Modified forms of ambroxol for therapeutic use

Abstract

The present invention relates to multiply deuterated analog forms of ambroxol and related compounds (including bromhexine and ambroxol salts, ambroxol hydrochloride), compositions comprising same, and methods of preventing and / or treating various diseases and medical conditions involving administration of multiply deuterated analogs of ambroxol and related compounds.The present invention relates to multiply deuterated analog forms of ambroxol and related compounds (including bromhexine, pharma- ceutically acceptable salts thereof, and pharma-ceutically acceptable salts of ambroxol, such as ambroxol hydrochloride), compositions comprising same, and methods of preventing and / or treating various diseases and medical conditions involving administration of multiply deuterated analogs of ambroxol and related compounds.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to modified forms of ambroxol, ambroxol hydrochloride and / or bromhexine suitable for use in a variety of therapeutic applications. [Background technology]

[0002] Discussion of the Prior Art Ambroxol, ambroxol hydrochloride and the structurally related "parent compound", bromhexine, are mucolytic agents that have been available in many countries since the late 1970s for use in the treatment of acute and chronic respiratory diseases and conditions associated with the production of excessive and / or highly viscous mucus.

[0003] By inducing lysosomal exocytosis via pH-dependent calcium release from acidic calcium stores (Fois G et al., Cell Calcium 58(6):628-637, 2015), ambroxol is thought to act to promote mucus clearance, for example by breaking down sputum and stimulating surfactant production by type II pneumocytes (Seiffert C et al., Toxicol Appl Pharmacol 203(1):27-35, 2005), thereby reducing mucus adhesion to the airway walls. In addition, ambroxol has been found to potently inhibit neuronal sodium channels, allowing for rapid analgesia, used for acute sore throats, especially when administered in the form of lozenges (de Mey C et al., Arzneimittel-Forschung 28(5a):889-898, 1978).

[0004] Recently, there has been widespread interest in repurposing ambroxol for many other medical applications. For example, it has been reported that ambroxol can act as a "molecular chaperone" for the lysosomal enzyme β-glucocerebrosidase (GCase, UniProtKB Entry P04062), thereby increasing the activity of this enzyme. This may mean that ambroxol may be suitable for the treatment of Gaucher disease, the most common lysosomal storage disease, which is caused by a deficiency of GCase (Maegawa GHB et al., J Biol Chem 284(35):23502-23516, 2009). Similarly, this ability to increase the activity of GCase may be beneficial for the treatment of Parkinson's disease (PD) in individuals with loss-of-function mutations in the glucocerebrosidase gene, GBA1 (McNeill A et al., Brain, 137(5):1481-1495, 2014). A recent report showed that daily administration of ambroxol was able to increase GCase activity in the brain of healthy non-human primates (Migdalska-Richards A et al., Synapse 71(7):e21967,2017). Therefore, ambroxol continues to be the subject of great research interest and effort.In the course of research to identify and develop new therapeutic methods and compositions based on ambroxol, the present inventors have designed novel modified forms of ambroxol (i.e., ambroxol analogues).It is believed that these modified compounds may provide one or more advantages over one or more of ambroxol, ambroxol hydrochloride and bromhexine, such as improved stability leading to an increased half-life and duration of action in the body. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Maegawa GHB et al. J Biol Chem (2009) 284(35):23502~23516 [Non-Patent Document 2] McNeill A et al., Brain, (2014) 137(5):1481-1495 Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention This Summary is provided to introduce in a simplified form a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the following Detailed Description, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0007] The present invention relates to multiply deuterated analog forms of ambroxol and related compounds (including bromhexine, pharma- ceutically acceptable salts thereof, and pharma- ceutically acceptable salts of ambroxol, such as ambroxol hydrochloride), compositions comprising same, and methods of preventing and / or treating various diseases and medical conditions involving administration of multiply deuterated analogs of ambroxol and related compounds.

[0008] More specifically, the present invention relates to, inter alia, multiply deuterated compounds according to formula I: [ka] [During the ceremony, R a is H, hydroxyl (OH), lower alkyl (e.g., C groups such as CH3 and CH2CH3), 1-3 alkyl), and lower alcohols (e.g., C 1-3 alcohol), optionally with one or more H atoms of any of the foregoing groups replaced with deuterium (D); R b is H, deuterium (D) and [ka] (In the formula, R e , R f and R g are independently selected from H and D; R c and R d is independently selected from H and D; R 1 ~R 14 each is independently selected from H and D; The polydeuterated compound contains at least two deuterium (D) atoms, provided that the compound is also bis-deuterated ambroxol A, shown below, [D 11 ]-Neither ambroxol B]; or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0009] Bis-deuterated Ambroxol A (ZW-001): [ka]

[0010] [D 11 ]-Ambroxol B: [ka]

[0011] It is contemplated that any or all H atoms in the compound of formula I may be replaced with deuterium atoms. In some embodiments, the compound comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 deuterium (D) atoms. Preferably, the compound comprises at least 3 deuterium (D) atoms.

[0012] In some embodiments, deuterium atoms are provided only on the ring structure of the compound of formula I. For example, the dibromophenylaniline ring may contain up to two deuterium (D) atoms, and the optionally substituted cyclohexyl ring may contain up to ten deuterium (D) atoms. Optionally, the amine substituent present on the dibromophenylaniline ring may contain one or two deuterium (D) atoms. That is, R c and R d At least one of, and preferably both of, is D.

[0013] In some other embodiments, deuterium atoms are provided on one or both of the ring structures of the compounds of formula I, as well as on the linking group between those ring structures. When deuterated, the linking group may contain one or two deuterium (D) atoms.

[0014] In some embodiments, R 11 and R 12 At least one, and preferably both, of the formula (I) are deuterium (D).

[0015] In some embodiments, one effect of multiple deuteration of a compound is resistance to metabolism (compared to a corresponding compound lacking deuteration), such as metabolism by cleavage (e.g., via oxidation) of the carbon-nitrogen covalent bond of the linking group between the ring structures of a compound according to Formula I.

[0016] In some particular embodiments, the compound is 1 ~R 10 Only in each of ;R 1 ~R 10 , R 11 and R 12 Only in each of ;R 1 ~R 10 , R 13 and R 14 or R 11 ~R 14 while in other particular embodiments, the compound is deuterated only at each of R 1 ~R 14Each of these is deuterated.

[0017] In some embodiments, the compound is a multiply deuterated analog of ambroxol or a multiply deuterated analog of bromhexine.

[0018] In some embodiments, the compound is a pharma- ceutically acceptable salt, preferably the hydrochloride salt, of a multiply deuterated analog of ambroxol or bromhexine.

[0019] The present invention also provides pharmaceutical compositions comprising a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof), as well as methods of preventing and / or treating various diseases and medical conditions in a subject comprising administering a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof).

[0020] More specifically, the present invention provides methods for preventing and / or treating diseases or medical conditions selected from the group consisting of respiratory diseases and conditions (e.g., bronchopulmonary diseases, and in particular diseases associated with the production of excessive and / or highly viscous mucus), including acute sore throat, pain associated with lysosomal storage diseases (LDS) such as Gaucher disease, neurological diseases and conditions involving dysfunction of autophagy (e.g., PD and other aging-related diseases) (Dockrill P., Science Alert, February, 2020).

[0021] Additionally, the present invention provides methods of treating, alleviating and / or stabilizing symptoms associated with neurological diseases and conditions, such as Parkinsonism (including PD and dementia with Lewy bodies), Alzheimer's disease (AD) and / or Pick's disease.

[0022] Additionally, the present invention provides a method for increasing the life expectancy of a subject. Specifically, the compounds of formula I can be used in (a) a method for treating, inhibiting, or reducing aging in a subject, (b) a method for treating, inhibiting, or reducing an age-related symptom or disease in a subject, and / or (c) a method for increasing the healthspan, lifespan, and / or mental acuity of a subject.

[0023] In preferred embodiments, the subject is a mammal, and in more preferred embodiments, the mammal is a human, a domestic animal (eg, a dog, cat, horse) or a livestock animal (eg, a cow or pig). [Brief description of the drawings]

[0024] The objects and features of the present invention can be better understood with reference to the following detailed description and the accompanying drawings.

[0025] [Figure 1-1] FIG. 1 provides structures of representative compounds of the present invention (compounds 1 to 14). [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0026] [Diagram 2] FIG. 2 provides the structures of ZW-001 (1) (bis-deuterated ambroxol A), ZW-002 (2) (also referred to herein as "compound 1"), ZW-003 (3) (also referred to herein as "compound 3") and ambroxol (4).

[0027] [Diagram 3] Figure 3 provides animal data showing that ambroxol can extend life span in a mouse animal model. Group 1 represents control animals that did not receive ambroxol; Group 2 represents animals that received 50 mg / kg (body weight) of ambroxol daily as a chow supplement starting at 2 months of age.

[0028] [Figure 4] Figure 4 shows results from mice treated with ambroxol at 7 months of age and tested for novel place recognition, a cognitive test to measure short-term working memory that involves the recognition of a familiar object found in an unfamiliar place (see Magen et al., Eur J Neurosci 35:870-882, 2012; Magen and Chesselet, J Parkinson's Disease 1:217-227, 2011). Discrimination index (DI) = (tnovel-tfamiliar) / (tnovel+tfamiliar) was used to assess the time spent exploring near the object in the novel place ("tnovel") compared to the overall total exploration time. Discrimination index scores greater than 0 are considered to indicate good place recognition memory.

[0029] [Figure 5-1] Figure 5 shows the effect of ambroxol on basal macroautophagy in mouse cells. Mouse fibroblasts (NIH3T3 cells) in culture expressing the tandem reporter mCherry-GFP-LC3 were exposed to the indicated concentrations of ambroxol in complete medium for 24 h. Panel A. Schematic of the analyzed autophagic compartments. Panels B-D. Number of autophagic vacuoles (AV) (panel B); autophagosomes (APG) (panel C); and autolysosomes (AUT) (panel D). All values ​​are mean + sem and quantification was performed on at least 2,500 cells per condition in three different experiments using high content microscopy. Differences from untreated (0 μM ambroxol) are significant at *p<0.05, **p<0.01 and ***p<0.001. [Figure 5-2] Same as above.

[0030] [Figure 6-1]6 shows the effect of deuterated ambroxol on human iPSC-derived neural cells. The compound designated ZW-001 in the figure is the compound otherwise referred to herein as bis-deuterated ambroxol A, and the compound designated ZW-002 in the figure is the compound otherwise referred to herein as compound 1. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0031] [Figure 7] 7 shows the effect of deuterated ambroxol on lysosomes, autophagosomes and TFEB gene expression. The compound designated ZW-001 in the figure is the compound otherwise referred to herein as bis-deuterated ambroxol A, and the compound designated ZW-002 in the figure is the compound otherwise referred to herein as Compound 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description I. Definition The following definitions are provided for specific terms used in the following specification description.

[0033] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0034] The present invention can "comprise" (open ended) or "consist essentially of" the components of the present invention as well as other components or elements described herein. As used herein, "comprise" means the recited elements, or their structural or functional equivalents, and any other element or elements not recited. The terms "having" and "comprise" should also be construed as open ended unless the context indicates otherwise. As used herein, "consist essentially of" means that the invention may include components in addition to those recited in the claims, but only if the additional components do not materially alter the basic and novel characteristics of the invention as recited in the claims.

[0035] As used herein, a "subject" is a vertebrate, preferably a mammal, more preferably a human, even more preferably a domestic animal such as a pet, or livestock. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sports animals, and pets. In other preferred embodiments, the "subject" is a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, monkey (e.g., monkey or ape), monkey (e.g., marmoset, baboon), or ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In other embodiments, non-human mammals, particularly mammals that are conventionally used as models to demonstrate therapeutic effects in humans (e.g., mice, primates, pigs, dogs, or rabbits) can be used. In preferred embodiments, an "individual" or "patient" (as in the subject of treatment) refers to mammals, particularly non-human primates, such as apes and monkeys, and most particularly humans.

[0036] As understood herein, an "effective amount" of a pharmaceutical composition of the present invention refers to an amount of the composition suitable for eliciting a therapeutically beneficial response in a subject, such as, for example, promoting mucus clearance associated with respiratory diseases and conditions, relieving pain associated with acute sore throat, providing amelioration of symptoms associated with lysosomal storage diseases (LSDs) and neurological diseases and conditions, or extending and / or increasing and / or improving healthspan, lifespan and / or mental acuity, such as increasing survival and / or healthy aging, and / or reducing pathological conditions and / or age-related diseases in a subject.

[0037] The term "dose" or "dosage" as used herein refers to physically discrete units suitable for administration to a subject, each dose containing a predetermined quantity of active pharmaceutical ingredient calculated to produce the desired response.

[0038] The term "about" or "approximately" means within an acceptable range of a particular value as determined by one of skill in the art, the acceptable range depending in part on the method of measuring or determining the value, e.g., the limitations of the measurement system. For example, "about" can mean within 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Unless otherwise indicated, the term "about" means within an acceptable range of error for a particular value, e.g., ±1-20%, preferably ±1-10%, and more preferably ±1-5%. In still further embodiments, "about" should be understood to mean + / - 5%.

[0039] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0040] All percentages and ratios used herein are by weight of the total composition unless otherwise specified herein. All temperatures are in degrees Celsius unless otherwise specified. All measurements made are at 25°C and normal pressure unless otherwise specified.

[0041] All ranges recited herein include endpoints, including those recited as a range "between" two values. Terms such as "about," "generally," "substantially," "approximately," etc., should be interpreted as not being absolute, but as modifying terms or values ​​that are not applicable in the prior art. As will be understood by those skilled in the art, such terms are defined by the context and the terms they modify. This includes at least the degree of expected experimental error, technique error, and instrument error for a given technique used to measure a value.

