., T. Kaminishi, B. Ochoa-Lizarralde, N. Dhimole, S. Zhou, JP Lopez-Alonso, SR Connell, and P. Fucini. 2015. 'Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome', Antimicrobial agents and chemotherapy, 59: 2849-54 [Figure 8A] ~ [Figure 8D] Figures 8A-8D show the effect of butyl ether minocycline on the loss of antibiotic activity compared to minocycline. [Figure 9] FIG. 9 shows a flow chart of the mechanism of action of minocycline on autophagy, inflammation, and angiogenesis. [Figure 10] FIG. 10 shows the intracellular signaling pathways of COX2, BNIP3, acidic vacuoles, and GFP-LC3 puncta. [Figure 11A] ~ [Figure 11C] 11A-11C show that the mass, purity, and structure of deaminodiacetylminocycline were confirmed using LC-MS (FIG. 11B) and HPLC (FIG. 11C), and further confirmed by NMR (not shown). [Figure 12A] ~ [Figure 12D]Figures 12A-12D show zone of inhibition and colony forming unit assays demonstrating the loss of antibiotic activity for deaminodiacetylminocycline in Figures 12A and 12C compared to minocycline in Figures 12B and 12D, respectively. [Figure 13] Figure 13 shows the basic experimental setup for rheumatoid arthritis. RA models were induced in male and female DBA / 1J mice with five monoclonal antibodies against type II collagen, followed by E. coli lipopolysaccharide adjuvant (CIA, Chondrex, Redmond, WA). Each group was treated with saline or 75 mg / kg CMM i.p. RA was quantified using caliper measurements, infrared heat signature, arthritis scoring, a hot plate (42°C), and the Von Frey filament assay. [Figure 14] Figure 14 shows that the CMM reduced paw swelling by approximately 50% as measured by caliper (mm). Dates are shown as a percentage increase over baseline measurements. [Figure 15] Figure 15 shows that CMM reduced pressure noxiousness in RA mice, as measured by the von Frey filament assay. In induced mice treated with saline alone, an increase in sensitivity of approximately 40% was observed after 21 days compared to baseline. This increase in sensitivity was minimal in the MMC-injected group. [Figure 16A] ~ [Figure 16B] Figure 16A shows that injection of Chondrex's antibody cocktail resulted in severe inflammation of the interphalangeal, metatarsal, and ankle joints of the forepaws, resulting in a loss of anatomical definition. MCC significantly reduced the arthritis score by approximately 70% compared to the induction group shown in Figure 16B, significantly reducing the severity of RA. Improvement was evident in both male and female mice. DETAILED DESCRIPTION OF THE INVENTION

[0021] While the making and using of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention, and do not delimit the scope of the invention.

[0022] To facilitate understanding of the present invention, several terms are defined below. Terms defined herein have meanings as commonly understood by one of ordinary skill in the art to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but are intended to encompass a general class of things of which individual examples may be used for illustration. While terminology herein is used to describe individual embodiments of the present invention, their use does not limit the invention, except as outlined in the claims.

[0023] The present invention relates to the modification of tetracyclines where R6' = H, such as doxycycline, minocycline, and tigecycline, by acetylation or other modifications such that the molecule eliminates most or all of its antibacterial properties while retaining the ability to reduce the negative characteristics associated with alcohol consumption, including, but not limited to, increased alcohol consumption, withdrawal symptoms, and pain sensitization and duration, and to reduce tobacco consumption. Furthermore, the novel molecules can be used to treat disorders that have a component of innate immunity. The present invention relates to any tetracycline that does not bind to its site of action in bacteria. Modifications of tetracyclines at R6' other than H may operate through different mechanisms, such as steric hindrance, stereochemistry, lack of hydrogen bonding, or transport into bacteria, but the modifications should be able to retain the ability to reduce increased alcohol consumption, withdrawal symptoms, increased pain sensitization and duration, and tobacco consumption.

