Compositions for treating neurodegenerative diseases and methods thereof

Abstract

Disclosed herein are compounds and methods of use thereof effective for the treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), a disease that affects nerve cells in the brain and spinal cord, eventually causing loss of muscle strength. The compounds of the disclosure include, cutamesine, a synthetic sigma receptor agonist selective for the 81 receptor, and also a chaperone protein of the central nervous system that plays a key role in the modulation of calcium ions and apoptosis, as well as a sapogenin, such as smilagenin, a non-peptide neurotrophic factor that aids in the reversal of free radical neurotoxicity.

Description

COMPOSITIONS FOR TREATING NEURODEGENERATIVE DISEASES AND METHODS THEREOFCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 654,685, filed on May 31, 2024, and U.S. Provisional Patent Application No. 63 / 728,539, filed on December 5, 2024, each of which is incorporated by reference in its entirety.BACKGROUND

[0002] Neurodegenerative diseases, characterized by the progressive loss of structure or function of neurons, affect numerous people around the world. For example, amyotrophic lateral sclerosis, also known as ALS, is a disease that affects nerve cells in the brain and spinal cord, eventually causing loss of muscle strength. Early death is common as the loss of muscle strength eventually hinders vital functions, such as breathing.INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.SUMMARY OF THE INVENTION

[0004] In some embodiments disclosed herein are combinations comprising a therapeutically-effective amount of cutamesine or a pharmaceutically-acceptable salt thereof and a therapeutically-effective amount of smilagenin. In some embodiments disclosed herein are pharmaceutical compositions comprising a) cutamesine, or a pharmaceutically acceptable salt thereof, and b) a sapogenin. In some embodiments disclosed herein are methods for treating ALS in a subject, the methods comprising administering a compound comprising: a) cutamesine, or a pharmaceutically acceptable salt thereof, and b) a sapogenin.

[0005] In some embodiments, disclosed herein are pharmaceutical compositions comprising cutamesine or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is a sublingual dosage form. In some embodiments disclosed herein are pharmaceutical compositions comprising cutamesine or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the soliddosage form is subjected to a shake test in which the solid dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5°C to disintegrate the solid dosage form in the water, then a time needed for disintegration of the solid dosage form into fine particles is less than sixty seconds.

[0006] In some embodiments, disclosed herein are pharmaceutical compositions comprising cutamesine or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the solid dosage form is subjected to a dissolution study in which the solid dosage form is placed in a medium of 2 mL of distilled water at 25 °C in a 15 mL glass funnel for sixty seconds, at which point the medium is drawn by vacuum through a 0.45 pm membrane into a collection tube, after which the medium is analyzed by HPLC, then a percent dissolution of cutamesine or the pharmaceutically acceptable salt thereof of about 80% to about 95% is obtained.

[0007] In some embodiments, disclosed herein are methods for treating a condition, the methods comprising orally administering to a subject in need thereof a therapeutically- effective amount of cutamesine or a pharmaceutically-acceptable salt thereof.

[0008] In some embodiments, disclosed herein are methods for treating a condition, the methods comprising sublingually administering to a subject in need thereof a therapeutically - effective amount of cutamesine or a pharmaceutically-acceptable salt thereof.FIGURE DESCRIPTIONS

[0009] Figure 1 shows an example schematic representation of an in vitro differentiation protocol used to generate iPSC-derived motor neurons. The time-course and combination of small molecules used for each step is shown. AA: Ascorbic Acid, CHIR: CHIR99021, SB: SB431542, RA: Retinoic Acid, Pur: Purmorphamine, VPA: Valproic Acid, CpdE: Compound E, IGF-1: Insulin Growth Factor 1, BDNF: Brain derived neurotrophic factor, CNTF: Ciliary Neurotrophic Factor.

[0010] Figure 2 shows an example schematic representation of a glutamate and viability assay using an ATP-based luminescence assay.

[0011] Figure 3A shows a graph of neuronal survival of primary wildtype motor neurons injured with glutamate after administration of different amounts of cutamesine or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0012] Figure 3B shows a graph of neurite network integrity of primary wildtype motor neurons injured with glutamate after administration of different amounts of cutamesine orriluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0013] Figure 3C shows a graph of abnormal TDP43 translocation in primary wildtype motor neurons injured with glutamate after administration of different amounts of cutamesine or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0014] Figure 3D shows the raw data used to make Figures 3A-3C.

[0015] Figure 3E shows a graph of neuronal survival of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0016] Figure 3F shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0017] Figure 3G shows a graph of abnormal TDP43 translocation in primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0018] Figure 3H shows the raw data used to make Figures 3E-3G.

[0019] Figure 4A shows a graph of neuronal survival of primary wildtype motor neurons injured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0020] Figure 4B shows a graph of neurite network integrity of primary wildtype motor neurons injured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0021] Figure 4C shows a graph of abnormal TDP43 translocation in primary wildtype motor neurons injured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0022] Figure 4D shows the raw data used to make Figures 4A-4C.

[0023] Figure 4E shows a graph of neuronal survival of primary SOD1-G93A motor neuronsinjured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0024] Figure 4F shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0025] Figure 4G shows a graph of abnormal TDP43 translocation in primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of smilagenin or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0026] Figure 4H shows the raw data used to make Figures 4E-4G.

[0027] Figure 5 shows a chromatogram and chromatographic parameters of a cutamesine peak at 230 nm.

[0028] Figures 6A-6B show the 2D (Figure 6A) and 3D (Figure 6B) UV spectra of the cutamesine peak. The spectra were recorded from 200 to 500 nm.

[0029] Figures 7A-7B show chromatograms of cutamesine standard solution (Figure 7A) and cutamesine standard solution in the presence of excipients (Figure 7B).

[0030] Figures 8A-8B show the linearity of cutamesine standard solutions (Figure 8A) and reconstituted standard solutions (Figure 8B).

[0031] Figures 9A-9L show results from the degradation study of cutamesine. Figure 9A shows the stress conditions of the degradation study. Cutamesine levels are expressed as the percent recovery versus the control. Figure 9B shows degradation under acidic hydrolysis conditions. Figure 9C shows chromatograms of stressed solutions under acidic hydrolysis conditions. Figure 9D shows degradation under basic hydrolysis conditions. Figure 9E shows chromatograms of stressed solutions under basic hydrolysis conditions. Figure 9F shows degradation under oxidizing conditions. Figure 9G shows chromatograms of stressed solutions under oxidizing conditions. Figure 9H shows degradation under photolytic conditions. Figure 91 shows chromatograms of stressed solutions under photolytic conditions. Figure 9J shows photolysis. Figure 9K shows chromatograms of stressed solutions under thermal conditions. Figure 9L shows a summary of the degradation results.

[0032] Figure 10 shows an example schematic diagram of the in vitro dissolution method.

[0033] Figures 11A-11C show a placebo tablet (Figure 11A) and cutamesine sublingual tablets at 1 mg (Figure 11B) and 3 mg (Figure 11C).

[0034] Figure 12 shows mass variation of cutamesine sublingual tablets.

[0035] Figure 13 shows uniformity of drug content in cutamesine tablets.

[0036] Figure 14A shows weight loss curves recorded during differential thermal analysis of sublingual cutamesine tablets. Figure 14B shows differential calorimetry curves recorded during differential thermal analysis of sublingual cutamesine tablets.

[0037] Figure 15A shows a chromatogram of an extracted 1 mg cutamesine sublingual tablet after thermal exposure at 70 °C. Figure 15B shows retention times and relative areas from the chromatogram of Figure 15A.

[0038] Figure 16 shows an example schematic of a cell culture insert.

[0039] Figure 17 shows an example schematic of a cell preparation method.

[0040] Figures 18A-18B show the sample preparation of a cutamesine solution (Figure 18A) and a cutamesine tablet (Figure 18B) for bioavailability assays.