[0042] As used herein, the term "and / or" when used in a list of two or more items means that any one of the recited characteristics may be present, or any combination of two or more of the recited features may be present. For example, if a composition of the invention is described as containing features A, B, and / or C, the composition may contain feature A only; B only; C only; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0043] As used herein, the term "lower alkyl" includes straight chain, branched, and cyclic alkyl groups having 1 to 8 carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, and the like).

[0044] As used herein, the term "lower alcohol" includes alcohol groups that contain straight-chain, branched, or cyclic alkyl groups having 1 to 8 carbon atoms and one or more hydroxyl (OH) groups (e.g., methanol, ethanol, propanol, etc.).

[0045] The term "pharmaceutically acceptable salt" as used herein refers to a salt that retains the desired biological activity of the compound of formula I, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compound of formula I may be prepared from inorganic or organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids can be selected from the organic acids of aliphatic, alicyclic, aromatic, heterocyclic, carboxylic and sulfonic classes, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkylsulfonic acid and arylsulfonic acid. Further information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co, Easton PA 1995.

[0046] The term "solvate" refers to any form of a compound of formula I resulting from solvation with a suitable solvent. Such forms may be, for example, crystalline solvates or complexes that may form between a solvent and a dissolved compound.

[0047] The term "prodrug" refers to a compound that is converted to a compound of formula I in a biological system, usually by metabolic means (e.g., hydrolysis, reduction or oxidation). For example, an ester prodrug of a compound of formula I containing a hydroxyl group may be convertible to a compound of formula I by hydrolysis in vivo. Suitable esters of a compound of formula I containing a hydroxyl group may be, for example, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-p-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. As another example, ester prodrugs of compounds of formula I containing a carboxy group may be converted to compounds of formula I by in vivo hydrolysis. Examples of ester prodrugs include those described in Leinweber FJ, Drug Metab Rev 18:379-439 (1987). Similarly, acyl prodrugs of compounds of formula I containing an amino group may be converted to compounds of formula I by in vivo hydrolysis. Examples of prodrugs of these and other functional groups, including amines, are described in Prodrugs: challenges and rewards, Valentino J Stella (ed), Springer, 2007.

[0048] A "derivative" compound, as that term is used herein, refers to a second compound derived from a first compound, such as a brominated version of a non-brominated parent compound. For example, ambroxol is a derivative of the parent compound bromhexine.

[0049] As used herein, specific positions of the compounds of formula I (e.g., R 1 , R 2The terms "deuterium," "D," and "deuterated," as used to describe hydrogen atoms located at (e.g., at) 100° C., 100° C., 15 ...

[0050] As used herein, "lifespan" refers to the time to death. As used herein, "healthy lifespan" or "healthy aging" refers to the period of time that can be lived without (or optimally without) serious disease. As used herein, "mental acuity" is a measure of a subject's cognitive abilities, such as concentration, attention span, and acuity.

[0051] As used herein, "nutrient sensing" is the ability of a cell to sense and respond to fluctuations in nutrient levels, as described in Efeyan et al., "Nutrient Sensing Mechanisms and Pathways," Nature 517:302-310 (2015), which is incorporated herein by reference in its entirety.

[0052] As used herein, "treating, inhibiting, and / or alleviating aging, age-related symptoms, and / or age-related diseases" and the like means reducing the risk of occurrence, delaying the onset, slowing the progression, and / or reducing the severity and / or manifestations of a symptom of aging and / or a degenerative disorder, including, but not limited to, preventing the occurrence, development, or progression of a symptom of aging and / or a degenerative disorder.

[0053] As used herein, the term "pharmaceutically acceptable carrier" means any carrier, diluent or excipient that is compatible with the other ingredients of the composition and not harmful to the intended subject to which the composition is administered.

[0054] Compounds of the Invention The compounds of the present invention are multiply deuterated analog forms of ambroxol and related compounds defined by Formula I, including bromhexine and ambroxol hydrochloride. Table 1 shows the structures of ambroxol (also known by the chemical name trans-4-((2-amino-3,5-dibromobenzyl)amino)cyclohexanol), bromhexine (also known by the chemical name 2-amino-3,5-dibromo-N-cyclohexyl-N-methylbenzenemethanamine), and ambroxol hydrochloride. Table 1: Structures of ambroxol, bromhexine and ambroxol hydrochloride [Table 1]

[0055] In some embodiments, the compound is selected from the multiply deuterated analogs of ambroxol and bromhexine shown in FIG. 1. One of the effects of the multiply deuterated compounds of these compounds (particularly compounds 3-5) is believed to be resistance (compared to the corresponding compounds lacking deuteration) to metabolism, such as metabolism by cleavage (e.g., via oxidation) of the carbon-nitrogen covalent bond of the linking group between the ring structures of the compounds according to formula I. In other words, one of the effects of the multiply deuterated compounds of these compounds (particularly compounds 3-5) is believed to be higher stability. Thus, in some preferred embodiments, the compound is selected from the multiply deuterated ambroxol analog compounds 3-5 shown in FIG. 1.

[0056] The compounds of the present invention can be used in methods of preventing and / or treating a variety of diseases and medical conditions, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, and can also be used in methods of increasing the life expectancy of a subject. In such methods, compounds of the present invention that exhibit greater stability (e.g., compared to corresponding compounds lacking deuteration) can, for example, exhibit one or more advantageous pharmacokinetic properties.

[0057] Preparation of the Compounds of the Invention The compounds of the present invention can be prepared by methods known to those skilled in the art of organic synthesis. For example, US Patent Application Publication No. 2004 / 0242700, the entirety of which is incorporated herein by reference, provides a synthetic protocol for the preparation of ambroxol. This protocol can be easily adapted to allow the synthesis of multiply deuterated analog forms of ambroxol and related compounds. In addition, a protocol for the synthesis of deuterium (D)-labeled ambroxol and related compounds is disclosed in Latli B et al., J Label Compd Radiopharm 53:15-23, 2010, the entirety of which is incorporated herein by reference, which can also be easily adapted to the preparation of compounds of formula I. For example, "Scheme 4" of Latli et al., 2010, can be adapted to the preparation of compounds of formula I that further include deuterium (D) on the dibromophenylalanine ring by replacing 2-amino-3,5-dibromobenzaldehyde with a deuterated analog of that compound. In addition, the compounds of the present invention can be prepared by methods such as those exemplified herein.

[0058] Salts of the Compounds of the Invention For compounds that typically contain acidic or basic groups (such as carboxyl or amino groups), such groups are not necessarily in free acid or free base form.When referring to the compounds of the present invention, the reference is intended to include the salt forms of the compounds.Therefore, within the scope of the present invention are salts of the compounds of formula I, particularly the salts of the multi-deuterated analogs of ambroxol and bromhexine.Preferred salts are pharmaceutically acceptable salts.

[0059] The term "salt" includes addition salts of free acids or free bases. The term "pharmaceutically acceptable salts" refers to salts that have a toxicity profile within a range that makes them useful in pharmaceutical applications. However, salts that are not pharmaceutically acceptable may have properties such as high crystallinity, which have utility in the practice of the present invention, for example, utility in the process of synthesizing, purifying, or formulating therapeutic compounds.

[0060] Suitable pharma- ceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Examples of inorganic acids include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Suitable organic acids can be selected from the aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, 4-hydroxybenzoic ... Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.All of these acid addition salts can be prepared from the compounds of formula I, for example, by reacting a suitable acid with a particular compound.

[0061] Suitable pharmaceutically acceptable base addition salts of the compound of formula I include, for example, metal salts, including alkali metal salts, alkaline earth metal salts and transition metal salts, for example, calcium salt, magnesium salt, potassium salt, sodium salt and zinc salt.Pharmaceutically acceptable base addition salts also include organic salts made from basic amines, such as N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.All of these base addition salts can be prepared from the compound of formula I, for example, by reacting a suitable base with a particular compound.

[0062] Methods of Using the Compounds of the Invention As mentioned above, the compounds of the present invention can be used in methods for preventing and / or treating various diseases and medical conditions, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, and can also be used in methods for increasing the life expectancy of a subject.For example, the compounds of formula I can be used in (a) methods for treating, inhibiting, or reducing aging in a subject, (b) methods for treating, inhibiting, or reducing age-related symptoms or age-related diseases in a subject, and / or (c) methods for increasing the healthspan, lifespan, and / or mental acuity of a subject.

[0063] In some other embodiments, the present invention more specifically relates to a method for preventing and / or treating a disease or medical condition in a subject, the disease or condition being selected from the group consisting of respiratory diseases and conditions (e.g., bronchopulmonary diseases, and in particular diseases associated with the production of excessive and / or highly viscous mucus), including acute sore throat, lysosomal storage diseases (LDS), such as Gaucher's disease, and pain associated with neurological diseases and conditions (e.g., Parkinson's disease (PD) and other aging-related diseases involving autophagy dysfunction), the method comprising administering to the subject an effective amount of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof). The present invention also relates to the use of a compound of formula I (or a pharma-ceutically acceptable salt, solvate or prodrug thereof) as a drug for preventing and / or treating a disease or medical condition selected from respiratory diseases and conditions, lysosomal storage diseases (LDS), and neurological diseases and conditions. Furthermore, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) in combination with a pharma- ceutically acceptable carrier for the treatment of a disease or medical condition selected from respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions.

[0064] In some other embodiments, the present invention relates to a method for extending the healthspan, lifespan and / or mental acuity of a subject, comprising administering to the subject an effective amount of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof).The present invention also relates to the use of a compound of formula I (or a pharma-ceutically acceptable salt, solvate or prodrug thereof) as a drug for extending life expectancy and / or alleviating aging or age-related diseases or symptoms.Furthermore, the present invention relates to a pharmaceutical composition for treatment extending life expectancy, comprising a therapeutically effective amount of a compound of formula I (or a pharma-ceutically acceptable salt, solvate or prodrug thereof) together with a pharma-ceutically acceptable carrier.

[0065] In some further embodiments, the present invention relates to long-term methods of inducing an increase and / or improvement in healthspan, lifespan and / or mental acuity in a subject, said methods comprising administering a therapeutically effective amount of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof), wherein the administration of the compound is for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In such methods, the subject's lifespan, healthspan, mental acuity and / or healthy aging is prolonged, improved or accelerated, preferably by up to about 10%, 20%, 30%, 40%, 50%, 60% or 70%, as compared to an untreated control subject.

[0066] Examples of age-related diseases and conditions include, but are not limited to, cardiovascular disease, metabolic syndrome, bone loss disorders, neurodegenerative diseases, pre-diabetes, diabetes, obesity, osteoporosis, coronary artery disease, cerebrovascular disease, heart attack, stroke, peripheral artery disease, aortic valve disease, stroke, mild cognitive impairment, pre-dementia, dementia, macular degeneration, and cataracts, thinning hair, graying hair, loss of motor function, reduced stamina, fatigue, increased susceptibility to infection, metabolic changes, biochemical changes, cardiac hypertrophy, heart failure, myocardial infarction, ischemia-reperfusion injury, inflammatory diseases, pro-inflammatory conditions, arthropathy, autoimmune diseases, and / or Alzheimer's Disease (AD).

[0067] In the methods of the invention, the compound may be administered to the subject at a daily dosage of about 20-500 mg / day, 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, or 250 mg-1500 mg / day. It may be necessary in some cases to use doses outside these ranges, and therefore higher or lower doses are also contemplated. The daily dosage may be divided, such as by dividing it into 2-4 equal doses per daily dose. Furthermore, for chronic administration, such as may be required in methods of inducing an increase and / or improvement in healthspan, lifespan and / or mental acuity in a subject, the compound is preferably administered at a dose of approximately 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 1200 mg / day, 1300 mg / day, 1400 mg / day, 1500 mg / day, 1600 mg / day, 1700 mg / day, 1800 mg / day, 1900 mg / day, 2000 mg / day, 2100 mg / day, 2200 mg / day, 2300 mg / day, 2400 mg / day, 2500 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, g / day, 1250 mg / day, 1300 mg / day, 1350 mg / day, or at doses of 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, 250 mg-1000 mg / day, or 1000 mg-1500 mg / day or less than 1000 mg / day, or at doses of approximately 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, and / or 4-12 mg / kg / day.

[0068] In some preferred embodiments, the methods of the invention may be particularly applicable to subjects including humans suffering from pain associated with acute sore throat, humans suffering from bronchopulmonary diseases associated with excessive and / or highly viscous mucus production, lysosomal storage diseases (LSDs) such as Gaucher disease, and humans suffering from neurological diseases and conditions such as Parkinsonism (including PD and dementia with Lewy bodies), AD, and / or frontotemporal dementia.

[0069] Parkinson's disease and other diseases with dysfunctional autophagy The ambroxol (and ambroxol hydrochloride and bromhexine) and multiply deuterated analogs of ambroxol of the present invention may be particularly applicable to the treatment of age-related diseases involving dysfunction of autophagy and chronic inflammation, such as Alzheimer's disease (AD) and Parkinson's disease (PD), and disease states characterized by chronic pain, including fibromyalgia.

[0070] As mentioned above, the ability of ambroxol to increase GCase activity may be beneficial for the treatment of PD in individuals with loss-of-function mutations in the glucocerebrosidase gene, GBA1 (McNeill A et al., 2014, supra), a major inherited risk factor for PD (Do J et al., Neurodegener, 14:36, 2019). Enhancement of GCase activity by ambroxol treatment is expected to increase the degradative capacity of lysosomes and aid in the clearance of intracellular aggregated α-synuclein (aSyn), a protein involved in the pathogenesis of PD (Migdalska-Richards A et al., 2016, supra). GCase deficiency is also associated with the lysosomal and mitochondrial dysfunction observed in PD (Brooker and Krainc, 2021 Essays in Biochemistry, 65(7), pp. 873-883), which may partially explain the improvement of lysosomal and mitochondrial function by ambroxol (Magalhaes et al., 2018 Scientific Reports, 8(1), p. 1385). Based on this promising preclinical data, the use of ambroxol is under investigation in clinical trials as a disease-modifying compound in PD (see, for example, Silveira CRA et al., BMC Neurol 19:20, 2019, and Mullin S et al., JAMA Neurol 77:427-434, 2020) and the lipid storage disorder Gaucher disease (Zimran, Altarescu and Elstein, 2013 Blood Cells, Molecules & Diseases, 50(2), pp.134-137; Narita et al., 2016 Annals of Clinical and Translational Neurology, 3(3), pp.200-215).