[0024] Only three pharmacotherapy treatments for alcohol use disorder (AUD) are FDA-approved, but none are widely used (<10% of AUD patients) or have shown strong long-term effects on reducing risky or dependent drinking (<20% sustained reduction in drinking). Unfortunately, approximately 10% of the U.S. population suffers from alcohol-induced withdrawal disorders (AUD), and over 5% of all medical disorders share the underlying problem of hazardous ethanol consumption. As a result, intoxication in general, and "alcoholism" (severe AUD) in particular, represent a significant clinical problem. Given limited pharmacotherapeutic options, there is a compelling need to continue developing new treatments across the spectrum of alcohol-induced withdrawal disorders (from mild to severe, as defined by the DSM-V classification). Indeed, because withdrawal symptoms can be fatal, improved treatments targeting heavy alcohol use and withdrawal symptoms are desirable. To date, medications targeting alcohol use have not provided protection against withdrawal, and medications used to alleviate withdrawal symptoms often have codependence with alcohol.

[0025] We recently demonstrated preclinical efficacy of tetracycline analogs for reducing high alcohol intake, withdrawal symptoms, and alcohol-mediated pain sensitization, and now have exciting preliminary data demonstrating the efficacy of an improved, chemically modified minocycline (CMM) (Bergeson, SE, H. Blanton, JM Martinez, DC Curtis, C. Sherfey, B. Seegmiller, PC Marquardt, JA Groot, CL Allison, C. Bezboruah, and J. Guindon. 2016. 'Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences', Alcoholism, clinical and experimental research, 40: 2506-15; Martinez, JM, JA Groot, DC Curtis, CL Allison, PC Marquardt, AN Holmes, DS Edwards, DR Trotter, PJ Syapin, DA Finn, and SE Bergeson. 2016. 'Effective Reduction of Acute Ethanol Withdrawal by the Tetracycline Derivative, Tigecycline, in Female and Male DBA / 2J Mice', Alcoholism, clinical and experimental research, 40: 2499-505, Bergeson, SE, MA Nipper, J. Jensen, ML Helms, and DA Finn. 2016."Tigecycline Reduces Ethanol Intake in Dependent and Non-Dependent Male and Female C57BL / 6J Mice," Alcoholism, Clinical and Experimental Research, 40: 2491-98; Syapin, PJ, JM Martinez, DC Curtis, PC Marquardt, CL Allison, JA Groot, C. Baby, YM Al-Hasan, I. Segura-Ulate, MJ Scheible, KT Nicholson, JL Redondo, DR Trotter, DS Edwards, and SE Bergeson. 2016. "Effective Reduction in High Ethanol Drinking by Semisynthetic Tetracycline Derivatives," Alcoholism, Clinical and Experimental Research, 40: 2482-90. Furthermore, the inventors have investigated the efficacy of tigecycline in reducing ethanol intake in dependent and non-dependent male and female C57BL / 6J mice. Tetracycline analogs, such as tetracycline, have previously been found to be effective against various aspects of alcohol use disorder (AUD), including abstinence, withdrawal symptoms, and increased sensitization and pain duration. (Bergeson, SE, H. Blanton, JM Martinez, DC Curtis, C. Sherfey, B. Seegmiller, PC Marquardt, JA Groot, CL Allison, C. Bezboruah, and J. Guindon. 2016. 'Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences', Alcoholism, clinical and experimental research, 40: 2506-15; Martinez, JM, JA Groot, DC Curtis, CL Allison, PC Marquardt, AN Holmes, DS Edwards, DR Trotter, PJ Syapin, DA Finn, and SE Bergeson.) 2016. 'Effective Reduction of Acute Ethanol Withdrawal by the Tetracycline Derivative, Tigecycline, in Female and Male DBA / 2J Mice', Alcoholism, clinical and experimental research, 40: 2499-505, Bergeson, SE, MA Nipper, J. Jensen, M. L. Helms, and D. A. Finn. 2016. 'Tigecycline Reduces Ethanol Intake in Dependent and Nondependent Male and Female C57BL / 6J Mice', Alcoholism, clinical and experimental research, 40: 2491-98、Syapin, P. J., J. M. Martinez, D. C. Curtis, P. C. Marquardt, C. L. Allison, J. A. Groot, C. Baby, Y. M. Al-Hasan, I. Segura-Ulate, M. J. Scheible, K. T. Nicholson, J. L. Redondo, D. R. Trotter, D. S. Edwards, and S. E. Bergeson. 2016. 'Effective Reduction in High Ethanol Drinking by Semisynthetic Tetracycline Derivatives', Alcoholism, clinical and experimental research, 40: 2482-90、Agrawal, R. G., J. A. Owen, P. S. Levin, A. Hewetson, A. E. Berman, S. R. Franklin, R. J. Hogue, Y. Chen, C. Walz, B. D. Colvard, J. Nguyen, O. Velasquez, Y. Al-Hasan, Y. A. Blednov, A. K. Fowler, P. J. Syapin, and S. E. Bergeson. 2014. 'Bioinformatics analyses reveal age-specific neuroimmune modulation as a target for treatment of high ethanol drinking', Alcoholism, clinical and experimental research, 38: 428-37, Agrawal, RG, A. Hewetson, CM George, PJ Syapin, and SE Bergeson. 2011. 'Minocycline reduces ethanol drinking', Brain, behavior, and immunity, 25 Suppl 1: S165-9).