[0041] Figure 19A shows a plate plan for a bioavailability assay. Figure 19B shows another plate plan for a bioavailability assay. PCT stands for placebo cutamesine tablet, CT stands for cutamesine tablet, and C stands for cutamesine.

[0042] Figure 20A shows resistance (R) and trans epithelial electrical resistance (TEER) values calculated for each condition reported in a plate plan. Figure 20B shows resistance (R) and trans epithelial electrical resistance (TEER) values calculated for each condition reported in another plate plan. R is the resistance to ion flux between the apical and basolateral sides of the cell monolayer and therefore the integrity of the intercellular tight junctions. TEER values represent the resistance per surface unit corrected by subtraction of the background (“Control without cell monolayer”). Under the experimental conditions described herein, the integrity of the cell monolayers was maintained along all tests.

[0043] Figure 21A shows kinetics results of the transfer of cutamesine through a SCC-4 cell monolayer. Figure 21B shows a graph of kinetic transfer results of cutamesine through a SCC-4 cell monolayer. Figure 21C shows additional kinetics results of the transfer of cutamesine through a SCC-4 cell monolayer. Figure 21D shows a graph of additional kinetic transfer results of cutamesine through a SCC-4 cell monolayer.

[0044] Figure 22A shows a graph of neuronal survival of primary wildtype motor neurons injured with glutamate after administration of increasing amounts of cutamesine (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (30 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0045] Figure 22B shows a graph of neuronal network integrity of primary wildtype motorneurons injured with glutamate after administration of increasing amounts of cutamesine (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (30 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0046] Figure 22C shows a graph of cytoplasmic accumulation of TDP43 in primary wildtype motor neurons injured with glutamate after administration of increasing amounts of cutamesine (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (30 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0047] Figure 22D shows the raw data used to make Figures 22A-22C.

[0048] Figure 22E shows a graph of neuronal survival of primary wildtype motor neurons injured with glutamate after administration of increasing amounts of smilagenin (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (100 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0049] Figure 22F shows a graph of neuronal network integrity of primary wildtype motor neurons injured with glutamate after administration of increasing amounts of smilagenin (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (100 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0050] Figure 22G shows a graph of cytoplasmic accumulation of TDP43 in primary wildtype motor neurons injured with glutamate after administration of increasing amounts of smilagenin (10 nM, 30 nM, 100 nM, 300 nM, 1 pM, 3 pM), cutamesine (100 nM) and smilagenin (300 nM), or riluzole (5 pM). Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0051] Figure 22H shows the raw data used to make Figures 22E-22G.

[0052] Figure 221 shows a graph of cytoplasmic accumulation of phosphorylated TDP43 in primary wildtype motor neurons injured with glutamate (5 pM, 20 min) after administration of increasing amounts of cutamesine (10 nM, 30 nM) and smilagenin (30 nM, 100 nM), or cutamesine and smilagenin (10 nM cutamesine and 30 nM smilagenin, 10 nM cutamesine and 100 nM smilagenin, 30 nM cutamesine and 30 nM cutamesine, and 30 nM cutamesine and 100 nM smilagenin. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0053] Figure 23A shows a graph of neuronal survival of primary SOD1-G93A motorneurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (30 nM or 100 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0054] Figure 23B shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (30 nM or 100 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0055] Figure 23C shows a graph of cytoplasmic accumulation of TDP43 in primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (30 nM or 100 nM), or both. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0056] Figure 23D shows the raw data used to make Figures 23A-23C.

[0057] Figure 23E shows a graph of neuronal survival of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (300 nM or 1000 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0058] Figure 23F shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (300 nM or 1000 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0059] Figure 23G shows a graph of cytoplasmic accumulation of TDP43 in primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (10 nM or 30 nM), smilagenin (300 nM or 1000 nM), or both. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0060] Figure 23H shows the raw data used to make Figures 23E-23G.

[0061] Figure 24A shows a graph of neuronal survival of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (30 nM or 100 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0062] Figure 24B shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (30 nM or 100 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0063] Figure 24C shows a graph of cytoplasmic accumulation of TDP43 in primarySOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (30 nM or 100 nM), or both. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0064] Figure 24D shows the raw data used to make Figures 24A-24C.

[0065] Figure 24E shows a graph of neuronal survival of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (300 nM or 1000 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0066] Figure 24F shows a graph of neurite network integrity of primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (300 nM or 1000 nM), or a combination thereof. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0067] Figure 24G shows a graph of cytoplasmic accumulation of TDP43 in primary SOD1-G93A motor neurons injured with glutamate after administration of different amounts of cutamesine (100 nM or 300 nM), smilagenin (300 nM or 1000 nM), or both. Data were calculated using a one-way ANOVA followed by Fisher’s LSD test, p<0.05 versus glutamate.

[0068] Figure 24H shows the raw data used to make Figures 24E-24G.

[0069] Figure 25 shows X-ray diffraction spectra of cutamesine polymorphic forms Form A- 1, Form A-2, and Form B acquired with an X-ray wavelength of 1.5406 A.

[0070] Figure 26 shows a differential scanning calorimetry curve of cutamesine polymorph Form A-l.

[0071] Figure 27 shows the thermogravimetric (TGA) graph of cutamesine polymorph Form A-l.

[0072] Figure 28 shows a differential scanning calorimetry curve of cutamesine polymorph Form B.

[0073] Figure 29 shows the thermogravimetric (TGA) graph of cutamesine polymorphic Form B.

[0074] Figure 30 shows the dynamic vapor sorption (DVS) spectrum of cutamesine polymorph Form A-l across two cycles of adsorption from 0-90% relative humidity (RH) and desorption from 90%-0% RH in 10% RH increments.

[0075] Figure 31 shows the isotherm plots for cutamesine polymorph Form A-l across the two DVS cycles.

[0076] Figure 32 shows representative images of cutamesine polymorph Form A-l taken during the DVS analysis at 0% RH of the first cycle, 90% RH of the first cycle, and 0% RHof the first cycle during desorption.

[0077] Figure 33 shows the X-ray diffraction spectra of cutamesine polymorph Form A-l acquired before and after DVS analysis acquired with an X-ray wavelength of 1.5406 A.

[0078] Figure 34 shows the dynamic vapor sorption (DVS) spectrum of cutamesine polymorph Form B across two cycles of adsorption from 0-90% relative humidity (RH) and desorption from 90%-0% RH in 10% RH increments.

[0079] Figure 35 shows the isotherm plots for cutamesine polymorph Form B across the two DVS cycles.

[0080] Figure 36 shows representative images of cutamesine polymorph Form B taken during the DVS analysis at 0% RH of the first cycle, 90% RH of the first cycle, and 0% RH of the first cycle during desorption.

[0081] Figure 37 shows the X-ray diffraction spectra of cutamesine polymorph Form B acquired before and after DVS analysis acquired with an X-ray wavelength of 1.5406 A.

[0082] Figure 38A shows an example schematic of an in vitro TDP-43 mislocalization imaging assay.

[0083] Figure 38B shows a graph of neurite length percentage compared to a control of mtSODl motor neurons injured with glutamate after administration of cutamesine (100 nM), smilagenin (300 nM), or a combination thereof.

[0084] Figure 38C shows a graph of cytosolic TDP43 percentage compared to a control of mtSODl motor neurons injured with glutamate after administration of cutamesine (100 nM), smilagenin (300 nM), or a combination thereof.

[0085] Figure 39A illustrates an experimental protocol of mice treated with cutamesine, smilagenin, and cutamesine and smilagenin. Figure 39B shows the results of a short combination trial in SOD1-G93A mice. Mice were treated daily from day 70 to day 105 with NeuroScores provided at day 105. NeuroScores range from 0 (non-symptomatic) to 6 (highly symptomatic). A score of 7 or 8 is end stage. Figures 39C-39E show volcano plots of differentially expressed genes from the lumbar spinal cord of SOD1-G93A mice treated with cutamesine (Figure 39C), smilagenin (Figure 39D), or both cutamesine and smilagenin (Figure 39E) versus a vehicle control. Lumbar spinal cord tissue was collected at day 105 of treatment.