[0071] However, the potentially beneficial biological activities of ambroxol are known to extend far beyond its GCase chaperone action. In particular, ambroxol exerts broad effects on the autophagy-endolysosomal network (AELN) (McNeill et al., 2014 Brain:A Journal of Neurology,137(Pt 5), pp.1481-1495; Fois et al., 2015 Cell Calcium,58(6), pp.628-637; Magalhaes et al., 2018, supra), induced immune responses (Beeh et al., 2008 European Journal of Medical Research,13(12), pp.557-562; Kern and Schwickert,2017 Journal of Pain Research,10,pp.1905-1929), and blockade of channels involved in chronic pain (Russo et al., 2022 Pain [Preprint]. Available at: https: / / doi.org / 10.1097 / j.pain.0000000000002693). Therefore, ambroxol may prove useful in treating a myriad of diseases that have defects in AELN, immune system or nociception as their central feature. For example, many neurodegenerative disorders, including PD and AD, show defects in the production or degradation of neurotoxic protein species in AELN, which may promote a chronic inflammatory environment in brain tissue.

[0072] Some of the mechanisms by which ambroxol exerts its influence on cellular processes have been elucidated. Ambroxol is an amphiphilic amine, which easily crosses cell membranes via passive diffusion and can directly access intracellular and intraorganelle spaces. Within the cell, ambroxol acts as a weak base, becoming protonated and being trapped in acidic intracellular compartments, including lysosomes and other structures of the late endolysosomal pathway (Magalhaes J et al., 2018, supra; and Fois G et al., 2015, supra). These properties explain the high bioavailability of ambroxol and its tendency to accumulate in lipid-rich organ systems such as the brain, lung and skin (Mullin S et al., 2020, supra). Within the cytoplasm, ambroxol acts as a potent scavenger and stabilizer of free radicals produced in the course of cellular metabolism and also as part of the innate immune response (Stetinova, Herout and Kvetina, 2004 Clinical and Experimental Medicine, 4(3), pp. 152-158). Finally, ambroxol reduces the Na+ associated with neuropathic pain. + v It has been shown to provide analgesia by directly blocking the 1.7 / 8 channel (Kern and Schwickert, 2017, supra).

[0073] The ambroxol amphiphilic entrapment in acidic compartments affects the AENL system in several ways. One of the important consequences of ambroxol protonation in lysosomes is the resulting deacidification of the lysosomal lumen (Mullin S et al., 2020, supra, and Lu S et al., PloS One 12:e0173771, 2017). This process mobilizes calcium stores within the organelle, leading to the activation of lysosome-associated transcription factor EB (TFEB). Activated TFEB relocalizes to the nucleus, where TFEB initializes the transcription of a gene network with key regulatory control over lysosomal biogenesis (Medina DL et al., Nat Cell Biol 17:288-299, 2015). Upregulation of this gene network results in an increased cellular ability to degrade proteins associated with neurodegenerative disorders, such as long-lived proteins (Sardiello et al., 2009 Science, 325(5939), pp. 473-477), especially hyperphosphorylated tau (Martini-Stoica et al., 2018 The Journal of Experimental Medicine, 215(9), pp. 2355-2377). Ambroxol effectively activates TFEB both in cell culture and in vivo. In cultures derived from the brain (Magalhaes et al., 2018, supra), skin (McNeill et al., 2014, supra), or immune-related tissues (Choi et al., 2018, Antimicrobial Agents and Chemotherapy, 62(9)), application of ambroxol results in upregulation of TFEB at the transcriptional, protein, and / or activation levels.

[0074] Relatedly, high-dose regimens of ambroxol can promote cellular autophagy to secretory regimens (see, for example, McNeill A et al., 2014, supra, and Silveira CRA et al., 2019, supra). Secretory autophagy is an alternative process to degradative autophagy, involving the packaging of normally functioning and defective proteins into membrane-bound organelles called autophagosomes and secretion outside the cell, which fuse with lysosomes to facilitate the degradation and recycling of autophagosomal contents (Rabouille C et al., J Cell Sci 125:5251-5255, 2012). Upregulation of autophagy-dependent secretion has been recognized as a potential pathway for disease modification in neurodegenerative disorders (Ponpuak M et al., Curr Opin Cell Biol,35:106-116, 2015). In PD, as well as AD, the aggregation-prone proteins aSyn (in PD; see, e.g., Dehay B et al., J Neurosci Off J Soc Neurosci 30:12535-12544, 2010) and beta-amyloid (in AD; see, e.g., Vickers JC et al., Exp Neurol 141:1-11, 1996) form degradation-resistant inclusions that accumulate in autophagic vacuoles. In both cases, protein aggregates cause dysregulation at multiple points along the cellular autophagy processing pathway, which may be alleviated by secretory offloading.

[0075] Ambroxol can directly engage the secretory autophagy system. In primary neuronal cultures from cells engineered to express PD pathology and from unmodified counterparts, ambroxol enhances the secretion of aSyn while clearing intracellular stores of aggregation-prone versions of this protein (Magalhaes J et al., 2018, supra). These results are further extended to AD pathology, where application of bromhexine to brain-derived cell cultures promotes the clearance of intracellular aggregation-prone tau (Chauhan S et al., Nat Commun 6:8620,2015). Taken together, these data indicate that ambroxol exerts beneficial effects on AELN by increasing their natural cellular degradation capacity while clearing toxic protein aggregates from cells, such as neurons, that have a lower natural degradation capacity.

[0076] Another important system through which ambroxol affects human health is the immune response. Epidemiological and preclinical studies have shown that inducing the immune response from an innate inflammatory response to an anti-inflammatory / adaptive state may be beneficial for multiple chronic disease states. For example, in many neurodegenerative disorders, longitudinal sampling of patients' serum and cerebrospinal fluid shows a progressive increase in inflammatory response markers such as cytokines including interleukin 1β, 6, 8, and tissue necrosis factor α, as well as activation of the NLRP3-associated inflammasome, which reflects loss of cognitive function (Heneka et al., 2015 The Lancet. Neurology, 14(4), pp. 388-405; Wang, Liu and Zhou, 2015 Translational Neurodegeneration, 4, p. 19). In agreement, variants in many genes with innate immune regulatory functions are associated with increased risk of AD and PD (Hollingworth et al., 2011 Nature Genetics, 43(5), pp. 429-435; Griciuc and Tanzi, 2021 Current Opinion in Neurology, 34(2), pp. 228-236). In contrast, upregulation of markers of anti-inflammatory and adaptive immunity, such as interferon gamma and interleukin-12, correlates with a reduced incidence of AD in older adults (Yang et al., 2022 Alzheimer's & Dementia, 18(4), pp. 645-653).

[0077] Neurodegenerative disorders represent a chronic inflammatory state that occurs when the immune system is unable to mitigate the cause of an immune attack over a long time frame. In the case of neurodegenerative disorders, these immune injuries may be partially caused by toxic aggregated proteins produced by cells, mainly in the brain. Chronic inflammation can also occur in the context of persistent infections in the body, such as Mycoplasma pneumoniae, and inappropriate immune activation by self-derived antigens.

[0078] With regard to inflammation, ambroxol has also been shown to profoundly reshape immune responses to both pathogenic and autologous threats. Studies across multiple organ systems, particularly the brain (Jiang et al., 2020, BioMed Research International, 2020, p.e8131286), lung (Takeda et al., 2016 Immune Network, 16(3), pp.165-175; Zhang et al., 2016; Kokai et al., 2021 Microorganisms, 9(4), p.880) and gut (Schneider et al., 2021 EMBO Molecular Medicine, 13(1), p.e12724; Cavalu et al., 2022 The FASEB Journal, 36(9), p.e22496), confirm that ambroxol acts to reduce pro-inflammatory responses to such threats while preserving adaptive aspects of the immune response. In particular, ambroxol has been shown to reduce the expression of pro-inflammatory cytokines such as interleukin 1β, 6, 8, 10 and tissue necrosis factor-α (Bianchi et al., 1990 Agents and Action, 31(3-4), pp. 275-279; Jang et al., 2003, Pharmacology & Toxicology, 92(4), pp. 173-179; Wang et al., 2011 Zhongguo Ying Yong Sheng Li Xue Za Zhi=Zhongguo Yingyong Shenglixue Zazhi=Chinese Journal of Applied Physiology, 27(2), pp. 231-235), as well as upstream pathways of inflammasome activation such as NF-κB (Cavalu et al., 2022, supra). This inhibition is due, at least in part, to ambroxol's ability to scavenge free radicals (Peroni et al., 2013 International Journal of Immunopathology and Pharmacology, 26(4), pp. 883-887) and may involve direct blockade of specific inflammation-related channels (Schneider et al., 2021, supra).

[0079] Unlike non-specific immunosuppressants such as nonsteroidal anti-inflammatory drugs, ambroxol maintains and in some cases enhances anti-inflammatory and adaptive immune system aspects. This includes upregulation of the anti-inflammatory cytokines interleukin 10 and 12 and adaptive immune-related interferon-gamma in lung tissue in response to pathogen and ovalbumin challenge (Takeda et al., 2016, supra; Kokai et al., 2021, supra). These immune effectors may explain the long-lasting clinical utility of ambroxol in chronic respiratory diseases such as chronic obstructive pulmonary disorder (Plomer and de Zeeuw, 2017 MMW Fortschritte der Medizin, 159(Suppl5), pp. 22-33). Moreover, they may explain preclinical findings of reduced inflammation in an ulcerative colitis model (Schneider et al., 2021, supra) and reduced microglial activation in an intracerebral hemorrhage model (Jiang et al., 2020, supra).

[0080] The above anti-inflammatory effects are likely related to ambroxol's effects on autophagy. Autophagy has been noted to play an important role in regulating immune responses. In particular, activation of TFEB function promotes the degradation of key mediators of inflammation, including inflammasome components such as NLRP3 and ASC (Shi et al., Nature Immunology, 13(3), pp. 255-263 (2012); Deretic, Immunity, 54(3), pp. 437-453 (2021)). Inactivation of inflammasomes by degradation attenuates inflammatory responses, including reduced production and release of pro-inflammatory cytokines such as IL-1β. Thus, the immunomodulatory function of ambroxol is likely downstream of its effects on autophagy.

[0081] Finally, ambroxol has potent analgesic properties. Mechanistically, this effect is mediated by Na+ receptors, which are preferentially expressed by neurons involved in nociception. vThis is due to ambroxol's ability to block voltage-gated sodium channels, including 1.7 (Leffler, Reckzeh and Nau, 2010 European Journal of Pharmacology, 630(1-3), pp. 19-28) and 1.8 (Weiser and Wilson, 2002 Molecular Pharmacology, 62(3), pp. 433-438) (Bennett et al., 2019 Physiological Reviews, 99(2), pp. 1079-1151). This mechanism at least partially explains the classical use of ambroxol as a treatment for moderate to severe pain associated with acute sore throat (Fischer et al., 2002 Arzneimittel-Forschung, 52(4), pp. 256-263). Furthermore, there has been growing interest in the use of ambroxol for the treatment of neuropathic pain (Russo et al., 2022, supra).

[0082] Therefore, in conclusion, without wishing to be bound by theory, it is believed that the above observations indicate that ambroxol may increase the degradation and / or secretion of toxic intracellular proteins, protein fragments, misfolded proteins, protein aggregates or debris associated with AELN disorders. Ambroxol further regulates immune responses to a less inflammatory state through the scavenging of free radicals and interaction with inflammation-related channels. Finally, ambroxol directly blocks channels that are upregulated in disease states characterized by chronic pain.

[0083] Related - Ambroxol and related compounds of the present invention (including pharma- ceutically acceptable salts of ambroxol, such as bromhexine and ambroxol hydrochloride), as well as ambroxol analogs, such as multiply deuterated analogs, offer considerable potential as effective treatments for neurological diseases and conditions, such as PD, AD and other aging-related diseases involving autophagy dysfunction, by allowing secretion of toxic aggregated proteins (e.g., aggregated aSyn, beta-amyloid and tau proteins) from diseased cells and maintaining organelle health despite autophagy dysfunction.

[0084] In some cases, it may be advantageous to administer ambroxol (or related compounds or multiply deuterated analogs of the invention) in combination therapy with agents intended, for example, to prevent and / or remove toxic aggregated proteins.

[0085] Thus, in another aspect of the present invention, there is provided a method for preventing, alleviating symptoms and / or stabilizing progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition (including Parkinson's disease (PD), Huntingdon's disease (HD) and frontotemporal degeneration (FTD)) in a subject, said method comprising administering to the subject an effective amount of ambroxol (or related compounds such as ambroxol hydrochloride and bromhexine) or a multiply deuterated analogue of the present invention in combination with one or more suitable anti-beta amyloid antibodies or fragments thereof.

[0086] Such combination therapy may produce synergistic effects, for example, in clearing aggregated beta-amyloid associated with AD.

[0087] The ambroxol (or related compound) or multiply deuterated analogue and anti-beta amyloid antibody or fragment thereof of the present invention may be administered, for example, in the same pharmaceutical composition or in separate pharmaceutical compositions. When administered in separate pharmaceutical compositions, the ambroxol (or related compound) or multiply deuterated analogue and anti-beta amyloid antibody or fragment thereof of the present invention may be administered simultaneously or sequentially in any order (e.g., within a few seconds, minutes, or even hours (e.g., 2 to 48 hours)).