[0026] Despite these promising results, however, we found that the effects of these tetracyclines were mediated through central nervous system (CNS) function and not through any alteration of resident flora. The presented data provide a key to understanding why tetracyclines can be modified to remove their antibiotic properties and still remain useful for treating AUD. Other literature suggests that the effects of alcohol may be mediated, at least in part, by bacteria or their components (Blednov, YA, JM Benavidez, C. Geil, S. Perra, H. Morikawa, and RA Harris. 2011. 'Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice', Brain, behavior, and immunity, 25 Suppl 1: S92-S105); for a recent review, see (Montesinos, J., S. Alfonso-Loeches, and C. See Guerri, 2016. 'Impact of the Innate Immune Response in the Actions of Ethanol on the Central Nervous System', Alcoholism, clinical and experimental research, 40: 2260-70. However, despite this debate in the literature, the present invention demonstrates for the first time that the action of known CMMs does not require their antibiotic properties.

[0027] Thus, the inventors recognized for the first time that the mechanism of action of CMM for use in alcohol use disorder, pain, and other disorders involving underlying inflammatory processes does not require the general antibiotic properties of tetracyclines. Thus, the inventors tested several tetracyclines to determine the structural or functional components that contribute to their therapeutic efficacy against AUD. As shown in Table 1, the C6' hydrogen is required, at least in part, to mediate positive effects on AUD-related traits, but is not known to bind to the A site of bacterial ribosomes (Schedlbauer, A., T. Kaminishi, B. Ochoa-Lizarralde, N. Dhimole, S. Zhou, JP Lopez-Alonso, SR Connell, and P. Fucini. 2015. 'Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome', Antimicrobial agents and chemotherapy, 59: 2849-54).

[0028] Table 1. Of the seven tetracyclines tested for AUD traits (Syapin, PJ, JM Martinez, DC Curtis, PC Marquardt, CL Allison, JA Groot, C. Baby, YM Al-Hasan, I. Segura-Ulate, MJ Scheible, KT Nicholson, JL Redondo, DR Trotter, DS Edwards, and SE Bergeson. 2016. 'Effective Reduction in High Ethanol Drinking by Semisynthetic Tetracycline Derivatives', Alcoholism, clinical and experimental research, 40: 2482-90), doxycycline, minoxidil, and cyclosporine were the most effective. Only iclin and tigecycline were effective. The gray highlighting indicates that the R6' group is the only difference between effective and ineffective tetracyclines. This, along with our unpublished data shown in Figures 1 and 2, indicates that the structure of the molecule can be modified to eliminate the bacterial ribosome-binding component. Eliminating the antimicrobial properties may reduce side effects and avoid the development of drug resistance.

[0029] [Table 1]

[0030] Compounds of the present invention have the following formula, including pharmaceutically acceptable salts, prodrugs, biologically active metabolites, and tautomers thereof, and are set forth in Table 2.