[0086] Figure 40 shows an example schematic of a Phase IB trial.

[0087] Figure 41A illustrates a schematic of co-culture experiments with spinal explants and neuromuscular junctions to assess muscle innervation.

[0088] Figure 41B illustrates the mean number of neuromuscular junctions of co-culturedspinal cord explants and myoblasts injured with glutamate following treatment with cutamesine, smilagenin, and cutamesine and smilagenin.

[0089] Figure 41C illustrates the mean area of neuromuscular junctions of co-cultured spinal cord explants and myoblasts injured with glutamate following treatment with cutamesine, smilagenin, and cutamesine and smilagenin.

[0090] Figure 41D illustrates the neurite network of co-cultured spinal cord explants and myoblasts injured with glutamate following treatment with cutamesine, smilagenin, and cutamesine and smilagenin.

[0091] Figure 41E illustrates the neurite network close to neuromuscular junctions of co- cultured spinal cord explants and myoblasts injured with glutamate following treatment with cutamesine, smilagenin, and cutamesine and smilagenin.DETAILED DESCRIPTION OF THE INVENTION

[0092] Amyotrophic lateral sclerosis (ALS) is a fatal disorder characterized by subtle onset of focal weakness, typically in the limbs but sometimes in bulbar muscles. ALS progresses to paralysis of almost all skeletal muscles. Significant clinico-pathological and genetic overlap exists between ALS and frontotemporal dementia (FTD) (e.g., frontotemporal lobar dementia (FTLD)). In ALS, death from respiratory paralysis is common within five years. The cellular pathology is focal at onset and spreads in a pattern which can suggest successive involvement of contiguous neuronal populations. Death of motor neurons occurs in conjunction with deposition of aggregated proteins in motoneurons and oligodendrocytes, and neuroinflammation. While most cases of ALS are sporadic (SALS), about 10% are inherited, usually dominantly (e.g., familial ALS (FALS)).

[0093] TDP-43 (Transactivating response element DNA binding protein 43 kDa) accumulates in cytoplasm of motor neurons in most cases of ALS. TDP-43 is a nuclear RNA- binding protein involved in several aspects of RNA processing that actively shuttles between the nucleus and cytoplasm. In ALS and frontotemporal dementia, TDP-43 is excluded from the nucleus. However, such cytoplasmic mislocalization is common in neuronal injury or stress. TDP-43 -positive inclusions can represent secondary pathology in some neurodegenerative disorders. Possible mechanisms of death in motor neurons in ALS include: a) glutamate-mediated excitotoxicity; b) decrease in neurotrophic factors (BDNF, GDNF) and associated signaling; c) mitochondrial alterations and oxidative damages; and d) abnormalities in cytoskeletal proteins resulting in neuronal atrophy and death.Compounds of the Disclosure

[0094] Described herein are compositions of cutamesine, sapogenins, pharmaceutically- acceptable salts thereof, and combinations thereof.

[0095] Cutamesine is a synthetic sigma receptor agonist selective for the ol receptor (SIGMAR1), a chaperone protein of the central nervous system that plays a key role in the modulation of calcium ions and apoptosis. Cutamesine has a molecular formula of C23H32N2O2 and a structure of:

[0096] Cutamesine has a core piperazine ring. Each nitrogen atom of the piperazine ring has a phenalkyl substituent. The phenethyl group has methoxy groups on the 3' and 4' locations of the phenyl ring. The phenpropyl group does not have any additional substituents.

[0097] Saponins are organic chemicals that can have a foamy quality when agitated in water. Saponins can be plant-derived or synthetically produced. Saponins are both water and fat soluble. Saponins can be classified into at least eleven categories, including dammaranes, tirucallanes, lupanes, hopanes, oleananes, taraxasteranes, ursanes, cycloartanes, lanostanes, cucurbitanes, and steroids.

[0098] Steroidal saponins are formed when hydrophilic glycoside moieties attach to a lipophilic steroidal group. Often found naturally as plant-specific metabolites essential to plant stress responses, steroidal saponins and their derivatives can be used as steroidal drugs.

[0099] In some steroidal saponins, the glycosyl group on the glycoside is replaced by a hydrogen atom, creating an aglycone. Sapogenins are aglycones of saponins. Sapogenins can comprise steroids or other triterpene frameworks as a primary organic feature. Non-limiting examples of sapogenins include tiggenin, geogitogenin, tokorogenin, diosgenin, hecogenin, smilagenin, yamogenin, solasodin, tomatidine, ketotigogenin, laxogenin, sarsasapogenin, and epismilagenin.

[0100] Smilagenin is a non-peptide neurotrophic factor that aids in the reversal of free radical neurotoxicity. Smilagenin is a steroidal sapogenin with a molecular formula of C27H44O3 and a structure of:Pharmaceutically-Acceptable Salts

[0101] The invention provides the use of pharmaceutically-acceptable salts of any compound described herein. Pharmaceutically-acceptable salts include, for example, acid-addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically- acceptable salt is an ammonium salt.

[0102] Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[0103] In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

[0104] Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N- methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, piprazole, imidazole, or pyrazine.

[0105] In some embodiments, an ammonium salt is a tri ethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N- ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, apiprazole salt, an imidazole salt, or a pyrazine salt.

[0106] Acid addition salts can arise from the addition of an acid to a compound of the invention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

[0107] In some embodiments, the salt is a hydrochloride salt, a dihydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccarate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt, or a maleate salt. In some embodiments, the salt is a dihydrochloride salt.

[0108] In some embodiments, the salt is a dihydrochloride salt, forming cutamesine dihydrochloride. Cutamesine dihydrochloride has a chemical formula of C23H32N2O2 • 2HC1 and a structure of:Cl-H

[0109] Cutamesine can be produced by the process below (3 step synthesis). In conversion 1, (3-bromopropyl)benzene) can react with 2,2'-azanediylbis(ethan-l-ol) in the presence of K2CO3 and ethanol to produce 2,2'-((3-phenylpropyl)azanediyl)bis(ethan-l-ol). In conversion 2, 2,2'-((3-phenylpropyl)azanediyl)bis(ethan-l-ol) can react with thionyl chloride in the presence of chloromethane to produce N,N-bis(2-chloroethyl)-3-phenylpropan-l -amine hydrochloride salt. In conversion 3, N,N-bis(2-chloroethyl)-3-phenylpropan-l -aminehydrochloride salt can react with 2-(3,4-dimethoxyphenyl)ethan-l -amine in the presence of K2CO3, Nal, and DMF, and then HC1 and ethanol to produce cutamesine dihydrochloride salt. In this process, the intermediate N,N-bis(2-chloroethyl)-3-phenylpropan-l -amine hydrochloride salt from conversion 2 is analogous to nitrogen mustard.Conversion 1 Conversion 2

[0110] The present disclosure provides a process for the synthesis of Cutamesine and Cutamesine salt that does not involve the use of any nitrogen mustard compound (four step synthesis, as shown below and in more detail in Example 9).Reaction 1 Reaction 2

[0111] In reaction one, 1-Boc-piperazine can be alkylated with an alkyl halide (3-bromopropyl)benzene in the presence of a base (e.g., K2CO3) and a solution (e.g., isopropanol) to produce / c / 7-butyl 4-(3-phenylpropyl)piperazine-l -carboxylate. In reaction two, / c / 7-butyl 4-(3-phenylpropyl)piperazine-l -carboxylate can be deprotected to remove the Boc protecting group in an acidic solution (e.g., HC1 solution and isopropanol) to form l-(3- phenylpropyl)piperazine dihydrochloride. In reaction three, 2-(3,4-dimethoxyphenyl)ethanol can react with -toluene sulfonyl chloride in the presence of a base (e.g., tetramethylethylenediamine) to form 3,4-dimethoxyphenylethyle tosylate. In reaction four, 3,4-dimethoxyphenylethyle tosylate and 1 -(3 -phenylpropyl )piperazine dihydrochloride can react in the presence of a base (e.g., K2CO3) to form cutamesine. The cutamesine can further react with HC1 to form cutamesine dihydrochloride salt. The cutamesine dihydrochloride salt can be recrystallized to improve the purity. In some embodiments, the purity of the cutamesine dihydrochloride salt can be at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.7%, at least about 99.8%, at least about 99.9%, or higher. In some embodiments, a process can produce at least about 1 g, at least about 10 g, at least about 100 g, at least 1000 g, at least about 10 kg, at least about 100 kg, or more cutamesine or a salt thereof. In some embodiments, a process can have a yield of cutamesine or a salt thereof of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or higher.Formulations