[0088] Anti-beta amyloid antibodies or fragments thereof may be selected from those known to those skilled in the art. Suitable antibodies may include human or humanized anti-Aβ monoclonal antibodies from the group consisting of bapineuzumab (Pfizer / Janssen Pharmaceuticals), solanezumab (Eli Lilly and Company), gantenerumab (Hoffmann-La Roche), crenezumab (Genentech), ponezumab (Pfizer), BAN2401 / lecanamab (BioArctic Neuroscience, AB / Eisai / Biogen) and aducanumab (Aduhelm™ Biogen) (see also van Dyck CH., Biol Psychiatry 83(4):311-319,2018). Suitable antibody fragments may include fragments such as Fab fragments and scFv antibodies targeting Aβ, including the scFv molecules described in Sebollela A., J Neurochem 142(6):934-937, 2017 and Zha J et al., Scientific Reports 6:36631, 2016.

[0089] In some embodiments, the method should be operated for the prevention, alleviation of symptoms, and / or delay of progression of AD or other diseases associated with pathological protein misfolding, aggregation and deposition, including Parkinson's disease (PD), Huntingdon's disease (HD) and frontotemporal degeneration (FTD). Thus, the method can, for example, prevent the onset, onset or progression of a disease or condition, or the onset, onset or progression of one or more symptoms or deleterious features of a disease or condition (e.g., toxic aggregation of Aβ protein). Since beta amyloid and tau aggregation are known to appear early in the development of Alzheimer's disease, therapeutic methods intended to prevent and / or remove toxic aggregates of Aβ or tau, as may be achieved by administering ambroxol (or related compounds) or multi-deuterated analogs of the present invention, offer great potential.

[0090] In a variant, the present invention may provide a method for preventing, alleviating symptoms, and / or slowing the progression of AD or other diseases associated with pathological protein misfolding, aggregation and deposition, including Parkinson's disease (PD), Huntingdon's disease (HD) and frontotemporal degeneration (FTD), comprising administering to a subject an effective amount of ambroxol (or related compounds such as ambroxol hydrochloride and bromhexine) or a multiply deuterated analog of the present invention. That is, the ambroxol or related compounds or multiply deuterated analog of the present invention may be used as the sole active agent.

[0091] In some embodiments of the method for preventing, alleviating symptoms of, and / or slowing the progression of AD or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD), subjects may be selected based on appropriate biomarkers indicative of patients at risk or in the early stages of development of AD or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD). In the case of AD, the biomarker may be a specific phosphorylated tau protein (p-tau), which has been found to be present at elevated levels in CSF and / or blood in the early or preclinical stages of AD (Suarez-Calvet M et al., EMBO Mol Med 12:e12921,2020). Thus, in one example, the method may further comprise selecting subjects by assaying for increased levels of p-tau217 (i.e., tau phosphorylated at the Thr-217 residue) and, more preferably, p-tau181 (i.e., tau phosphorylated at the Thr-181 residue) in a suitable sample (e.g., a CSF, whole blood or plasma sample). These biomarkers can accurately distinguish Aβ-positive from Aβ-negative cognitively unimpaired subjects, and furthermore, the p-tau181 biomarker has been shown to be mildly but significantly increased in the preclinical stages of AD (Suarez-Calvet M et al., 2020, supra). Other p-tau tau isoforms, such as p-tau231 (Ashton, NJ et al., Acta Neuropathol 1-16 (2021)) or p-tau235 (Lantero-Rodriguez, J. et al., Embo Mol Med 13, e15098 (2021)), can be assayed similarly.Other potential biomarkers include glucose metabolism or aggregated proteins such as beta amyloid or tau measured by positron emission tomography (PET) (Therriault, J. et al., Nat Aging 1-10 (2022) doi:10.1038 / s43587-022-00204-0. Iaccarino, L., et al., “Journal of Alzheimer's Disease, 59, pp 603-614 (2017)). Retinal imaging-based biomarkers, including amyloid deposits and thinning of the nerve fiber layer, may also be used for early diagnosis of AD (Snyder, PJ et al., Alzheimers Dement. Diagn. Assess. Dis. Monit. 4, 169-178 (2016); Koronyo, Y. et al., JCI Insight 2, (2017)).

[0092] Other suitable biomarkers indicative of patients at risk for or in the early stages of development of AD or other amyloidoses include, for example, the ε4 allele of the ApoE gene and / or the pyroglutamic acid-modified amyloid beta protein, N3pG. Plasma NFL (neurofilament light chain) is elevated in many neurodegenerative diseases. It is not specific to a particular neurodegenerative disease, but instead reflects disease activity and severity. For example, it is elevated in multiple sclerosis patients with active ongoing demyelination, but is decreased when patients are treated with immunosuppressants (Barro, C. & Zetterberg, H. Acta Neurol Scand (2021) doi:10.1111 / ane.13415). Plasma exosomes carrying neuronal proteins also appear to be promising biomarkers for diagnosing several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and head injury (Rastogi, S. et al., Int J Mol Sci 22, 440 (2021)).

[0093] In some embodiments of the method for preventing, alleviating symptoms of, and / or slowing the progression of AD or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD), subjects may be selected based on genotyping of at least one gene or locus indicative of a patient at risk for AD or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD). In the case of AD, the genotyping may be of the gene encoding apolipoprotein E (ApoE). Subjects carrying the ε4 allele of ApoE are at increased risk for AD compared to subjects carrying the more common ε3 allele, whereas the ε2 allele reduces the risk (Liu CC et al., Nat Rev Neurol. 9(2):106-118, 2013). Suitable methodologies for performing ApoE genotyping are known to those skilled in the art and include, for example, suitable PCR protocols (see, for example, Zhong L et al., Mol Neurodegener. 11:2, 2016). However, in other embodiments, subjects may be selected based on genotyping of p-tau217, p-tau181 and / or N3pG.

[0094] In some embodiments of the method for preventing and / or treating Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation and deposition, including Parkinson's disease (PD), Huntingdon's disease (HD) and frontotemporal degeneration (FTD), the ambroxol (or related compounds) or multiply deuterated analogs of the present invention are (i) a dosage that provides a peak concentration in the subject's serum that is greater than 1 μM, e.g., 2-50 μM, 2-25 μM, or 10-20 μM; (ii) a dose that provides a peak concentration in brain tissue of a subject that is greater than 3 μM, e.g., between 5 and 50 μM, between 5 and 25 μM, or between 10 and 20 μM; or (iii) a dosage in the range of about 250 mg to 1000 mg / day or 750 mg to 1000 mg / day It may be preferable to administer to the subject a relatively high daily dose selected from: as such daily doses may be necessary to enable the ambroxol (or related compounds) or multiply deuterated analogs of the invention to upregulate the secretion of toxic aggregated proteins (e.g., aggregated Aβ) from diseased cells.

[0095] Pharmaceutical Compositions In one aspect, the present invention includes a composition comprising a therapeutically effective amount of a compound of formula I (e.g., a compound shown in FIG. 1) or a pharmaceutically acceptable salt, solvate or prodrug thereof, together with a pharmaceutically acceptable carrier, for preventing and / or treating various diseases and medical conditions in a subject, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, or for extending the life expectancy of a subject.For example, a composition comprising a compound of formula I (or a pharmaceutically acceptable salt, solvate or prodrug thereof) can be used to (a) treat, inhibit, or reduce aging in a subject, (b) treat, inhibit, or reduce age-related symptoms or age-related diseases in a subject, and / or (c) increase the healthspan, lifespan and / or mental acuity of a subject.

[0096] The compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) may be administered in the form of a pharmaceutical composition in combination with a pharma- ceutically acceptable carrier. In such compositions, the compound of formula I may comprise 0.1 to 99.99 weight percent.

[0097] The compound of formula I is preferably administered with a pharma- ceutically acceptable carrier selected based on the selected route of administration and standard pharmaceutical practice. The compound of formula I can be formulated into dosage forms according to standard practice in the field of pharmaceutical preparations. See Alphonso Gennaro, ed. Remington's Pharmaceutical Sciences, 18th Edition (1990), Mack Publishing Co., Easton, Pa. Suitable dosage forms can include, for example, tablets, capsules, liquids, parenteral solutions, lozenges, suppositories, or suspensions. Suitable examples of the preparation of oral, topical, suppository, and parenteral formulations of ambroxol, bromhexine, or other ambroxol derivatives that can be readily adapted to the compounds of the present invention are disclosed, for example, in Examples 1-8 of WO 2005 / 007146, or its equivalent, U.S. Patent Application Publication No. 2005 / 00148747, which are incorporated herein by reference.

[0098] In another aspect, the present invention provides the use of a compound of formula I in the preparation of a medicament for preventing and / or treating various diseases and medical conditions in a subject, including respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, or for extending the life expectancy of a subject.

[0099] For parenteral administration, the compound of formula I may be mixed with a suitable carrier or diluent, such as water, oil (especially vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or glycols such as propylene glycol or polyethylene glycol, or plant extracts as supplements. Solutions for parenteral administration preferably contain a water-soluble salt of the active agent. Stabilizers, antioxidants and preservatives may also be added. Suitable antioxidants include sulfites, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. Compositions for parenteral administration may take the form of aqueous or non-aqueous solutions, dispersions, suspensions or emulsions.

[0100] For oral administration, the compound of formula I (or its pharma- ceutically acceptable salt, solvate or prodrug) can be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules or other suitable oral dosage forms.For example, the compound of formula I can be combined with at least one excipient, such as a filler, binder, humectant, disintegrant, dissolution retarder, absorption promoter, wetting agent, absorbent or lubricant.According to one embodiment of the tablet, the compound of formula I can be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into a tablet by conventional tableting methods.

[0101] For oral administration, the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) may be provided in a liquid oral pharmaceutical composition. Liquid oral dosage forms can offer unique advantages over solid dosage forms, such as tablets and capsules. For example, in many cases, the amount of active ingredient needed to treat a particular disease or condition requires multiple tablets or capsules, one or more times per day. Ingestion of multiple tablets or capsules includes not only the active pharmaceutical ingredient, but also multiple doses of excipients used in the formulation of the tablets and capsules. Such excipients, such as oils and alcohols, are generally not well tolerated by many patients and commonly cause gastric discomfort. In addition, liquid oral dosage forms are easier for patients to comply with than solid dosage forms, since delivery of the active pharmaceutical ingredient is achieved with only one or two doses per day. In addition, liquid oral dosage forms provide rapid absorption of the active pharmaceutical ingredient from the gastrointestinal tract. Additionally, liquid oral dosage forms may utilize flavoring and / or palatability agents to further promote patient acceptance and compliance.

[0102] Thus, in some preferred embodiments, the composition of the present invention is a liquid oral pharmaceutical composition. Such compositions may be particularly suitable for preventing and / or treating lysosomal storage diseases (LSDs), such as Gaucher's disease, or neurological diseases or conditions, such as PD. In some embodiments, the composition comprises a "high loading" of the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) to deliver an effective amount in a small volume (e.g., a 15 mL dose, twice a day or more). A high drug loading liquid oral pharmaceutical composition according to the present invention may provide at least one of the following advantages: (1) improved adsorption in the gastrointestinal tract; (2) maintenance of effective blood levels over 24 hours; (3) reduced undesirable side effects of excipients compared to solid dosage forms; (4) reduced number of doses required; (5) improved taste and mouthfeel.

[0103] In one particular embodiment of the high drug load liquid oral pharmaceutical composition of the present invention, the composition comprises: (i) a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof); and (ii) at least one pharma- ceutically acceptable excipient, wherein the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) is in the form of a granule having a granule core comprising from about 60 to about 97 weight percent of the active pharmaceutical ingredient and from about 3 to about 40 weight percent of the excipient, said weight percents being based on the total weight of the granule core.

[0104] In another embodiment of the high drug load liquid oral pharmaceutical composition of the present invention, the composition comprises: (i) a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof); (ii) at least one pharma- ceutically acceptable excipient; and (iii) a diluent, wherein the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) is in the form of a granule having a granule core comprising about 60 to about 97 weight percent of the active pharmaceutical ingredient and about 3 to about 40 weight percent of the excipient, said weight percents being based on the total weight of the granule core; the granule core is coated with (iv) a water-soluble seal coating in an amount to provide a weight gain of about 0.5 to about 5%, and (v) an enteric coating in an amount to provide a weight gain of about 0.5 to about 50%.

[0105] Such high drug load liquid oral pharmaceutical compositions may be prepared, for example, by a method comprising the steps of: (a) preparing granules having a granule core comprising about 60 to about 97 weight percent of the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) and about 3 to about 40 weight percent of at least one pharma- ceutically acceptable excipient, said weight percent being based on the total weight of the granule core; (b) coating the granules with a water-soluble seal coating in an amount that results in a weight gain of about 0.5 to about 5%; (c) coating the granules prepared in step (b) with an enteric coating in an amount that results in a weight gain of about 0.5 to about 50%; and (d) preparing liquid suspensions and liquid suspension formulations comprising the enteric coated granules prepared in step (c), wherein the liquid suspension formulations comprise a suspending agent, a vehicle for enhancing the stability of the high drug load liquid oral pharmaceutical composition, and a diluent.