[0031] [Table 2] JPEG2025169433000016.jpg215169 JPEG2025169433000017.jpg212166 JPEG2025169433000018.jpg213166 JPEG2025169433000019.jpg213170JPEG2025169433000020.jpg220166 JPEG2025169433000021.jpg221167JPEG2025169433000022.jpg177170

[0032] [ka] JPEG2025169433000024.jpg234170JPEG2025169433000025.jpg55170

[0033] Example 1 Reduction of ethanol consumption by chemically modified minocycline compounds. Alcohol use disorder (AUD) is the third leading cause of preventable disease in the United States, yet fewer than 20% of patients who undergo pharmacological intervention with currently approved drugs achieve remission. Furthermore, the antimicrobial drug minocycline has shown promise as a potential treatment for AUD due to its non-antimicrobial mechanism of action. However, the antimicrobial properties of minocycline pose a problem when used in non-infectious diseases. The inventors have successfully modified minocycline to remove its antimicrobial properties while maintaining its therapeutic potential for AUD.

[0034] Figure 1 shows the proposed mechanism by which ethanol consumption is reduced. Figure 2 shows the basic strategy for generating novel compounds for the treatment of alcohol use disorder (AUD) with low or no antimicrobial activity. Figure 3 shows that minocycline inhibits bacterial protein synthesis by binding to the 30S subunit of the bacterial ribosome.

[0035] Figures 4A-4C show the following: Figure 4A) Zone of inhibition assay shows that CMM lost its antibacterial activity against E. coli after 24 hours of cell culture. Figure 4B) Colony-forming unit assay shows that CMM lost its antibacterial activity against E. coli after 24 hours of cell culture. Figure 4C) Colony-forming unit assay shows that CMM lost its antifungal activity against Candida albicans after 48 hours of cell culture. MINO = minocycline, BeMAc = butyl ether monoacetate minocycline, DeDiAc = deaminodiacetyl minocycline, TRI = triacetyl minocycline.

[0036] Figures 5A-5C show the following: Figure 5A) CMM rapidly reduced ethanol consumption in the drinking-in-the-dark (DID) model of AUD. MINO, DeDiAc, and TRI reduced ethanol consumption by more than 50% via intraperitoneal injection (left). MINO, DeDiAc, TRI, and BeMAc slightly reduced ethanol consumption via gavage (right). Figure 5B) Gavage dose-response curve for DeDIAc in female mice. Figure 5C) Gavage dose-response curve for DeDIAc in male mice. MINO = minocycline, BeMAc = butyl ether monoacetate minocycline, DeDiAc = deaminodiacetyl minocycline, TRI = triacetyl minocycline. Compared to controls * p<.05.

[0037] These results are consistent with the hypothesis that CMM maintains its ability to reduce ethanol consumption even after its antimicrobial activity is lost. CMM is unlikely to contribute to antibiotic resistance or gastrointestinal side effects with long-term use. Deaminodiacetylminocycline significantly reduced ethanol consumption after both intraperitoneal and oral administration, and thus emerged as a promising lead compound.

[0038] Example 2 Targeted carbon chain length modifications alter the therapeutic properties of minocycline to reduce alcohol consumption. Alcohol use disorder (AUD) is a chronic, relapsing brain disorder characterized by compulsive alcohol use, inability to control alcohol consumption, and negative emotional states when not using alcohol. Minocycline has been shown to have anti-inflammatory effects, and numerous preclinical studies have suggested that its off-target properties may be useful in the treatment of many diseases. The inventors have previously shown that minocycline reduces high alcohol consumption.

[0039] However, minocycline exerts strong antibiotic effects on healthy gut microbiota, potentially limiting its therapeutic efficacy in non-infectious diseases. We have prepared, validated, and tested chemically modified minocycline (CMM) analogs with ether carbon chains. The modifications were C1-4, i.e., methyl-, ethyl-, propyl-, and butyl-ether additions to the -OH of the phenolic carbocycle.