[0112] A pharmaceutical composition of the disclosure can provide a therapeutically - effective amount of cutamesine or a pharmaceutically-acceptable salt thereof. A pharmaceutical composition of the disclosure can provide a therapeutically-effective amount of smilagenin. A pharmaceutical composition of the disclosure can provide a therapeutically- effective amount of a combination of smilagenin and cutamesine. A pharmaceutical composition of the disclosure can provide a therapeutically-effective amount of a combination of cutamesine, or a pharmaceutically-acceptable salt thereof, and smilagenin. A pharmaceutical composition of the disclosure can provide a therapeutically-effective amount of a combination of cutamesine, or a pharmaceutically-acceptable salt thereof, and a sapogenin.

[0113] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a solid dosage form. Alternatively, in some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a liquid dosage form. Alternatively, insome embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a sublingual dosage form.

[0114] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of from about 0. 1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0. 1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 5 mg, from about 0.5 mg to about 10 mg, from about 0. 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, or from about 95 mg to about 100 mg.

[0115] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of about 0. 1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg. In someembodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of about 3 mg. In some embodiments, cutamesine dihydrochloride is present in a formulation in an amount of about 3 mg. In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of at most about 3 mg. In some embodiments, cutamesine dihydrochloride is present in a formulation in an amount of at most about 3 mg. In some embodiments, cutamesine or a pharmaceutically- acceptable salt thereof is present in a formulation in an amount of about 6 mg. In some embodiments, cutamesine dihydrochloride is present in a formulation in an amount of about 6 mg. In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof is present in a formulation in an amount of at most about 6 mg. In some embodiments, cutamesine dihydrochloride is present in a formulation in an amount of at most about 6 mg.

[0116] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof (e.g., cutamesine dihydrochloride) is present in a formulation in a concentration of at least about 1 nM, at least about 2 nM, at least about 3 nM, at least about 4 nM, at least about 5 nM, at least about 6 nM, at least about 7 nM, at least about 8 nM, at least about 9 nM, at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1 pM, at least about 2 pM, at least about3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about 18 pM, at least about 19 pM, or at least about 20 pM.

[0117] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof (e.g., cutamesine dihydrochloride) is present in a formulation in a concentration of at most about 20 pM, at most about 19 pM, at most about 18 pM, at most about 17 pM, at most about 16 pM, at most about 15 pM, at most about 14 pM, at most about 13 pM, at most about 12 pM, at most about 11 pM, at most about 10 pM, at most about 9 pM, at most about 8 pM, at most about 7 pM, at most about 6 pM, at most about 5 pM, at most about 4 pM, at most about 3 pM, at most about 2 pM, at most about 1 pM, at most about 950 nM, at most about 900 nM,at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 90 nM, at most about 80 nM, at most about 70 nM, at most about 60 nM, at most about 50 nM, at most about 40 nM, at most about 30 nM, at most about 20 nM, at most about 10 nM, at most about 9 nM, at most about 8 nM, at most about 7 nM, at most about 6 nM, at most about 5 nM, at most about 4 nM, at most about 3 nM, at most about 2 nM, or at most about 1 nM. In some embodiments, the molarity of the cutamesine formulation is determined by the molarity of the cutamesine salt (e.g., cutamesine dihydrochloride). Alternatively, in some embodiments, the molarity of the cutamesine formulation is determined by the molarity of the cutamesine base.

[0118] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof (e.g., cutamesine dihydrochloride) is present in a formulation in an amount of from about 0.1 mg / mL to about 100 mg / mL, from about 0.1 mg / mL to about 1 mg / mL, from about 0.1 mg / mL to about 5 mg / mL, from about 5 mg / mL to about 10 mg / mL, from about 10 mg / mL to about 15 mg / mL, from about 15 mg / mL to about 20 mg / mL, from about 20 mg / mL to about 25 mg / mL, from about 25 mg / mL to about 30 mg / mL, from about 30 mg / mL to about 35 mg / mL, from about 35 mg / mL to about 40 mg / mL, from about 40 mg / mL to about 45 mg / mL, about 45 mg / mL to about 50 mg / mL, from about 50 mg / mL to about 55 mg / mL, from about 55 mg / mL to about 60 mg / mL, from about 60 mg / mL to about 65 mg / mL, from about 65 mg / mL to about 70 mg / mL, from about 70 mg / mL to about 75 mg / mL, about 75 mg / mL to about 80 mg / mL, from about 80 mg / mL to about 85 mg / mL, from about 85 mg / mL to about 90 mg / mL, from about 90 mg / mL to about 95 mg / mL, or from about 95 mg / mL to about 100 mg / mL. In some embodiments, the concentration of the cutamesine formulation is determined by the concentration of the cutamesine salt (e.g., cutamesine dihydrochloride). Alternatively, in some embodiments, the concentration of the cutamesine formulation is determined by the concentration of the cutamesine base.

[0119] In some embodiments, cutamesine or a pharmaceutically-acceptable salt thereof (e.g., cutamesine dihydrochloride) is present in a formulation in an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of the cutamesine formulation is determined by the concentration of the cutamesine salt (e.g., cutamesine dihydrochloride). Alternatively, in some embodiments, the concentration of the cutamesine formulation is determined by the concentration of the cutamesine base.

[0120] In some embodiments, smilagenin is present in a solid dosage form. In some embodiments, smilagenin is present in a formulation in an amount of from about 0.1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0.1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 to about 105 mg, from about 105 mg toabout 110 mg, from about 110 mg to about 115 mg, from about 115 mg to about 120 mg, from about 120 mg to about 125 mg, from about 125 mg to about 130 mg, from about 130 to me about 135 mg, from about 135 mg to about 140 mg, from about 140 mg to about 145 mg, from about 145 mg to about 150 mg from about 150 mg to about 155 mg, from about 155 mg to about 160 mg, from about 160 mg to about 165 mg, from about 165 mg to about 170 mg, from about 170 mg to about 175 mg, from about 175 mg to about 180 mg, from about 180 mg to about 185 mg, from about 185 mg to about 190 mg, from about 190 mg to about 195 mg, or from about 195 mg to about 200 mg.

[0121] In some embodiments, smilagenin is present in a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg. In some embodiments, smilagenin is present in an amount of 90 mg. In some embodiments, smilagenin is present in an amount of at most 90 mg. In some embodiments, smilagenin is present in an amount of 180 mg. In some embodiments, smilagenin is present in an amount of at most 180 mg.

[0122] In some embodiments, smilagenin is present in a formulation in a concentration of at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about90 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1 pM, at least about 2 pM, at least about 3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, at least about 10 pM, at least about 11 pM, at least about 12 pM, at least about 13 pM, at least about 14 pM, at least about 15 pM, at least about 16 pM, at least about 17 pM, at least about 18 pM, at least about 19 pM, or at least about 20 pM.

[0123] In some embodiments, smilagenin is present in a formulation in a concentration of at most about 20 pM, at most about 19 pM, at most about 18 pM, at most about 17 pM, at most about 16 pM, at most about 15 pM, at most about 14 pM, at most about 13 pM, at most about 12 pM, at most about 11 pM, at most about 10 pM, at most about 9 pM, at most about 8 pM, at most about 7 pM, at most about 6 pM, at most about 5 pM, at most about 4 pM, at most about 3 pM, at most about 2 pM, at most about 1 pM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about100 nM, at most about 90 nM, at most about 80 nM, at most about 70 nM, at most about 60 nM, at most about 50 nM, at most about 40 nM, at most about 30 nM, at most about 20 nM, or at most about 10 nM.