[0106] The compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) is preferably in the form of granules having a particle size of about 100 microns to about 500 microns, more preferably about 100 to about 300 microns, about 150 to about 350 microns, about 200 to about 350 microns, and even more preferably the particle size is about 100 to about 200 microns, about 150 to about 250 microns, about 200 to about 300 microns, about 200 to about 400 microns, about 250 to about 350 microns, about 250 to about 450 microns, about 300 to about 400 microns, about 300 to about 500 microns, about 350 to about 450 microns, and / or about 400 to about 500 microns after micronization. Particle sizes within these ranges are coatable and have been found to provide ease of swallowing without encouraging the user to chew. For example, in a preferred embodiment, a particle size of about 250 to about 350 microns has been found to balance the ability to coat and avoid a harsh "mouthfeel". Particle size can be measured by laser light scattering, for example, using a Malvern Mastersizer Apparatus MS 2000 equipped with a HydroS dispersion unit. For example, micronization of the compound of formula I can be carried out in the dry state using dry mills such as cutting mills, pin / cage mills, hammer mills, jet mills, fluidized bed jet mills and ball mills.

[0107] A preferred excipient that can be used to prepare the granule core is a binder. The binder can be any water-soluble pharma- ceutically acceptable polymer. In one embodiment, the compound of formula I (or its pharma-ceutically acceptable salt, solvate or prodrug) is in the form of a powder, and the binder will necessarily bind it together due to the poor cohesive properties of most powders. Preferably, the binder is selected from povidone (polyvinylpyrrolidone), copovidone (vinylpyrrolidone-vinyl acetate copolymer), microcrystalline cellulose, powdered cellulose, crystalline cellulose, siliconized microcrystalline cellulose, cellulose derivatives such as hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose, starch, pregelatinized starch, polymethacrylates, compressible sugar, sucrose and sugar alcohols such as mannitol, sorbitol, maltitol and xylitol, and mixtures thereof. More preferably, the binder is hydroxypropylcellulose (Klucel LF).

[0108] Granules used in high-load liquid oral pharmaceutical compositions are preferably prepared by direct spheronization, which includes preparing a granule core containing about 60 to about 97 weight percent (preferably about 75 to about 97 weight percent, more preferably about 90 to about 97 weight percent, and about 3 to about 40 weight percent of a binder, preferably about 3 to about 25 weight percent, more preferably about 3 to about 10 weight percent) of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof), the weight percent being based on the total weight of the granule core. Granulation can be carried out under high shear (mixer granulation) or in a fluidized bed (fluidized bed granulation).

[0109] The granule cores are optionally seal coated with a water-soluble seal coating in an amount that results in about 0.5 to about 5% weight gain, more preferably about 0.5 to about 3% weight gain, even more preferably about 0.5 to about 2% weight gain, even more preferably about 0.5 to about 1% weight gain, even more preferably about 1 to about 2% weight gain, even more preferably about 1 to about 3% weight gain, even more preferably about 1 to about 4% weight gain, more preferably about 2 to about 3% weight gain, even more preferably about 2 to about 4% weight gain, more preferably about 1% weight gain, more preferably about 2% weight gain, more preferably about 3% weight gain, and / or more preferably about 4% weight gain. Application of a water-soluble seal coating has been found to produce smooth, uniform granules that are more amenable to enteric coating. Preferred water-soluble polymers include hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (sodium and calcium salts), ethylcellulose, methylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, hydroxypropylcellulose (HPC), L-HPC (low-substituted HPC), polyvinylpyrrolidone, polyvinyl alcohol, polymers of acrylic acid and its salts, vinylpyrrolidone-vinyl acetate copolymers (e.g., Kollidon® VA64, BASF), gelatin, guar gum, partially hydrolyzed starch, alginates and xanthan. Most preferably, the water-soluble polymer is hydroxypropylmethylcellulose. The seal coating is preferably applied by a bottom spray fluidized bed coater equipped with a Wurster column.

[0110] In addition to or in the absence of a seal coating, the granule cores may optionally be coated with an enteric coating, either directly on the granule cores or on a seal coating previously applied to the granule cores. Preferably, the enteric coating is applied in an amount that results in a weight gain of about 0.5 to about 50%, a weight gain of about 1 to about 40%, a weight gain of about 2 to about 30%, a weight gain of about 3 to about 20%, or a weight gain of about 4 to about 10%, based on the total weight of the granule cores. More preferably, the enteric coating is applied in an amount that results in a weight gain of about 0.5 to about 5%, a weight gain of about 1 to about 4%, or a weight gain of about 2 to about 3%, based on the total weight of the granule cores. The enteric coating is preferably applied by a bottom spray fluid bed coater equipped with a Wurster column.

[0111] The enteric coating comprises a polymer selected from an acrylate polymer or an aqueous cellulose dispersion. A combination of an acrylate polymer and / or an aqueous cellulose dispersion can also be used. Preferably, the acrylate polymer is selected from polymethacrylate methyl methacrylate copolymer (Eudragit® L-100), polyethyl acrylate methyl methacrylate trimethylammonioethyl methacrylate chloride copolymer (Eudragit® RL-100, RS-100), polymethacrylate ethyl acrylate copolymer (Eudragit® L30D-55), ethyl acrylate methyl methacrylate trimethylammonioethyl methacrylate chloride copolymer (Eudragit® RL30D), ethyl acrylate methyl methacrylate trimethylammonioethyl methacrylate chloride copolymer (Eudragit® RS30D), and polyethyl acrylate methyl methacrylate copolymer (Eudragit® NE30D). More preferably, the acrylate polymer is polymethacrylate ethyl acrylate copolymer (Eudragit® L30D-55). The enteric coating may be applied by a bottom spray fluid bed coater, preferably equipped with a Wurster column. The enteric coating may increase the delivery of the active pharmaceutical ingredient to regions of the subject's digestive tract where the pH is between about 4.5 and about 6.5. The enteric coating may also increase the delivery of the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) to the proximal small intestine or the mid-small intestine or both. Additionally, the enteric coating may increase the delivery of the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) to one or more of the duodenum, jejunum, or mid-ileum. Preferably, the enteric coating begins to dissolve in aqueous solution at a pH of about 4.5 to about 5.5.

[0112] The vehicle that enhances the stability of the high drug-loaded liquid oral pharmaceutical composition may preferably be a protective colloid.

[0113] The high-load liquid oral pharmaceutical composition of the present invention may further contain a plasticizer.Preferred plasticizers are diethyl phthalate, dibutyl phthalate, triethyl citrate and glycerol.Combinations of plasticizers can also be used.More preferably, the plasticizer is triethyl citrate.

[0114] In one embodiment, the high-load liquid oral pharmaceutical composition is in the form of a solution. In another embodiment, the high-load liquid oral pharmaceutical composition is in the form of a suspension. To form a suspension, the enteric coated granules can be combined with a liquid suspension formulation. The liquid suspension formulation can include a suspending agent, a vehicle for improving the stability of the high-drug-load liquid oral pharmaceutical composition, and a diluent. The suspending agent and the vehicle for improving the stability of the high-drug-load liquid oral pharmaceutical composition can be the same or different, since many suspending agents also function as stability enhancers.

[0115] Examples of suspending agents include, but are not limited to, methylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, gum tragacanth, and glycerol monostearate. More preferably, the vehicle that enhances the stability of the liquid oral pharmaceutical composition is a protective colloid. Examples of protective colloids include, but are not limited to, hydroxymethylcellulose, sodium carboxymethylcellulose, polyvinyl alcohol, gelatin, and polyvinylpyrrolidone. A combination of protective colloids may also be used. A preferred protective colloid is a mixture of cellulose gum, xanthan gum, and carrageenan. Examples of diluents include, but are not limited to, water, alcohol (e.g., methyl alcohol, ethyl alcohol, propyl alcohol, i-propyl alcohol, etc.), acetone, glycerin, oil (e.g., castor oil), any other pharma- ceutically acceptable diluent, or a mixture thereof. Most preferably, water is used as the suspension or solution diluent. In addition, a pH adjuster and / or an antioxidant may also be used.

[0116] In some particular embodiments of the liquid suspension formulations of the invention, the formulation comprises microcrystalline cellulose and sodium carboxymethylcellulose (Avicel RC-591), gum (CP Kelco), and water; or, microcrystalline cellulose and sodium carboxymethylcellulose (Avicel RC-591), cellulose gum, xanthan gum, and carrageenan (Ticaloid Ultrasmooth), and water.

[0117] According to a preferred embodiment, the liquid suspension formulation comprises about 0.5% to about 3% of at least one suspending agent; about 0.5% to about 1% of at least one protective colloid; and about 98% of diluent, the weight percentages being based on the weight of the liquid suspension formulation. More preferably, the protective colloid is used in an amount of about 0.1 to 0.5 weight percent. In some specific embodiments, the liquid suspension formulation comprises about 1.5% of at least one suspending agent; about 0.2% of at least one protective colloid; and about 98% of diluent.

[0118] Preferably, the high drug load liquid oral pharmaceutical composition in the form of a suspension has a viscosity of less than about 5 Pa·s. More preferably, the high drug load liquid oral pharmaceutical composition in the form of a suspension has a viscosity of less than about 3 Pa·s, and most preferably less than about 1 Pa·s.

[0119] In some embodiments, the high drug load liquid oral pharmaceutical composition is used to prevent and / or treat a lysosomal storage disease selected from Gaucher disease (including types 1, 2 and 3), Pompe disease (including infantile and late-onset) and Fabry disease, or to prevent and / or treat (e.g., alleviate symptoms of) PD. Other lysosomal storage diseases that can be prevented and / or treated with the high drug load liquid oral pharmaceutical composition include GMI-gangliosidosis, Tay-Sachs disease, Sandhoff disease, Niemann-Pick disease, Krabbe disease, Farber disease, metachromatic leukodystrophy, Hurler-Shy disease, Hunter disease, Sanfilippo disease A, Sanfilippo disease B, Sanfilippo disease C, Sanfilippo disease D, Morquio disease A, Morquio disease B, Maroteaux-Lamy disease, Sly disease, alpha-mannosidosis, beta-mannosidosis, fucosidosis, sialidosis and Schindler-Kanzaki disease.

[0120] In some more specific embodiments, the high drug load liquid oral pharmaceutical composition is used to prevent and / or treat a lysosomal disease selected from Gaucher disease type 1, type 2 and type 3. In some further specific embodiments, the high drug load liquid oral pharmaceutical composition is used to prevent and / or treat a subject with a mutation in glucocerebrosidase (GCase) (e.g., a mutation in β-glucocerebrosidase, and the subject may also have either Gaucher disease and / or Parkinson's disease). The mutation in β-glucocerebrosidase may be selected from: a. Point mutations at any one of the following positions: D140H, V15L, G46E, K79N, R119Q, P122S, R131L, K157Q, N188S, Y212H, F213I, F216V, F216Y, H225Q, F251L, R257E, P289L, A309V, H311R, W312C, Y323I, G325 R, E326K, C342G, R353G, R359X(termination), S364T, N370S, L371V, G377S, V394L, V398F, P401L, D409H, D409V, P415R, L444P, A456P, V460V, R463C, G478S or R496H and / or any combination thereof; b. Point mutation at L444P; c.Point mutation at N370S; d.Point mutation at E326K; e.point mutations at L444P, A456P, and V460V; f.Point mutations at D140H and E326K; g.Point mutations at H255Q and D409H; guanine insertion at h.84GG; i. a splice site mutation in intron 2 (IVS2DS+lG-A), resulting in skipping of exon 2; j. 1 bp deletion in the GCase gene (1023delC in the genomic sequence); k. a 55-bp deletion in the GCase gene (nucleotides 5879 to 5933 in genomic DNA); l. homozygous 259C-T transition (1763 in genomic DNA); m. A homozygous 1-bp deletion in the GCase gene, resulting in a frameshift and premature truncation of the protein in exon 6; and n. A G to A substitution at the first position of the splice site in intron 10 of the GCase gene, resulting in an insertion of the first 11 base pairs of IVSl 0 and a deletion of the first 11 base pairs of exon 11.

[0121] In some embodiments, the high drug load oral liquid pharmaceutical composition can also be administered to a subject undergoing enzyme replacement therapy (e.g., combination therapy). Examples of such enzyme replacement therapy include, but are not limited to, recombinant glucocerebrosidase, such as imiglucerase, velaglucerase, taliglucerase alfa (ELELYSO®) and / or eliglustat (CERDELGA®). The high drug load oral liquid pharmaceutical composition can be administered simultaneously, sequentially, or at different times with enzyme replacement therapy.

[0122] In some embodiments, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharmaceutically acceptable salt thereof for treating subjects with misfolded and / or mistransported glucocerebrosidase. In a further embodiment, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharmaceutically acceptable salt thereof for treating or preventing lysosomal storage disease in subjects. In yet a further embodiment, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharmaceutically acceptable salt thereof for treating subjects with mutations in glucocerebrosidase. In yet a further embodiment, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharmaceutically acceptable salt thereof for treating subjects with mutations in β-glucocerebrosidase. Also, in some preferred embodiments, the mutation of β-glucocerebrosidase is selected from N370S, L444P, and / or E326K. In a further embodiment, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharma- ceutically acceptable salt thereof for treating a subject suffering from Gaucher's disease. In a further embodiment, the high drug load oral liquid pharmaceutical composition comprises a multi-deuterated analog of ambroxol or a multi-deuterated analog of bromhexine or a pharma-ceutically acceptable salt thereof for treating a subject suffering from PD.

[0123] The pharmaceutical compositions of the present invention can also be formulated in unit dosage form, each dosage containing from about 50 to about 1000 mg, more typically from about 250 to about 500 mg, of a compound of formula I per unit dose. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined amount of a compound of formula I calculated to produce the desired therapeutic effect, in association with a suitable pharma- ceutically acceptable carrier.

[0124] In a further preferred embodiment, the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) is administered as a daily dose in several doses over a given period, e.g., a period of a week or more. For example, daily doses of about 20-500 mg / day, 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, or 250 mg-1000 mg / day may be utilized. It may be necessary in some cases to use doses outside these ranges, and therefore higher or lower doses are also contemplated. The daily dose may be divided, such as by dividing it into 2-4 equal doses per day.