[0040] The purity and molecular formula of the CMM analogs were initially identified by thin-layer chromatography (TLC), followed by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry (LC-MS). The antibacterial effects of the CMM analogs were evaluated in Escherichia coli cell cultures using the zone of inhibition (ZOI) and colony-forming unit (CFU) assays. Ethanol and water consumption were assessed in male and female C57BL / 6J mice using the drinking-in-the-dark (DID) and two-bottle choice (2BC) methods. A series of four CMM analogs was found to progressively lose antimicrobial activity and reduce ethanol consumption with increasing chain length. Butyl ether minocycline may be used to treat AUD.

[0041] Figure 6 shows the screening method for modified minocycline, and the purity and structure of the CMM analogs were confirmed using HPLC, NMR, and LC-MS. The loss of antibiotic activity was confirmed by ZOI and CFU assays, and alcohol consumption was tested in C67BL / 6J mice. Figure 7 shows the chemical modification sites of minocycline and, for reference, the structure of ribosome dissociation induced by minocycline. Schedlbauer, A., T. Kaminishi, B. Ochoa-Lizarralde, N. Dhimole, S. Zhou, JP Lopez-Alonso, SR Connell, and P. Fucini. 2015. 'Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome', Antimicrobial agents and chemotherapy, 59: 2849-54. Figures 8A-8D show the effect of butyl ether minocycline on the loss of antibiotic activity compared to minocycline.

[0042] Table 3 shows the drinking results, showing that butyl ether minocycline (100 mg / kg, ip) significantly reduced alcohol consumption.

[0043] [Table 3]

[0044] This example demonstrates that chemical modification of minocycline by adding a carbon chain to the phenolic -OH group prevented bacterial ribosome binding and successfully reduced the associated antibiotic activity. Of the four compounds tested, all compounds showed a reduction in alcohol consumption, with butyl ether minocycline showing the greatest reduction in alcohol consumption.

[0045] In this example, the carbon chain length correlated with reduced alcohol consumption, suggesting that the addition of the chemical may not only be related to the loss of antimicrobial activity, but may also have an anti-inflammatory or other unknown positive mechanism of action (relative to alcohol consumption).

[0046] Example 4 Chemically modified minocycline as a novel treatment for rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory joint disorder that affects approximately 1% of the population. There is no cure, but several medications can promote remission, all of which have significant side effects. Minocycline is known for its anti-inflammatory, immunomodulatory, and chondroprotective properties, making it an excellent choice for the treatment of RA. It is believed to play an important role in the treatment of RA. However, in the long term, negative side effects on the gastrointestinal microbiota are a hindrance. We have created a new chemically modified minocycline (CMM) analogue that eliminates antibiotic activity. The CMM analogue was tested as a therapeutic agent in a mouse RA model and was found to significantly alleviate symptoms.

[0047] FIG. 9 shows a flow chart of the mechanism of action of minocycline on autophagy, inflammation, and angiogenesis.

[0048] FIG. 10 shows the intracellular signaling pathways of COX2, BNIP3, acidic vacuoles, and GFP-LC3 puncta.

[0049] 11A-11C show that the mass, purity, and structure of deaminodiacetylminocycline were confirmed using LC-MS (FIG. 11B) and HPLC (FIG. 11C), and further confirmed by NMR (not shown).

[0050] Figures 12A-12D show zone of inhibition and colony forming unit assays demonstrating the loss of antibiotic activity for deaminodiacetylminocycline in Figures 12A and 12C compared to minocycline in Figures 12B and 12D, respectively.

[0051] Figure 13 shows the basic experimental setup for rheumatoid arthritis. RA models were induced in male and female DBA / 1J mice with five monoclonal antibodies against type II collagen, followed by E. coli lipopolysaccharide adjuvant (CIA, Chondrex, Redmond, WA). Each group was treated with saline or 75 mg / kg CMM i.p. RA was quantified using caliper measurements, infrared heat signature, arthritis scoring, a hot plate (42°C), and the Von Frey filament assay.

[0052] Figure 14 shows that the CMM reduced paw swelling by approximately 50% as measured by caliper (mm). Dates are shown as a percentage increase over baseline measurements.

[0053] Figure 15 shows that CMM reduced pressure noxiousness in RA mice, as measured by the von Frey filament assay. In induced mice treated with saline alone, an increase in sensitivity of approximately 40% was observed after 21 days compared to baseline. This increase in sensitivity was minimal in the MMC-injected group.