[0124] In some embodiments, a formulation of cutamesine and smilagenin comprises a ratio of one-part cutamesine to one-part smilagenin. For example, a formulation containing about 100 mg of cutamesine or a pharmaceutically-acceptable salt thereof can comprise about 100 mg of smilagenin. In some embodiments, the ratio can be based on the number, or moles, of a cutamesine compound compared to the number, or moles, of a smilagenin compound (cutamesine: smilagenin). In some embodiments, the ratio can be based on the mass of a cutamesine compound compared to the mass of a smilagenin compound. In some embodiments, the ratio can be based on the mass of the parent ion of a pharmaceutically- acceptable salt (e.g., cutamesine dihydrochloride). In some embodiments, the ratio can be based on the mass of the whole salt of a pharmaceutically-acceptable salt of cutamesine (e.g., cutamesine dihydrochloride). In some embodiments, the ratio can be based on the mass of theparent ion, the whole salt, or the pharmaceutically-acceptable salt of cutamesine. Additional non-limiting ratios can be about 20:about 1; about 19.9:about 1; about 19.8:about 1; about 19.7:about 1; about 19.6:about 1; about 19.5:about 1; about 19.4:about 1; about 19.3:about 1; about 19.2:about 1; about 19.1 :about 1; about 19:about 1; about 18.9:about 1; about 18.8:about 1; about 18.7:about 1; about 18.6:about 1; about 18.5:about 1; about 18.4:about 1; about 18.3:about 1; about 18.2:about 1; about 18.1:about 1; about 18:about 1; about 17.9:about 1; about 17.8:about 1; about 17.7:about 1; about 17.6:about 1; about 17.5:about 1; about 17.4:about 1; about 17.3:about 1; about 17.2:about 1; about 17.1 :about 1; about 17:about 1; about 16.9:about 1; about 16.8:about 1; about 16.7:about 1; about 16.6:about 1; about 16.5:about 1; about 16.4:about 1; about 16.3:about 1; about 16.2:about 1; about 16.1 :about 1; about 16:about 1; about 15.9:about 1; about 15.8:about 1; about 15.7:about 1; about 15.6:about 1; about 15.5:about 1; about 15.4:about 1; about 15.3:about 1; about 15.2:about 1; about 15.1 :about 1; about 15:about 1; about 14.9:about 1; about 14.8:about 1; about 14.7:about 1; about 14.6:about 1; about 14.5:about 1; about 14.4:about 1; about 14.3:about 1; about 14.2: about 1; about 14.1 :about 1; about 14:about 1; about 13.9:about 1; about 13.8:about 1; about 13.7:about 1; about 13.6:about 1; about 13.5:about 1; about 13.4:about 1; about 13.3:about 1; about 13.2:about 1; about 13.1:about 1; about 13:about 1; about 12.9:about 1; about 12.8:about 1; about 12.7:about 1; about 12.6:about 1; about 12.5:about 1; about 12.4:about 1; about 12.3:about 1; about 12.2:about 1; about 12.1 :about 1; about 12:about 1; about 11.9:about 1; about 11.8:about 1; about 11.7:about 1; about 11.6:about 1; about 11.5:about 1; about 11.4:about 1; about 11.3:about 1; about 11.2:about 1; about l l. l :about 1; about l l :about 1; about 10.9:about 1; about 10.8:about 1; about 10.7:about 1; about 10.6:about 1; about 10.5:about 1; about 10.4:about 1; about 10.3:about 1; about 10.2:about 1; about 10.1 :about 1; about 10:about 1; about 9.9:about 1; about 9.8:about 1; about 9.7:about 1; about 9.6:about 1; about 9.5:about 1; about 9.4:about 1; about 9.3:about 1; about 9.2:about 1; about 9.1:about 1; about 9:about 1; about 8.9:about 1; about 8.8:about 1; about 8.7:about 1; about 8.6:about 1; about 8.5:about 1; about 8.4:about 1; about 8.3:about 1; about 8.2:about 1; about 8.1 :about 1; about 8:about 1; about 7.9:about 1; about 7.8:about 1; about 7.7:about 1; about 7.6:about 1; about 7.5:about 1; about 7.4:about 1; about 7.3:about 1; about 7.2:about 1; about 7.1 :about 1; about 7:about 1; about 6.9:about 1; about 6.8:about 1; about 6.7:about 1; about 6.6:about 1; about 6.5:about 1; about 6.4:about 1; about 6.3:about 1; about 6.2:about 1; about 6.1 :about 1; about 6:about 1; about 5.9:about 1; about 5.8:about 1; about 5.7:about 1; about 5.6:about 1; about 5.5:about 1; about 5.4:about 1; about 5.3:about 1; about 5.2:about 1; about 5.1 :about 1; about 5:about 1; about 4.9:about 1; about 4.8:about 1;about 4.7:about 1; about 4.6:about 1; about 4.5:about 1; about 4.4:about 1; about 4.3:about 1; about 4.2:about 1; about 4.1 :about 1; about 4:about 1; about 3.9:about 1; about 3.8:about 1; about 3.7:about 1; about 3.6:about 1; about 3.5:about 1; about 3.4:about 1; about 3.3:about 1; about 3.2:about 1; about 3.1 :about 1; about 3:about 1; about 2.9:about 1; about 2.8:about 1; about 2.7:about 1; about 2.6:about 1; about 2.5:about 1; about 2.4:about 1; about 2.3:about 1; about 2.2:about 1; about 2.1 :about 1; about 2:about 1; about 1.9:about 1; about 1.8:about 1; about 1.7:about 1; about 1.6:about 1; about 1.5:about 1; about 1.4:about 1; about 1.3:about 1; about 1.2:about 1; about l.l :about 1; about l:about 1; about l :about 5, about l :aboutlO; about l :about!5; about 1: about 20; about 1: about 25; about 1: about 30; about 1: about 40; about 1 : about 45; about 1 : about 50; about 1 : about 55; about 1 : about 60; about 1 : about 65; about l :about 70; about l :about 75; about l :about 80; about l :about 85; about l :about 90; about l :about 95; about l :about 100; about l:about 105; about l :about 110; about l:about 115; about l:about 120; about l :about 125; about l:about 130; about l :about 140; about l :about 145; about l:about 150; about l :about 155; about l:about 160; about l :about 165; about l :about 170; about l:about 175; about l :about 180; about l:about 185; about l :about 190; about l:about 195; or about l :about 200 cutamesine:smilagenin. In some embodiments, a formulation of cutamesine and smilagenin comprises a ratio of 3: 180 cutamesine:smilagenin. In some embodiments, a formulation of cutamesine and smilagenin comprises a ratio of 1 : 15 cutamesine: smilagenin. In some embodiments, a formulation of cutamesine and smilagenin comprises a ratio of 1:30 cutamesine: smilagenin. In some embodiments, a formulation of cutamesine and smilagenin comprises a ratio of 1:60 cutamesine: smilagenin.

[0125] A formulation that is disclosed herein can be made more soluble, for example, by the addition of an additive or agent. A non-limiting example of a solubilizing agent includes an organic solvent. Non-limiting examples of organic solvents include alcohols, for example, C1-C4 linear alkyl, C3-C4 branched alkyl, ethanol, ethylene glycol, glycerin, 2-hydroxypropanol, propylene glycol, maltitol, sorbitol, xylitol; substituted or unsubstituted aryl, and benzyl alcohol.