[0125] The pharmaceutical compositions of the present invention can also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes and / or microspheres to provide the desired release profile.

[0126] In general, a controlled release preparation is a pharmaceutical composition that can release an active ingredient at a rate required to maintain a constant pharmacological activity for a desired period of time. Such dosage forms deliver the drug to the body over a predetermined period of time, thus maintaining drug levels in the therapeutic range for a longer period of time than conventional non-controlled formulations.

[0127] US Patent No. 5,674,533 discloses a liquid dosage form controlled release pharmaceutical composition for administering moguisteine, a potent peripheral antitussive. US Patent No. 5,059,595 describes the controlled release of active agents by the use of gastro-resistant tablets for the treatment of organic psychiatric disorders. US Patent No. 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-inflammatory drug with potent analgesic properties. US Patent No. 5,120,548 discloses a controlled release drug delivery device made of a swellable polymer. US Patent No. 5,073,543 describes a controlled release formulation containing trophic factors entrapped by ganglioside-liposome vehicles. US Patent No. 5,639,476 discloses a stable solid controlled release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known to be used in controlled release formulations. US Patent No. 5,354,566 discloses controlled release powders containing active ingredients. US Patent No. 5,733,566 describes the use of polymeric microparticles to release antiparasitic compositions. Any or all of these techniques can be adapted to the controlled release of the compound of formula I (or its pharma-ceutically acceptable salt, solvate or prodrug).

[0128] The controlled release of the compound of formula I (or its pharma- ceutically acceptable salt, solvate or prodrug) can also be stimulated by various inducers, such as pH, temperature, enzymes, water, or other physiological conditions or compounds. The mechanism of drug release is varied. For example, in one embodiment, the release-controlling component can swell and form porous openings large enough to release the active ingredient after administration to a patient. The term "controlled release component" in the context of the present invention is defined herein as one or more compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and / or microspheres, that facilitate the controlled release of the compound of formula I (or its pharma- ceutically acceptable salt, solvate or prodrug) in a pharmaceutical composition. In another embodiment, the controlled release component is biodegradable and is induced by exposure to an aqueous environment, pH, temperature, or enzymes in the body. In another embodiment, a sol-gel can be used in which the active ingredient is incorporated into a sol-gel matrix that is solid at room temperature. This matrix is ​​implanted into a subject, preferably a human or other mammal, whose body temperature is high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the subject.

[0129] Particularly interesting is the composition comprising the compound of the present invention suitable for administration in the nasal cavity or by inhalation.Thus, the compound of formula I can be formulated for administration in the nasal cavity or by inhalation typically in the form of dry powder from a dry powder inhaler (alone, as a mixture, for example, in the dry blend with lactose, mannitol, dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose or trehalose in anhydrous or monohydrate form, preferably monohydrate, or as a mixture of component particles mixed with, for example, phospholipid), or as an aerosol spray from a pressurized container, pump, spray, nebulizer (preferably the nebulizer that uses electrohydrodynamics to generate fine mist) or nebulae, with or without the use of suitable propellants such as dichlorofluoromethane.

[0130] The pressurized container, pump, spray, atomizer, or nebulae contains, for example, a solution or suspension of a compound of formula I (or a pharma- ceutically acceptable salt, solvate, or prodrug thereof) in ethanol (optionally aqueous ethanol) or an alternative agent suitable for dispersing, solubilizing, or prolonging the release of the compound, one or more propellants as a solvent, and optionally a surfactant, such as sorbitan trioleate or oligolactic acid.

[0131] Prior to use in a dry powder or suspension formulation, the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) is micronized to a size suitable for delivery by inhalation (typically less than 5 microns).This can be accomplished by any suitable comminuting method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[0132] Suitable solution formulations of the compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) for use in nebulizers that use electrohydrodynamics to generate a fine mist may contain 1 μg to 20 mg of compound per actuation, with actuation volumes varying from 1 μL to 100 μL. A typical formulation may contain the compound of formula I, propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol. Capsules, blisters and cartridges (e.g. made of gelatin or HPMC) for use in inhalers or insufflators may be formulated to contain a powder mix of the compound of formula I (or a pharma-ceutically acceptable salt, solvate or prodrug thereof) and a suitable powder base, such as lactose or starch, and a performance modifier, such as L-leucine, mannitol, or magnesium stearate.

[0133] The formulation for inhalation / intranasal administration of the compound of formula I (or its pharma- ceutically acceptable salt, solvate or prodrug) can be formulated to be immediate release and / or modified release. Modified release formulations include delayed release formulations, sustained release formulations, pulsed release formulations, controlled dual release formulations, targeted release formulations and programmed release formulations. Sustained or controlled release can be obtained, for example, by using poly(D,L-lactic-co-glycolic acid).

[0134] Administration of Compounds of the Invention In some preferred embodiments, the compound of the present invention is orally administered to patients.However, the compound can be administered by any route, including rectal administration, pulmonary administration, sublingual administration and parenteral administration.Parenteral administration includes, for example, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intranasal administration, intravaginal administration, intravesical administration (e.g., administration into the bladder), intradermal administration, transdermal administration, topical administration or subcutaneous administration.

[0135] Dosage intervals can be once a week, twice a week, every other day, once a day, typically once, twice, three times or four times a day, and doses are administered at equal intervals throughout the day and night to ensure that drug is always present.However, those skilled in the art will recognize that treatment schedules can be optimized for any given subject, and administration of the compound of formula I (or its pharma-ceutically acceptable salt, solvate or prodrug) can occur less frequently than once a day.Treatment can be carried out for as long as required.

[0136] The specific dose of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) required to elicit a therapeutically beneficial response in a subject will, of course, be determined by the particular circumstances of the individual subject, including the subject's size, weight, age, and sex, as well as the route of administration of the compound.

[0137] For example, daily dosages of about 20-500 mg / day, 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, or 250 mg-1000 mg / day may be utilized. However, in some cases it may be necessary to use dosages outside these ranges, and therefore higher or lower doses are also contemplated. The daily dosage may be divided, such as into two to four equal doses per day. Furthermore, for chronic administration, such as may be required in methods of inducing an increase and / or improvement in healthspan, lifespan and / or mental acuity in a subject, the compound is preferably administered at a dose of approximately 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 1200 mg / day, 1300 mg / day, 1400 mg / day, 1500 mg / day, 1600 mg / day, 1700 mg / day, 1800 mg / day, 1900 mg / day, 2000 mg / day, 2100 mg / day, 2200 mg / day, 2300 mg / day, 2400 mg / day, 2500 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, g / day, 1250 mg / day, 1300 mg / day, 1350 mg / day, or at doses of 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-500 mg / day, 250 mg-1000 mg / day, or 1000 mg-1500 mg / day or less than 1000 mg / day, or at doses of approximately 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, and / or 4-12 mg / kg / day.

[0138] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention, optionally having associated therewith a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of the manufacture, use or sale for human administration.

[0139] Without further explanation, it is believed that one of ordinary skill in the art can readily make and utilize a compound of formula I (e.g., a compound shown in FIG. 1) or a pharma- ceutically acceptable salt, solvate, or prodrug thereof, and practice the methods of the present invention, using the foregoing description and the following illustrative examples. The following examples point out some preferred embodiments of the present invention, but should not be construed as limiting the present disclosure in any way. Furthermore, although the present invention has been described herein with reference to embodiments, it should be understood that such embodiments and examples provided herein are merely illustrative of the principles and applications of the present invention. Thus, it should be understood that numerous modifications can be made to the illustrative embodiments and examples, and that other configurations can be devised without departing from the spirit and scope of the present invention, as defined, for example, by the following claims. All patent applications, patents, literature, and references cited herein are incorporated herein by reference in their entirety. EXAMPLES

[0140] Working Example The invention will be further described with reference to the following examples, it being understood that the following are merely examples and that modifications of detail may be made whilst still falling within the scope of the invention.

[0141] Example 1: Compound synthesis and metabolic assays Multiply deuterated compounds 2, 4 and 5 (shown in FIG. 1) can be synthesized by adapting one or more synthetic schemes described in Latli B et al., J Label Compd Radiopharm 53:15-23, 2010. For example, compound 2 can be synthesized by reacting 2-amino-3,5-dibromo-benzaldehyde with 2-amino-3,5-dibromo-4,6-[ 2H2]-benzaldehyde (i.e., replacing the hydrogen on the benzene ring with two deuterium atoms) and reacting the described compound with polydeuterated 4-aminocyclohexanol. Dideutero-2-amino-3,5-dibromobenzaldehyde can be obtained from several starting materials by methods known to those skilled in the art, including, but not limited to, the aldehyde used in the first step. 14 Starting from a perdeuterated anthranilic acid analog of C-labeled anthranilic acid, the method includes that presented in "Scheme 2" of Latli et al., 2010.

[0142] Example 1.1 - Synthesis of Bis-Deuterated Ambroxol A (ZW-001) HCl Salt

[0143] The compound bis-deuterated ambroxol A HCl salt (denoted as ZW-01 in Scheme 1, although ZYW-001 is used herein) was prepared according to Scheme 1. [ka]

[0144] Reagents and conditions: a) MeOH (solvent and reagents), H2SO4 (2.3 equiv), 65 °C, 24 h; b) NaOMe (5 mol%), NaBD4 (3.0 equiv), MeOH, 25 °C, 7 h; c) MnO2 (1.3 equiv), toluene, 80 °C, 80 min; d) trans-4-aminocyclohexanol (1 equiv), NaBD4 (3.0 equiv), EtOH, 80 °C, 5 h.

[0145] Preparation of intermediate 2: To a solution of 2-amino-3,5-dibromobenzoic acid 1 (7.0 g, 23.73 mmol) in MeOH (120 ml) was added concentrated H2SO4 (3 ml, 54.00 mmol). The reaction mixture was refluxed for 24 h (reaction completion monitored by TLC). After the allotted time, the reaction mixture was cooled to 0° C. and saturated sodium hydroxide solution was added until pH 5.5 (measured by pH paper), followed by saturated NaHCO3(水溶液) was added until a pH of 7.5 was reached, and a white solid precipitated from the reaction mixture. The mixture was filtered and the residue was washed with ice-cold MeOH (25 ml). The filtrate was extracted twice with 50 ml of dichloromethane. The combined organic extracts were dried (Na2SO4, 75 g), filtered and concentrated to give an off-white crystalline solid 2, which was used without further purification (3.3 g, 66%). MS (ESI): m / z 309 (M+H). + .

[0146] Preparation of intermediate 3: A 50 ml two-necked round bottom flask equipped with a magnetic stir bar and fitted with a rubber septum was charged with 2 (1.5 g, 5 mmol), methanol (10 ml), and 5 mol% NaOMe (13.5 mg, 0.25 mmol) at 25 °C. The reaction was stirred under nitrogen for 20 min and powdered NaBD4 (627 mg, 15 mmol) was added in one portion with constant stirring. The mixture was stirred under nitrogen at 25 °C for an additional 7 h. The progress of the reaction was followed by TLC. The reaction was quenched by the addition of 10 ml of methanol. The resulting solution was evaporated under reduced pressure on a rotary evaporator and the residue was resuspended in 30 ml of dichloromethane and filtered through Celite. The filtrate was dried over anhydrous sodium sulfate (50 g) and the solvent was evaporated on a rotovap to give the crude reaction product 4 (0.75 g, 53%), which was used in subsequent reactions without purification. MS(ESI): m / z 284(M+H) + .

[0147] Preparation of intermediate 4: A solution of crude 3 (0.75 g, 2.65 mmol) in toluene (30 ml) was treated with manganese(IV) oxide (1.63 g, 18.5 mmol, 88%) and the reaction mixture was stirred at 80° C. for 80 min. Upon cooling to ambient temperature, the mixture was filtered through Celite. The Celite pad was washed thoroughly with toluene (100 ml) and the filtrate was concentrated to give a pink solid (0.48 g, 64%) 4, which was used in the subsequent reaction without purification. MS (ESI): m / z 281 (M+H). + .

[0148] Preparation of ZW-001 HCl salt: A mixture of 4 (0.48 g, 1.7 mmol) and trans-4-aminocyclohexanol (0.2 g, 1.7 mmol) in ethanol (15 ml) was heated at 80° C. for 5 h and allowed to cool to ambient temperature. Sodium borodeuteride (0.22 g, 5.1 mmol) was added and stirring at ambient temperature was continued for 2 h, after which saturated NH (水溶液) (5 ml). After removal of the solvent, the solid was resuspended in dichloromethane and filtered through Celite. The Celite pad was rinsed with 100 ml of dichloromethane and the filtrate was concentrated under reduced pressure to give an off-white solid. Elution through a flash column (silica gel 60, 230-400 mesh, 7% MeOH in EtOAc) gave an off-white crystalline solid which was converted to the HCl salt (90 mg, 12%). MS(ESI) m / z: 380.9 (M+H) + . Chemical purity (LCMS):>99%. 1 H NMR (d4-methanol) δ (ppm): 1.35 (m, 2H), 1.5 (m, 2H), 2.05 (m, 2H), 2.25 (m, 2H), 3.2 (m, 1H), 3.55 (m, 1H), 7.45 (s, 1H), 7.65 (s, 1H).

[0149] Example 1.2 - Synthesis of compound ZW-002

[0150] Compound ZW-002 (also referred to herein as compound 1 and designated ZW-02 in Scheme 2) was prepared according to Scheme 2. [ka]

[0151] Reagents and conditions: a) di-tert-butyl azodicarboxylate (2 equiv), Ag2O (5 mol%), HO, 0 °C, 30 min; b) HCl (7.1 equiv), MeOH, 55 °C, 24 h; c) acetic anhydride (1.5 equiv), HO, 55 °C, 12 h; d) D2 (500 psi), 5% Rh / Al2O3 (40 wt%), CD3OD, 72 h; e) KOH (2.3 equiv), HO, 100 °C, 6 h; f) NaBH4 (2.0 equiv), EtOH, 80 °C, 3 h. g) HCl (2 equiv), CHCl, 5 min.