[0054] Figure 16A shows that injection of Chondrex's antibody cocktail resulted in severe inflammation of the interphalangeal, metatarsal, and ankle joints of the forepaws, resulting in a loss of anatomical definition. MCC significantly reduced the arthritis score by approximately 70% compared to the induction group shown in Figure 16B, significantly reducing the severity of RA. Improvement was evident in both male and female mice.

[0055] Injection of a Chondrex 5-monoclonal antibody cocktail followed by LPS adjuvant induced severe RA symptoms in a DBA / 1J mouse model. CMM (75 mg / kg, i.p., every other day) reduced the severity of RA symptoms. Because CMM was shown to have no antibiotic activity, it may provide a long-term solution for RA patients without affecting the gut microbiota.

[0056] Examples of salts that may be used with the compounds of the present invention include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, besylate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharinate, sucralose ... These include carboxylate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and / or pamoate salts.

[0057] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0058] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are encompassed by the claims.

[0059] All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0060] In the claims and / or description, when used in conjunction with the word "comprises," the word "a The use of "" or "an" may indicate "one" but does not exclude "one or more," "at least one," or "one or more." The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists among the subject matter studied.

[0061] As used in this specification and claims, "comprising" (and all forms of "comprising", such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") The term "form" is inclusive or open-ended and does not include additional, unlisted forms. In any of the embodiments of the compositions and methods provided herein, "comprising" may be used interchangeably with "consisting essentially of" or "including." This specification may be replaced with "consisting of" or "consisting of." When used in this document, the phrase "consisting essentially of" is a specified integer or step, as well as a feature or It is required that the functionality is not substantially affected. As used herein, the term "consisting" is used to indicate the presence of only a stated whole (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of wholes (e.g., feature, element, characteristic, property, method / process step, or limitation).

[0062] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" refers to at least one of A, B, C, AB, AC, BC, or ABC, and, if order is important in a particular context, BA, CA, CB, CBA, BCA. , ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms are expressly included, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is generally no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0063] As used herein, without limitation, approximation words such as "about," "substantial," or "substantially" refer to a condition that, when so modified, is understood not necessarily to be absolute or complete, but that would be considered by one of ordinary skill in the art to be close enough to warrant specifying the condition as existing. The degree to which the description can vary will depend on how large a change can occur and still allow one of ordinary skill in the art to recognize the modified feature as still possessing the desired characteristics and capabilities of the unmodified feature. Generally, but subject to the preceding discussion, numerical values ​​herein modified by approximation words such as "about" can vary by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15% from the stated value.

[0064] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods, and in the steps or sequence of steps of the methods, described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0065] In order to assist the Patent Office, and any reader of any patent issued based on this application, in interpreting the claims appended hereto, Applicants wish to note that unless the words "means for" or "step for" are expressly used in a particular claim, Applicants do not intend any of the appended claims to be subject to 35 U.S.C. 112, paragraph 6, 112, paragraph (f), or the equivalent, as they exist on the filing date of this application.

[0066] For each claim, each dependent claim may depend on each independent claim and each preceding dependent claim, so long as the preceding claim has the appropriate antecedent for that claim term or element.