[0126] A formulation as disclosed herein can comprise a therapeutic compound (e.g., cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof) and an additive in a ratio of 1: 1 therapeutic compound: additive. For example, a formulation containing about 100 mg of a therapeutic compound can comprise about 100 mg of an additive. In another embodiment, the ratio can be based on the number, or moles, of a therapeutic compound compared to the number, or moles, of an additive. Additional non-limiting ratios can be about 20:about 1; about 19.9:about 1; about 19.8:about 1; about 19.7:about 1; about 19.6:about 1; about 19.5:about 1; about 19.4:about 1; about 19.3:about 1; about 19.2:about 1; about 19.1 :about 1; about 19:about 1; about 18.9:about 1; about 18.8:about 1; about 18.7:about 1; about 18.6:about 1; about 18.5:about 1; about 18.4:about 1; about 18.3:about 1; about 18.2:about 1; about 18.1:about 1; about 18:about 1; about 17.9:about 1; about 17.8:about 1; about 17.7:about 1; about 17.6:about 1; about 17.5:about 1; about 17.4:about 1; about 17.3:about 1; about 17.2:about 1; about 17.1 :about 1; about 17:about 1; about 16.9:about 1; about 16.8:about 1; about 16.7:about 1; about 16.6:about 1; about 16.5:about 1; about 16.4:about 1; about 16.3:about 1; about 16.2:about 1; about 16.1 :about 1; about 16:about 1; about 15.9:about 1; about 15.8:about 1; about 15.7:about 1; about 15.6:about 1; about 15.5:about 1; about 15.4:about 1; about 15.3:about 1; about 15.2:about 1; about 15.1 :about 1; about 15:about 1; about 14.9:about 1; about 14.8:about 1; about 14.7:about 1; about 14.6:about 1; about 14.5:about 1; about 14.4:about 1; about 14.3:about 1; about 14.2: about 1; about 14.1 :about 1; about 14:about 1; about 13.9:about 1; about 13.8:about 1; about 13.7:about 1; about 13.6:about 1; about 13.5:about 1; about 13.4:about 1; about 13.3:about 1; about 13.2:about 1; about 13.1:about 1; about 13:about 1; about 12.9:about 1; about 12.8:about 1; about 12.7:about 1; about 12.6:about 1; about 12.5:about 1; about 12.4:about 1; about 12.3:about 1; about 12.2:about 1; about 12.1 :about 1; about 12:about 1; about 11.9:about 1; about 11.8:about 1; about 11.7:about 1; about 11.6:about 1; about 11.5:about 1; about 11.4:about 1; about 11.3:about 1; about 11.2:about 1; about l l. l :about 1; about l l :about 1; about 10.9:about 1; about 10.8:about 1; about 10.7:about 1; about 10.6:about 1; about 10.5:about 1; about 10.4:about 1; about 10.3:about 1; about 10.2:about 1; about 10.1 :about 1; about 10:about 1; about 9.9:about 1; about 9.8:about 1; about 9.7:about 1; about 9.6:about 1; about 9.5:about 1; about 9.4:about 1; about 9.3:about 1; about 9.2:about 1; about 9.1:about 1; about 9:about 1; about 8.9:about 1; about 8.8:about 1; about 8.7:about 1; about 8.6:about 1; about 8.5:about 1; about 8.4:about 1; about 8.3:about 1; about 8.2:about 1; about 8.1 :about 1; about 8:about 1; about 7.9:about 1; about 7.8:about 1; about 7.7:about 1; about 7.6:about 1; about 7.5:about 1; about 7.4:about 1; about 7.3:about 1; about 7.2:about 1; about 7.1 :about 1; about 7:about 1; about 6.9:about 1; about 6.8:about 1; about 6.7:about 1; about 6.6:about 1; about 6.5:about 1; about 6.4:about 1; about 6.3:about 1; about 6.2:about 1; about 6.1 :about 1; about 6:about 1; about 5.9:about 1; about 5.8:about 1; about 5.7:about 1; about 5.6:about 1; about 5.5:about 1; about 5.4:about 1; about 5.3:about 1; about 5.2:about 1; about 5.1 :about 1; about 5:about 1; about 4.9:about 1; about 4.8:about 1; about 4.7:about 1; about 4.6:about 1; about 4.5:about 1; about 4.4:about 1; about 4.3:about 1;about 4.2:about 1; about 4.1 :about 1; about 4:about 1; about 3.9:about 1; about 3.8:about 1; about 3.7:about 1; about 3.6:about 1; about 3.5:about 1; about 3.4:about 1; about 3.3:about 1; about 3.2:about 1; about 3.1 :about 1; about 3:about 1; about 2.9:about 1; about 2.8:about 1; about 2.7:about 1; about 2.6:about 1; about 2.5:about 1; about 2.4:about 1; about 2.3:about 1; about 2.2:about 1; about 2.1 :about 1; about 2:about 1; about 1.9:about 1; about 1.8:about 1; about 1.7:about 1; about 1.6:about 1; about 1.5:about 1; about 1.4:about 1; about 1.3:about 1; about 1.2:about 1; about l.l :about 1; about l:about 1; about l :about 10; about l :about 20; about 1 : about 30; about 1 : about 40; about 1 : about 50; about 1 : about 60; about 1 : about 70; about 1 : about 80; about 1 : about 90; about 1 : about 100; about 1: about 110; about 1 : about 120; about l :about 130; about l:about 140; about l :about 150; about l:about 160; about l :about 170; about l;about 180; about l :about 190; about l:about 200; about l :about 210; about l :about 220; about l:about 230; about l :about 240; about l:about 250: about l :about 260; about l :about 270; about l:about 280; about l :about 290: about l:about 300; about l :about 310; about l:about 320; about l :about 330; about l:about 340; about l :about 350; about l :about 360; about l:about 370; about l :about 380; about l:about 390; or about l :about 400.

[0127] A formulation as disclosed herein can comprise a therapeutic compound (e.g., cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof) as an active ingredient. In some embodiments, there can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more active ingredients in a composition.

[0128] In some embodiments, the active ingredient(s) can comprise at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100% of the total composition. In some embodiments, the active ingredient(s) can comprise at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at mostabout 35%, at most about 30%, at most about 25%, at most about 20% at most about, 19%, at most about 18%, at most about 17%, at most about 16%, at most about 15%, at most about 14%, at most about 13%, at most about 12%, at most about 11%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0. 1% or less of the total composition.

[0129] An additive or agent can increase the solubility of the formulation by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 225%, about 250%, about 275%, about 300%, about 325%, about 350%, about 375%, about 400%, about 450%, or about 500%.

[0130] A formulation disclosed herein can be stable for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about one year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more. A formulation disclosed herein can be stable, for example, at about 0 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50 °C, about 60 °C, about 70 °C, or about 80 °C.

[0131] A pharmaceutical composition of the invention can be a combination of any pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, or excipients. Pharmaceutical compositions can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulations can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a compound described herein can be manufactured, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or compression processes.

[0132] The pharmaceutical compositions can include at least one pharmaceutically- acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. The methods and pharmaceutical compositions described herein include the use of crystalline forms (also known as polymorphs), and active metabolites of these compounds having the same type of activity.

[0133] In some embodiments, crystalline forms of a compound can be characterized by techniques including, but not limited to, X-ray diffraction, X-ray powder diffraction, single crystal X-ray diffraction, infrared spectroscopy, Raman spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and dynamic vapor sorption. In some embodiments, a crystalline form of a compound can have altered stability, altered solubility, altered bioavailability, or altered formulation properties relative to different form of a compound.

[0134] A pharmaceutical composition can comprise one or more carriers. Non-limiting examples of pharmaceutically-acceptable carriers include saline solution, Ringer's solution, and dextrose solution. The pH of the solution can be from about 5 to about 8 or can be from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof where the matrices are in the form of shaped articles, such as films, liposomes, microparticles, and microcapsules.

[0135] A pharmaceutical composition can comprise one or more fillers. Non-limiting examples of fillers can include lactose, sucrose, mannitol starch, magnesium stearate, glucose, cellulose, and calcium carbonate. In some embodiments, a filler can be mannitol starch. In some embodiments, a filler can be lactose.