[0152] Preparation of intermediate 6: A mixture of phenol-d5 (5, 5.0 g, 49.9 mmol) and di-tert-butyl azodicarboxylate (30.0 g, 100 mmol) was added to a 250 ml round bottom flask containing a stir bar and sealed with a Teflon lined cap. Water (150 ml) was then introduced. The resulting mixture was stirred for 15 min and then cooled to 0° C. at which point Ag2O (0.58 g, 5 mol%) was added and the resulting mixture was stirred vigorously for 30 min. At the allotted time, the reaction mixture was added to 500 ml of water and extracted four times with 100 ml of ethyl acetate. The organic portions were combined and dried over Na2SO4 (300 g) and then filtered. The solvent was then removed in vacuo to give crude yellow solid 6 (13 g, 80%) which was used without further purification. MS (ESI): m / z 217 (fragment) + .

[0153] Preparation of intermediate 7: Intermediate 6 (10.0 g, 30 mmol) was added to 150 ml of methanol and stirred for 30 min. After the allotted time, 54 ml (215 mmol) of 4N HCl in dioxane was added and the reaction mixture was stirred at 55° C. for 24 h, forming a suspended solid. The mixture was cooled to room temperature and filtered. The residue was washed with 200 ml of ethyl acetate. The resulting light purple solid (2.5 g, 74%), 7, was dried in a vacuum oven at 35° C. for 16 h and used in the next step without further purification. MS (ESI): m / z 114 (M+H) + .

[0154] Preparation of intermediate 8: A solution of 7 (2.5 g, 22 mmol) in water (90 ml) was treated with 3.25 ml (34 mmol) of acetic anhydride and the mixture was heated to 55° C. with stirring for 30 min, then cooled to room temperature and stirred for 12 h. After the allotted time, the mixture was extracted with ethyl acetate (3×100 ml). The organic fractions were combined, washed with brine (50 ml), dried over Na2SO4 (200 g), filtered and concentrated in vacuo to give 1.3 g of a brown solid (38%) 8, which was used in the subsequent step without purification. MS (ESI) m / z: 156.1 (M+H). + .

[0155] Preparation of intermediate 9: A mixture of 8 (1.3 g, 8.4 mmol) and 5% Rh / Al2O3 (0.6 g) in deuterated methanol (15 ml) was stirred in a high pressure reactor under 500 psi of deuterium gas for 72 h. The mixture was filtered through a short pad of Celite, rinsed with methanol, and concentrated in vacuo to give 1.43 g of a semi-solid, which was used in the next step without purification. MS (ESI) m / z: 168.1 (M+H). + .

[0156] Preparation of intermediate 10: A solution of 9 (1.43 g, 8.5 mmol) in aqueous KOH (10 ml, 2N) was heated to gentle reflux for 6 h. The progress of the reaction was monitored by MS (ESI). Upon completion, the reaction mixture was cooled to room temperature, saturated with NaCl (3.5 g), then extracted three times with 20 ml of CHCl:IPA (3:1), dried over NaSO (150 g), and concentrated in vacuo to give 0.3 g (28%) of a viscous semi-solid 10, which was used in the next step without further purification.

[0157] Preparation of intermediate 11: A mixture of crude 4 (0.5 g, 1.8 mmol) and 10 (0.25 g, 2.2 mmol) in ethanol (25 ml) was heated at 80° C. for 3 h and allowed to cool to ambient temperature. Sodium borohydride (0.17 g, 4.5 mmol) was added and stirring at ambient temperature was continued for 2 h, after which saturated NH4Cl (水溶液)(5 ml). After removal of the solvent, the solid was resuspended in dichloromethane and filtered through Celite. The Celite pad was rinsed with 100 ml of dichloromethane and the filtrate was concentrated under reduced pressure to give 11 as an off-white solid (480 mg, 70%) as a mixture of cis / trans isomers. The solid was subjected to SFC conditions to separate the isomers. MS(ESI): m / z 389(M+H) + .

[0158] Preparation of ZW-002 HCl salt: A solution of ZW-002 free base (145 mg, 0.4 mmol) in dichloromethane (5 ml) was treated with 4N HCl in dioxane (0.2 ml, 0.8 mmol). After stirring the solution at room temperature for 5 min, hexane was added (15 ml) and the resulting solution was kept at 2° C. for 24 h, whereupon ZW-002 HCl salt crystallized. After filtration, a white solid (100 mg, 60%) was collected and dried in vacuum at 35° C. overnight. MS (ESI) m / z: 388.9 (M+H). + . Chemical purity (LCMS):>99%. 1 H NMR (d4-methanol) δ (ppm): 4.25 (s, 2H), 7.45 (s, 1H), 7.65 (s, 1H).

[0159] Example 1.3 - Synthesis of compound ZW-003

[0160] Compound ZW-003 (also referred to herein as compound 3 and designated ZW-03 in Scheme 3) was prepared according to Scheme 3. [ka]

[0161] Reagents and conditions: a) NaBD4 (2.0 equiv), EtOH, 80 °C, 3 h; b) HCl (2 equiv), CH2Cl2, 5 min.

[0162] Preparation of intermediate 12: A mixture of crude 4 (0.5 g, 1.8 mmol) and 10 (0.27 g, 2.2 mmol) in ethanol (25 ml) was heated at 80° C. for 3 h and allowed to cool to ambient temperature. Sodium borodeuteride (0.23 g, 5.4 mmol) was added and stirring at ambient temperature was continued for 2 h, after which saturated NH4Cl (水溶液) (5 ml). After removal of the solvent, the solid was resuspended in dichloromethane and filtered through Celite. The Celite pad was rinsed with 100 ml of dichloromethane and the filtrate was concentrated under reduced pressure to give 12 as an off-white solid (600 mg, 87%) as a mixture of cis / trans isomers. The solid was subjected to SFC conditions to separate the isomers. MS(ESI): m / z 391(M+H) + .

[0163] Preparation of ZW-003 HCl salt: A solution of ZW-003 free base (82 mg, 0.2 mmol) in dichloromethane (5 ml) was treated with 4N HCl in dioxane (0.1 ml, 0.4 mmol). After stirring the solution at room temperature for 5 min, hexane was added (15 ml) and the resulting solution was kept at 2° C. for 24 h, whereupon ZW-002 HCl salt crystallized. After filtration, an off-white solid (60 mg, 70%) was collected and dried overnight in vacuum at 35° C. MS (ESI) m / z: 391.0 (M+H). + . Chemical purity (LCMS):>99%. 1 H NMR (d4-methanol) δ (ppm): 7.45 (s, 1H), 7.65 (s, 1H).

[0164] The stability of the multiply deuterated compounds can be readily tested by performing routine in vitro metabolic assays (e.g., using microsomal incubations to achieve CYP3A4-dependent metabolism); following the disappearance of the assayed compound (e.g., compound 2) and the formation of expected metabolite compounds (e.g., carboxylic acid and / or dibromoanthranilic acid). In vitro metabolic assays of the multiply deuterated compounds can be performed, for example, in comparison to ambroxol and / or one or more other suitable comparator compounds. The multiply deuterated compounds are expected to provide resistance to metabolism in such assays, as indicated, for example, by slower disappearance of the assayed compound and / or slower formation of metabolites.

[0165] Example 2: Metabolic stability of ambroxol and analogues in human liver microsomes The metabolic stability of ambroxol and deuterated analogues was studied in the presence of human liver microsomes to assess the effect of deuteration at different sites on the metabolic stability of ambroxol.

[0166] Study design - Test compounds (ambroxol and deuterated analogues) were dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM stock solutions. Compounds were added to preparations of human liver microsomes (200 μL per incubation at a protein concentration of 1 mg / mL) to give a final compound concentration of 1 μM. Samples were incubated at 37°C in the presence of a reduced nicotinamide adenine dinucleotide phosphate (NADPH) regenerating system (NADP, 1 mM, pH 7.4; glucose-6-phosphate, 5 mM, pH 7.4; glucose-6-phosphate dehydrogenase, 1 unit / mL). A positive control (midazolam, 1 μM, 0.1 mg / mL protein) was incubated simultaneously to ensure that the microsome preparation was performing as expected. Samples were taken at t=0, 30, 60 and 120 min and quenched by the addition of acetonitrile.

[0167] Quenched samples were analyzed by liquid chromatography coupled with tandem mass spectrometry detection (LC / MS-MS). The concentration of test compound at each time point was converted to % remaining relative to the concentration of compound at time t=0 (taken as 100% value). All data points were included in the data processing. The elimination rate constant (k el , min -1 ) fits experimental data to a single exponential decay equation (A t =A0e -kelt , where At is the remaining % at time t, A0 is 100%, k el The in vitro half-life (t 1 / 2 ) into the formula: t 1 / 2 =ln(2) / k el Based on k el The elimination rate constant was determined from the in vitro intrinsic clearance CL int-ミクロソーム It was also used to calculate (μL / min / mg protein).

[0168] Results - Compound structures are shown in Figure 2. The estimated in vitro elimination half-lives (t 1 / 2 ) and in vitro intrinsic clearance (CL int-ミクロソーム ) are shown in Table 2 and compared with the values ​​obtained for the parent compound, ambroxol. Table 2: In vitro elimination half-life (t 1 / 2 ) and intrinsic clearance (CL int-ミクロソーム ) [Table 2]

[0169] As shown in Table 2, deuteration of ambroxol results in an improvement of stability against metabolism, since the elimination half-life of the deuterated compound is increased by about 50% relative to ambroxol, regardless of the deuteration scheme. Interestingly, deuteration of the cyclohexyl ring without deuteration of the methylene group of the aminodibromobenzyl moiety (compound ZW-002) results in the same improvement of stability as deuteration of the methylene group (compound ZW-001). In contrast, deuteration of both the methylene group and the cyclohexyl ring (compound ZW-003) does not result in a synergistic or additive stabilizing effect, since the stability of this compound is characterized by the same elimination half-life.

[0170] Example 3: Prospective studies with compound 2 showing activity in improving lifespan Compound 2 is a multiply deuterated analog of ambroxol. As a representative example of the compounds of the present invention, this compound can be investigated for its lifespan improving activity as follows.

[0171] Two-month-old weaned male BDF1 mice, bred from pups, housed 1–4 animals per cage and fed Teklad 7013 NIH-31 rodent chow and water ad libitum, are divided into two approximately equal groups of 22–25 mice. One group continues to receive Teklad chow as before (control); the other group is switched to Teklad chow formulated with 300 mg of compound 2 per kg of chow. This formulation was designed to deliver 50 mg / kg / day (weight basis) to each mouse in the treatment group, based on the average ad libitum chow consumption of adult male BDF1 mice. Mice are maintained on this diet until natural death or until they reach 16 months of age, at which point the experiment is terminated. During the course of the experiment, all mice are periodically removed from their cages and handled in the process of being subjected to various sensorimotor, cognitive and / or behavioral tests.

[0172] Figure 3 provides animal data from the experiment described in the previous paragraph, in which ambroxol was used instead of compound 2. This shows that ambroxol can extend lifespan in a mouse animal model. Group 1 represents control animals that did not receive ambroxol; group 2 represents animals that received 50 mg / kg ambroxol as a chow supplement daily, starting at 2 months of age. The data also suggest that not only did ambroxol increase lifespan, but that when sacrificed, the surviving animals in group 2 appeared, on average, to be at least as healthy as the animals in group 1, indicating that healthspan was extended in parallel with lifespan. Compound 2 is expected to achieve similar results.

[0173] Example 4: Prospective studies with compound 2 showing activity in improving tremor in a mouse model of PD Compound 2 can be tested for its tremor-ameliorating activity in a mouse animal model of Parkinson's disease as follows.

[0174] Select 6-OHDA mice (i.e., mice injected into the striatum with 6-hydroxydopamine) that exhibit resting tremor. Administer 3 groups of mice, with at least 10 mice in each group, with a "high dose" (group 1: 150 mg / kg / day of compound 2), a "low dose" (group 2: 50 mg / kg / day of compound 2), or no administration of compound 2 (control group) and monitor the tremor of the mice daily using electromyography or force plate-based measurements (Bekar L et al., Nat Med 14:75-80,2008) to evaluate the effect of compound 2 on resting tremor.

[0175] Example 5: Prospective study with Compound 2 showing effects on aggregated Aβ in a mouse model of AD Compound 2 can be examined for its activity against amyloid β peptide (Aβ) aggregate deposits in a mouse animal model of Alzheimer's disease as follows.

[0176] Select an AD mouse model that shows a marked increase in the production of Aβ (e.g., mice containing the "Swedish mutation" in human amyloid precursor protein (APP)). Using three groups of mice; a "high dose" group (150 mg / kg / day of compound 2), a "low dose" group (50 mg / kg / day of compound 2), and a control group (no compound 2), an analysis can be performed after an appropriate treatment period for the relative occurrence of diffuse and fibrillar deposits of aggregated Aβ, e.g., by comparing silver staining or Aβ immunohistochemistry with Congo Red or Thioflavin S histology (Jankowsky J et al., Mol Neurodegen, 12:89, 2017).

[0177] Example 6: Prospective study using compound 2 showing activity in improving cognitive function Compound 2 can be tested for its activity in improving cognitive function in a mouse animal model as follows.

[0178] Three groups of mice, each containing at least 13 mice, are subjected to cognitive acuity testing at month 7. The three groups of mice are a "high dose" group (150 mg / kg / day of Compound 2), a "low dose" (50 mg / kg / day of Compound 2), and a control group (no Compound 2).