[0067] References Agrawal, RG, A. Hewetson, CM George, PJ Syapin, and SE Bergeson. 2011. 'Minocycline reduces ethanol drinking', Brain, behavior, and immunity, 25 Suppl 1: S165-9. Agrawal, R. G., J. A. Owen, P. S. Levin, A. Hewetson, A. E. Berman, S. R. Franklin, R. J. Hogue, Y. Chen, C. Walz, B. D. Colvard, J. Nguyen, O. Velasquez, Y. Al-Hasan, Y. A. Blednov, A. K. Fowler, P. J. Syapin, and S. E. Bergeson. 2014. 'Bioinformatics analyses reveal age-specific neuroimmune modulation as a target for treatment of high ethanol drinking', Alcoholism, clinical and experimental research, 38: 428-37. Bergeson, S. E., H. Blanton, J. M. Martinez, D. C. Curtis, C. Sherfey, B. Seegmiller, P. C. Marquardt, J. A. Groot, C. L. Allison, C. Bezboruah, and J. Guindon. 2016. 'Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences', Alcoholism, clinical and experimental research, 40: 2506-15. Bergeson, S. E., M. A. Nipper, J. Jensen, M. L. Helms, and D. A. Finn. 2016. 'Tigecycline Reduces Ethanol Intake in Dependent and Nondependent Male and Female C57BL / 6J Mice', Alcoholism, clinical and experimental research, 40: 2491-98. Blednov, Y. A., J. M. Benavidez, C. Geil, S. Perra, H. Morikawa, and R. A. Harris. 2011. 'Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice', Brain, behavior, and immunity, 25 Suppl 1: S92-S105. Martinez, J. M., J. A. Groot, D. C. Curtis, C. L. Allison, P. C. Marquardt, A. N. Holmes, D. S. Edwards, D. R. Trotter, P. J. Syapin, D. A. Finn, and S. E. Bergeson. 2016. 'Effective Reduction of Acute Ethanol Withdrawal by the Tetracycline Derivative, Tigecycline, in Female and Male DBA / 2J Mice', Alcoholism, clinical and experimental research, 40: 2499-505. Montesinos, J., S. Alfonso-Loeches, and C. Guerri. 2016. 'Impact of the Innate Immune Response in the Actions of Ethanol on the Central Nervous System', Alcoholism, clinical and experimental research, 40: 2260-70. Rhodes, J. S., K. Best, J. K. Belknap, D. A. Finn, and J. C. Crabbe. 2005. 'Evaluation of a simple model of ethanol drinking to intoxication in C57BL / 6J mice', Physiology & Behavior, 84: 53-63. Schedlbauer, A., T. Kaminishi, B. Ochoa-Lizarralde, N. Dhimole, S. Zhou, J. P. Lopez-Alonso, S. R. Connell, and P. Fucini. 2015. 'Structural characterization of an alternative mode of tigecycline binding to the bacterial ribosome', Antimicrobial agents and chemotherapy, 59: 2849-54. Syapin, P. J., J. M. Martinez, D. C. Curtis, P. C. Marquardt, C. L. Allison, J. A. Groot, C. Baby, Y. M. Al-Hasan, I. Segura-Ulate, M. J. Scheible, K. T. Nicholson, J. L. Redondo, D. R. Trotter, D. S. Edwards, and S. E. Bergeson. 2016. 'Effective Reduction in High Ethanol Drinking by Semisynthetic Tetracycline Derivatives', Alcoholism, clinical and experimental research, 40: 2482-90.

Claims

1. A tetracycline molecule having substantially no antibacterial activity or substantially no antifungal activity, selected from any of the following compounds, and pharmaceutically acceptable salts thereof: 【Chemistry 1】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】

2. 10. The molecule of claim 1, which has reduced or substantially no antibacterial activity, or has reduced or substantially no antifungal activity, or both.

3. 10. The molecule of claim 1, wherein the substance use disorder (SUD) is alcohol use disorder (AUD), and wherein the molecule is provided in an amount that inhibits alcohol use disorder (AUD).

4. 10. The molecule of claim 1, wherein the molecule is placed in a pharmaceutically acceptable buffer, excipient, filler, or carrier, or formulated into a composition for oral, enteral, intramuscular, parenteral, intravenous, or intraperitoneal administration.

5. A pharmaceutical composition comprising the tetracycline molecule of claim 1, and pharmaceutically acceptable salts thereof.

6. Use of one or more tetracycline molecules described in claim 1, having reduced or no antimicrobial activity, in an amount effective to improve or eliminate substance use disorder (SUD), in the manufacture of a medicament provided to a subject for treating SUD.

7. 7. The use of claim 6, wherein the molecule has reduced or substantially no antibacterial activity, or has reduced or substantially no antifungal activity, or both.

8. 7. The use of claim 6, wherein the molecule is provided in a pharmaceutically acceptable buffer, excipient, filler, or carrier, or is formulated into a composition for oral, enteral, intramuscular, parenteral, intravenous, or intraperitoneal administration.

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