[0136] In some embodiments, a filler (e.g., mannitol starch or lactose) is present in a composition in an amount of present in a formulation in an amount of from about 0.1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0.1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg toabout 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 150 mg, from about 100 mg to about 200 mg, from about 100 mg to about 250 mg, from about 100 mg to about 300 mg, from about 150 mg to about 200 mg, from about 150 mg to about 250 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg , from about 250 mg to about 400 mg, from about 300 mg to about 400 mg, or from about 300 mg to about 400 mg.

[0137] In some embodiments, a filler (e.g., mannitol starch or lactose) is present in a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, or about 400 mg.

[0138] A pharmaceutical composition can comprise one or more binders. Non-limiting examples of binders can include starch, sodium alginate, gelatin, polyvinyl pyrrolidone (PVP), methylcellulose, hydroxy propyl methyl cellulose (PHMC), polymethacrylate, sodium carboxy methyl cellulose, polyethylene glycol (PEG), methylcellulose, lactose, sucrose, sorbitol, xylitol, or mannitol. In some embodiments, a binder can be mannitol. In someembodiments, a binder can be lactose.

[0139] In some embodiments, a binder (e.g., mannitol or lactose) is present in a formulation in an amount of from about 0.1 mg to about 200 mg, from about 10 mg to about 200 mg, from about 10 mg to about 150 mg, from about 50 mg to about 150 mg, from about 100 mg to about 150 mg, from about 10 mg to about 100 mg, from about 10 mg to about 50 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0.1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 150 mg, from about 100 mg to about 200 mg, from about 100 mg to about 250 mg, from about 100 mg to about 300 mg, from about 150 mg to about 200 mg, from about 150 mg to about 250 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg , from about 250 mg to about 400 mg, from about 300 mg to about 400 mg, or from about 300 mg to about 400 mg.

[0140] In some embodiments, a binder (e.g., mannitol or lactose) is present in a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 101 mg, about 102 mg, about 103 mg, about 104 mg, about 105 mg, about 106 mg, about 107 mg, about 108 mg, about 109 mg, about 110 mg, about 111 mg, about 112 mg, about 113 mg, about 114 mg, about 115 mg, about 116 mg, about 117 mg, about 118 mg, about 119 mg, about 120 mg, about 121 mg, about 122 mg, about 123 mg, about 124 mg, about 125 mg, about 126 mg, about 127 mg, about 128 mg, about 129 mg, about 130 mg, about 131 mg, about 132 mg, about 133 mg, about 134 mg, about 135 mg, about 136 mg, about 137 mg, about 138 mg, about 139 mg, about 140 mg, about 141 mg, about 142 mg, about 143 mg, about 144 mg, about 145 mg, about 146 mg, about 147 mg, about 148 mg, about 149 mg, or about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, or about 400 mg.

[0141] A pharmaceutical composition can comprise one or more superdisintegrants. Nonlimiting examples of superdisintegrants can include croscarmellose, crospovidone, sodium starch glycolate, or magnesium aluminum silicate. In some embodiments, a superdisintegrant can be sodium starch glycolate.

[0142] In some embodiments, a superdisintegrant (e.g., sodium starch glycolate) is present in a formulation in an amount of from about 0.1 mg to about 100 mg, from about 5 mg to about 50 mg, from about 5 mg to about 15 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0. 1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0.1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, or from about 95 mg to about 100 mg.

[0143] In some embodiments, a superdisintegrant (e.g., sodium starch glycolate) is present in a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg.

[0144] A pharmaceutical composition can comprise one or more lubricants. Non-limiting examples of lubricants can include magnesium stearate, stearic acid, calcium stearate, sodium stearyl fumarate, polyethylene glycol, silicone dioxide, talc, or beeswax. In some embodiments, a lubricant can be magnesium stearate.

[0145] In some embodiments, a lubricant (e.g., magnesium stearate) is present in a formulation in an amount of from about 0. 1 mg to about 100 mg, from about 10 mg to about 100 mg, from about 10 mg to about 80 mg, from about 20 mg to about 100 mg, from about 20 mg to about 60 mg, from about 20 mg to about 40 mg, from about 1 mg to about 20 mg, from about 40 mg to about 50 mg, from about 0. 1 mg to about 1 mg, from about 0.5 mg to about 1 mg, from about 0.5 mg to about 10 mg, from about 0. 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, or from about 95 mg to about 100 mg.

[0146] In some embodiments, a lubricant (e.g., magnesium stearate) is present in a formulation in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg.

[0147] Pharmaceutical formulations can include additional carriers, thickeners, diluents, buffers, preservatives, and surface-active agents in addition to the compounds disclosed herein. An excipient can fdl a role as simple and direct as being an inert fdler, or an excipient as used herein can be part of a pH stabilizing system or coating to insure delivery of the ingredients safely to the stomach or other desired target.

[0148] A pharmaceutical composition comprising cutamesine or a pharmaceutically- acceptable salt thereof can be sublingual formulation. The formulation provides sufficient solubility for cutamesine or a pharmaceutically acceptable salt thereof to be incorporated into the sublingual formulation at therapeutically-effective doses and sublingually delivered. The formulation can be an oral disintegrating formulation of cutamesine or a pharmaceutically- acceptable salt thereof. The formulation can be a liquid solution of cutamesine or a pharmaceutically-acceptable salt thereof. The formulation can be a solid dosage form of cutamesine or a pharmaceutically-acceptable salt thereof.

[0149] A pharmaceutical composition comprising smilagenin or a pharmaceutically- acceptable salt thereof can be solid dosage formulation.

[0150] A pharmaceutical preparation can be administered orally. For an oral administration, pharmaceutical compositions can be formulated by combining the active compounds withpharmaceutically-acceptable carriers or excipients. Such carriers can be used to formulate tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a subject.

[0151] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can contain an excipient such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or to characterize different combinations of active compound doses.

[0152] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In some embodiments, the capsule comprises a hard gelatin capsule comprising one or more of pharmaceutical, bovine, and plant gelatins. A gelatin can be alkaline-processed. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers can be added. All formulations for oral administration are provided in dosages suitable for such administration.

[0153] For buccal or sublingual administration, the compositions can be tablets, lozenges, or gels. For example, a composition can be in a solid dosage form.

[0154] A solid dosage form is a pharmaceutically acceptable composition that is safe for oral administration to subjects (e.g., humans), in which case all excipients in the dosage form are to be used in the oral formulation. A solid dosage form can be an immediate release tablet, an immediate release capsule, a controlled release tablet, a controlled release capsule, a fastdissolving form, a chewable form, a sachet, etc. In some embodiments, the solid dosage form is a tablet.

[0155] A solid dosage form tablet can comprise a single-layer tablet. Alternatively, a solid dosage form tablet can comprise a double-layer tablet. A solid dosage form can comprise a round tablet, a square tablet, a rectangular tablet, a circular tablet, a spherical tablet, an ovoid tablet, a triangular tablet, or a polygonal tablet. A solid dosage form tablet can be convex onone side. A solid dosage form tablet can be convex on two sides (e.g., biconcave). Alternatively, a solid dosage form tablet can be concave one side. A solid dosage form tablet can be concave on two sides (e.g., biconcave).

[0156] A solid dosage form tablet can have a diameter from about 1 mm to about 15 mm, from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, from about 3 mm to about 4 mm, from about 4 mm to about 5 mm, from about 5 mm to about 6 mm, from about 6 mm to about 7 mm, from about 7 mm to about 8 mm, from about 8 mm to about 9 mm, from about 9 mm to about 10 mm, from about 10 mm to about 11 mm, from about 11 mm to about 12 mm, from about 12 mm to about 13 mm, from about 13 mm to about 14 mm, from about 14 mm to about 15 mm, from about 1 mm to about 3 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5 mm, from about 4 mm to about 6 mm, from about 5 mm to about 7 mm, from about 6 mm to about 8 mm, from about 7 mm to about 9 mm, from about 8 mm to about 10 mm, from about 9 mm to about 11 mm, from about 10 mm to about 12 mm, from about 11 mm to about 13 mm, from about 12 mm to about 14 mm, or from about 13 mm to about 15 mm. In some examples, a solid dosage form tablet can have a diameter from about 7 mm to about 9 mm. In some examples, a dosage form tablet can have a diameter from about 8 mm to about 8.2 mm.