[0179] The results of such an experiment using ambroxol instead of compound 2 are shown in Figure 4. A dose-related improvement in cognitive acuity was observed, with the "high dose" group showing the best cognitive acuity results, the "low dose" group showing results between the control and "high dose" animals, and the control group showing baseline cognitive acuity results. The resulting doses are significantly lower than those previously observed to effectively promote GCase chaperone activity in mice (e.g., Migdalska-Richards et al., Ann Neurol 80:766-775,2016).

[0180] Example 7: Prospective studies with compound 2 showing induction of macroautophagy Compound 2 can be examined for its ability to induce macroautophagy in mouse cells in culture as follows.

[0181] Mouse fibroblasts (NIH3T3) were obtained from the American Type Culture Collection (ATCC). Cells are maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO) at 37 °C and 5% CO2 in the presence of 10% newborn calf serum (NCS), 50 μg / ml penicillin, and 50 μg / ml streptomycin. Cells plated in glass-bottom 96-well plates are treated for the indicated times and fixed before images are acquired using a high-content microscope (Operetta, PerkinElmer). For example, images of nine different fields of view per well can be captured, resulting in an average of 2,500-3,000 cells. Nuclei and puncta are identified using the manufacturer's software. The number of particles / puncta per cell can be quantified using the "particle identifier" function in the cytosolic region after thresholding on non-saturated images. In all cases, the focal plane thickness is set to 0.17 μm and the section with the largest nuclear diameter is selected for quantification. Values ​​can be presented as the number of puncta per cell section, which represents 10-20% of the total puncta per cell under the acquisition conditions. Macroautophagy activity in intact cells is measured upon transduction with lentivirus carrying an mCherry-GFP-LC3 tandem construct (Kimura S et al., Autophagy 3(5):452-460, 2007). Cells are plated in glass-bottom 96-well plates and fluorescence is read in both channels. Puncts positive for both fluorophores correspond to autophagosomes, whereas puncta positive only for the red fluorophore correspond to autolysosomes. Autophagy flux is determined as the conversion of autophagosomes (yellow puncta) to autolysosomes (only red puncta).

[0182] FIG. 5 shows the results of an equivalent experiment using ambroxol instead of compound 2. It appears that ambroxol may inhibit nutrient sensing apart from its pharmacological activity as a GCase chaperone. By interfering with nutrient sensing, ambroxol may induce a response by the animal's cells that favors the organism entering a nutrient-restricted or fasted state, which may result in the organism shifting into a "catabolic signaling" mode characterized by lysosomal biogenesis and autophagy induction, resulting in improved lifespan, healthspan, and / or cognitive acuity (see, e.g., Efeyan et al., Nature, 517:302-310, 2015) (incorporated herein by reference in its entirety). Thus, ambroxol and its multi-deuterated analogs and related compounds may systemically inhibit nutrient sensing, which may result in the entire organism entering a catabolic signaling mode.

[0183] The human equivalent doses (HED) calculated from the "low" and "high" mouse doses in this study are approximately 4 mg / kg / day and 12 mg / kg / day, which is approximately 250 mg / day or 750 mg / day for an average 62.5 kg human. Thus, in a preferred embodiment, the dosage is approximately 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 300 mg / day, 350 mg / day, 400 mg / day, 450 mg / day, 500 mg / day, 550 mg / day, 600 mg / day, 650 mg / day, 700 mg / day, 750 mg / day, 800 mg / day, 850 mg / day, 900 mg / day, 950 mg / day, 1000 mg / day, 1050 mg / day, 1100 mg / day, 1150 mg / day, 1200 mg / day, or 50-150 mg / day, 50-200 mg / day, 50-250 mg / day, 250-50 Long-term administration of a compound of formula I (or a pharma- ceutically acceptable salt, solvate or prodrug thereof) administered at a dose of 0 mg / day, 250 mg-1000 mg / day, or less than 1000 mg, or at a dose of approximately 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day, 11 mg / kg / day, 12 mg / kg / day, and / or 4-12 mg / kg / day, may be expected to be effective in improving healthspan, longevity and / or mental acuity.

[0184] Example 8: Formulation of multiply deuterated analogs into high drug-loading liquid oral pharmaceutical compositions and their properties Granule preparation - suitable granules of Compound 2 (as a representative example of a compound of the present invention) can be prepared as follows: Micronized Compound 2 in powder form is placed in a rotor granulator (GXR-35 rotor granulator, Freund-Vector Corporation) and a binder solution of hydroxypropyl cellulose (HPC-Klucel LF) is sprayed onto the ambroxol HCl powder to form granule cores. Additional Compound 2 powder is then co-sprayed with the binder solution to grow spheres. As layers of Compound 2 are added, the particles become more spherical. The resulting spheres may contain 97% Compound 2 and 3% HPC by weight. The particle size (x) of approximately 350 microns is approximately 1.5 microns. 50 ) and a density of about 0.7 g / ml can be achieved.

[0185] Applying a water-soluble seal coating to the granules - Compound 2 granules can be seal coated to produce a smooth, uniform substrate using a bottom spray fluid bed coater equipped with a Wurster column. The batch size can be approximately 750 grams. A suitable seal coating contains 9.1% by weight hypromellose (HPMC); 0.9% by weight triethyl citrate (TEC); and 90% by weight water. Granules can be seal coated to a 2% weight gain.

[0186] Applying an enteric coating to the granules - The seal coated Compound 2 granules can be enteric coated using a bottom spray fluid bed coater equipped with a Wurster column. The batch size can be approximately 750 grams. A suitable enteric coating contains 58.0% by weight Eudragit L30D55; 0.9% by weight triethyl citrate (TEC); 8.7% by weight Plasacryl T20; and 32.5% by weight water. The seal coated granules can be enteric coated to a weight gain of 35%.

[0187] Dissolution properties - The enteric coated granules can be tested for their enteric dissolution properties. The dissolution parameters of equivalent granules containing ambroxol hydrochloride are shown in Table 3. Table 3: Solubility parameters [Table 3]

[0188] Example 9: Study on the effect of deuterated ambroxol on human iPSC-derived neural cells The effect of deuterated ambroxol on human iPSC-derived neural cells was investigated and the results are provided in Figure 6. Human iPSC-derived neural cells were grown in culture for 14 days. Lysosomes were stained with "Lysotracker" and cells were fixed and imaged using confocal microscopy. Lysosomal size was measured using IMARIS software (Bitplane).

[0189] Example 10: Study of the effect of deuterated ambroxol on lysosomes, autophagosomes and TFEB gene expression The effect of deuterated ambroxol on lysosomes, autophagosomes and TFEB gene expression was examined and the results are provided in Figure 7. Mouse neuroblastoma (N2A) cells were treated with solvent alone (DMSO), 10 micromolar concentrations of ambroxol or deuterated ambroxol compounds D1 or D2 for 3 days. Cells were extracted and mRNA was quantified by qPCR.

[0190] The data obtained in Examples 9 and 10 show that: All cells grow in the presence of the compound All compounds increase lysosomal size in human neurons and in human neurons with Alzheimer's disease-causing mutations From this, it can be inferred that the function of not only β-glucocerebrosidase (UniProt Entry P04062) ("GBA"), but also lysosomal genes in general has been improved. All compounds increased the expression of lysosomal and autophagosomal genes, as well as the expression of TFEB. Some of these increases are significant improvements over the original ambroxol compound.

[0191] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0192] While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. Formula I: 【Transformation 8】 I or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein R ａ is selected from H, hydroxyl (OH), lower alkyl, and lower alcohol, optionally with one or more H atoms of any of the foregoing groups replaced with deuterium (D); R ｂ is H, deuterium (D) and 【Chemistry 9】 wherein R ｅ , R ｆ and R ｇ is independently selected from H and D; R ｃ and R ｄ is independently selected from H and D; R １ ~R １４ each is independently selected from H and D; The multiply deuterated compound comprises at least two deuterium (D) atoms, However, the compound is one of the following: 【Chemistry 10】 In the case of bis-deuterated ambroxol A shown in [D １１ ] - nor ambroxol B, A multiply deuterated compound or a pharmaceutically acceptable salt, solvate or prodrug thereof.

2. 10. The compound of claim 1, or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising at least three deuterium (D) atoms.

3. 10. The compound of claim 1, or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising at least 10 deuterium (D) atoms.

4. 2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein the deuterium atoms are provided only on the ring structure of the compound of formula I.

5. R １１ and R １２ 5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein at least one of: is deuterium (D).

6. R １１ and R １２ and R are deuterium (D), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

7. The compound is R １ ~R １０ Only in each of the following; １ ~R １０ , R １１ and R １２ Only in each of the following; １ ~R １０ , R １３ and R １４ or R １１ ~R １４ 5. The compound of claim 1, wherein each of the following is deuterated only at each of:

8. The compound is R １ ~R １４ 5. The compound of claim 1, wherein each of the following is deuterated:

9. 5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the compound is a multiply deuterated analog of ambroxol or a multiply deuterated analog of bromhexine.

10. The compound is selected from the group consisting of: 【Chemistry 11】 【Chemistry 12】 10. The compound of claim 1 selected from: or a pharmaceutically acceptable salt, solvate or prodrug thereof.

11. 10. A pharmaceutical composition comprising a compound of claim 1 (or a pharmaceutically acceptable salt, solvate or prodrug thereof), optionally in combination with a pharmaceutically acceptable carrier.

12. The composition of claim 11 , wherein the composition is a liquid oral pharmaceutical composition.

13. 13. The composition of claim 12, comprising a high load of the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) according to any one of claims 1 to 4, wherein the composition comprises: (i) the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); and (ii) at least one pharmaceutically acceptable excipient, wherein the compound of formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in the form of a granule having a granule core comprising from about 60 to about 97 weight percent of the active pharmaceutical ingredient and from about 3 to about 40 weight percent of the excipient, wherein the weight percents are based on the total weight of the granule core.

14. 13. The composition of claim 12, comprising a high load of the compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) according to any one of claims 1 to 4, the composition comprising: (i) the compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof); (ii) at least one pharmaceutically acceptable excipient; and (iii) a diluent, wherein the compound of Formula I (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) is in the form of granules having a granule core comprising from about 60 to about 97 weight percent of an active pharmaceutical ingredient and from about 3 to about 40 weight percent of the excipient, said weight percents being based on the total weight of the granule core; and the granule core is coated with (iv) a water-soluble seal coating in an amount to provide a weight gain of from about 0.5 to about 5%, and (v) an enteric coating in an amount to provide a weight gain of from about 0.5 to about 50%.

15. 10. A composition for preventing and / or treating a disease or medical condition in a subject selected from the group consisting of respiratory diseases and conditions, lysosomal storage diseases (LSDs), and neurological diseases and conditions, comprising a compound of claim 1 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof).

16. 16. The composition of claim 15, wherein the disease or medical condition to be prevented and / or treated is a bronchopulmonary disease, or Gaucher's disease, Pompe's disease or Fabry's disease, or Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease or frontotemporal dementia.

17. A composition for use in a method, the composition comprising a compound of claim 1 (or a pharmaceutically acceptable salt, solvate or prodrug thereof), wherein the method is for increasing life expectancy in a subject, or for treating, inhibiting or alleviating aging in a subject, or for treating, inhibiting or alleviating an age-related symptom or disease in a subject, or for increasing healthspan, lifespan and / or mental acuity in a subject.

18. 10. A composition for preventing and / or treating, alleviating symptoms of, and / or delaying progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition (including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD)) in a subject, comprising ambroxol (or related compounds such as ambroxol hydrochloride and bromhexine) or a compound according to claim 1 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof), wherein the composition is administered in combination with one or more suitable anti-beta amyloid antibodies or fragments thereof.

19. 10. A composition for preventing, alleviating symptoms of, and / or slowing the progression of Alzheimer's disease (AD) or other diseases associated with pathological protein misfolding, aggregation, and deposition, including Parkinson's disease (PD), Huntington's disease (HD), and frontotemporal degeneration (FTD), comprising ambroxol (or related compounds such as ambroxol hydrochloride and bromhexine) or a compound of claim 1 (or a pharmaceutically acceptable salt, solvate, or prodrug thereof).

20. 20. The composition of claim 18 or 19, wherein the subject is selected by assaying for biomarkers indicative of patients at risk or in the early stages of development of AD or other diseases associated with pathological protein misfolding, aggregation and deposition, including Parkinson's disease (PD), Huntington's disease (HD) and frontotemporal degeneration (FTD).

21. 21. The composition of claim 20, wherein the subject is selected by assaying for phosphorylated tau protein (p-tau), which is indicative of patients at risk for or in the early stages of AD development.

22. 20. The composition of claim 18 or 19, wherein the subject is selected by genotyping at least one gene or locus indicative of a patient at risk for AD or other diseases associated with pathological protein misfolding, aggregation and deposition, including Parkinson's disease (PD), Huntington's disease (HD) and frontotemporal degeneration (FTD).

23. 23. The composition of claim 22, wherein the subject is selected by genotyping of the ApoE gene, p-tau217, p-tau181, p-tau231, p-tau235 and / or N3pG.

24. 24. The composition of claim 23, wherein the subject is selected by genotyping the ε4 allele of the ApoE gene, p-tau217, p-tau181, p-tau231, p-tau235 and / or N3pG.

25. The ambroxol (or related compound) or the compound (or a pharmaceutically acceptable salt, solvate or prodrug thereof) is (i) a dose that provides a peak concentration in the subject's serum of greater than 1 μM, e.g., 2-50 μM, 2-25 μM, or 10-20 μM; (ii) a dose that provides a peak concentration in brain tissue of said subject that is greater than 3 μM, e.g., 5-50 μM, 5-25 μM, or 10-20 μM; or (iii) a dosage in the range of about 250 mg to 1000 mg / day or 750 to 1000 mg / day; 20. The composition of claim 18 or 19, wherein the composition is administered to the subject in a daily dose selected from the group consisting of:

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