[0157] A solid dosage form tablet can have a thickness from about from about 1 mm to about 10 mm, from about 1 mm to about 2 mm, from about 1.5 mm to about 2.5 mm, from about 2 mm to about 3 mm, from about 2.5 mm to about 3.5 mm, from about 3 mm to about 4 mm, from about 3.5 mm to about 4.5 mm, from about 4 mm to about 5 mm, from about 4.5 mm to about 5.5 mm, from about 5 mm to about 6 mm, from about 5.5 mm to about 6.5 mm, from about 6 mm to about 7 mm, from about 6.5 mm to about 7.5 mm, from about 7 mm to about 8 mm, from about 7.5 mm to about 8.5 mm, from about 8 mm to about 9 mm, from about 8.5 mm to about 9.5 mm, from about 9 mm to about 10 mm, from about 1 mm to about 3 mm, from about 2 mm to about 4 mm, from about 3 mm to about 5 mm, from about 4 mm to about 6 mm, from about 5 mm to about 7 mm, from about 6 mm to about 8 mm, from about 7 mm to about 9 mm, or from about 8 mm to about 10 mm. In some examples, a solid dosage form tablet can have a thickness from about 2 mm to about 4 mm. In some examples, a dosage form tablet can have a thickness from about 2.7 mm to about 2.9 mm.

[0158] Dissolution tests (e.g., a shake test) can be performed on a solid dosage form to simulate the sublingual cavity. A solid dosage form can be subjected to a shake test in which the solid dosage form is placed in water and the shaken to disintegrate the solid dosage form in the water. A solid dosage form can disintegrate into fine particles during a shake test inless than about 20 seconds, less than about 25 seconds, less than about 30 seconds, less than about 35 seconds, less than about 40 seconds, less than about 45 seconds, less than about 50 seconds, less than about 55 seconds, less than about 60 seconds, less than about 65 seconds, less than about 70 seconds, less than about 75 seconds, less than about 80 seconds, less than about 85 seconds, or less than about 90 seconds. In some embodiments, a solid dosage form can disintegrate into fine particles during a shake test in less than about 60 seconds. In some embodiments, a solid dosage form can disintegrate into fine particles during a shake test in less than about 40 seconds. In some embodiments, a solid dosage form can disintegrate into fine particles during a shake test in about 33 ± 5 seconds.

[0159] Thermal analysis can be performed on a solid dosage form to determine stability of the solid dosage form. A dosage form (e.g., a sublingual dosage form) can be heated. The resulting thermograph from the thermal analysis can show an endothermic peak from about 145-170 °C. Alternatively, the resulting thermograph from the thermal analysis can show an endothermic peak from about 156-161 °C.

[0160] In practicing the methods of treatment or use provided herein, therapeutically- effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors. The compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures.

[0161] In some embodiments, the therapeutically-effective amounts of the compounds described herein can be administered as a single composition. Alternatively, or in addition to, the therapeutically-effective amounts of the compounds described herein can be administered as separate compositions.

[0162] If the compounds described herein are administered as separate compositions, the first composition and the second composition can be administered simultaneously. Alternatively, the first composition and second composition can be administered at different timepoints. In some embodiments, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or about 24 hours elapses between administration of the first composition and the administration of the second composition.

[0163] A composition of the invention can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the compounds to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that drug release rates and drug release profiles can be matched to physiological and chronotherapeutic requirements or, alternatively, has been formulated to effect release of a drug at a programmed rate. Nonlimiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gelforming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.

[0164] In some, a controlled release formulation is a delayed release form. A delayed release form can be formulated to delay a compound's action for an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more compounds, for example, for about 4, about 8, about 12, about 16, or about 24 hours.

[0165] A controlled release formulation can be a sustained release form. A sustained release form can be formulated to sustain, for example, the compound's action over an extended period of time. A sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., provide a physiologically-effective blood profile) over about 4, about 8, about 12, about 16, or about 24 hours.

[0166] Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0167] The disclosed methods include administration of a cutamesine compound, a smilagenin compound, a pharmaceutically-acceptable salt thereof, or a combination thereof, in combination with a pharmaceutically-acceptable carrier. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects inthe subject.

[0168] The cutamesine compound, smilagenin compound, pharmaceutically-acceptable salt thereof, or combination thereof herein can be formulated into pharmaceutical compositions composed of one or more pharmaceutically-acceptable carriers. See e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, Pa., which discloses carriers and methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compound described herein and which is incorporated by reference herein.

[0169] The disclosed methods relate, for example, to sublingual administration of cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof as part of a pharmaceutical composition. In some embodiments, a method of treating a disease of a subject by administering the oral and / or sublingual formulation is provided. In some embodiments, the method comprises providing an oral and / or sublingual formulation as described herein having an effective amount of cutamesine, smilagenin, or a pharmaceutically-acceptable salt, solvate, anomer, hydrate, or prodrug thereof, and administering the formulation to a subject to treat the diseased state.Pharmaceutical Compositions

[0170] Pharmaceutical compositions containing the compounds described herein can be administered for prophylactic or therapeutic treatments. In therapeutic applications, the compositions can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, reduce, lessen, or ameliorate the disease or condition. Compounds can also be administered to lessen or reduce a likelihood of developing, contracting, or worsening a condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject's health status, weight, response to the drugs, and the judgment of the treating physician.

[0171] Multiple therapeutic agents can be administered in any order or simultaneously. If simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms, for example, as multiple separate pills or injections. The compounds can be packed together or separately, in a single package or in a plurality of packages. One or all of the therapeutic agents can be given in multiple doses. If not simultaneous, the timing between the multiple doses can vary.

[0172] Compounds and compositions of the invention can be packaged as a kit. In some embodiments, the invention provides a kit comprising a compound disclosed herein, or apharmaceutically-acceptable salt thereof, and written instructions on use of the kit in the treatment of a condition described herein. In some embodiments, the invention provides a kit comprising a compound disclosed herein, or a pharmaceutically-acceptable salt thereof, and written instructions on use of the kit in the treatment of a condition described herein.

[0173] The compounds described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a compound can vary. For example, the compounds can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen or reduce a likelihood of the occurrence of the disease or condition. The compounds and compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the compounds can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein.

[0174] A compound can be administered as soon as is practical after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments, the length of time a compound can be administered can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. The length of treatment can vary for each subject.

[0175] Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosagecan be in the form of a package containing discrete quantities of the formulation. Nonlimiting examples are packaged injectables, vials, or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0176] A cutamesine compound described herein can be present in a composition in a range of from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, from about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, from about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, or from about 250 mg to about 300 mg. In some embodiments, a cutamesine compound is a cutamesine salt compound. In some embodiments, a cutamesine compound is a cutamesine dihydrochloride compound.

[0177] A cutamesine compound described herein can be present in a composition in an amount of about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, or about 300 mg. In some embodiments, a cutamesine compound is a cutamesine salt compound. In some embodiments, a cutamesine compound is a cutamesine dihydrochloride compound.

[0178] A smilagenin compound described herein can be present in a composition in a range of from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, from about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg,from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, from about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, or from about 250 mg to about 300 mg.

[0179] A smilagenin compound described herein can be present in a composition in an amount of about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, or about 300 mg.

[0180] A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can achieve a lower therapeutic dose than that of orally administered cutamesine. A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can achieve a therapeutic dose that is about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160, about 170, about 180, about 190, or about 200 mg less than that of an amount of orally administered cutamesine or a pharmaceutically-acceptable salt thereof that has a similar or same therapeutic effect.

[0181] A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can produce a rapid therapeutic onset of action. A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can produce a therapeutic onset of action within about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 1 hour.

[0182] A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can produce a therapeutic onset of action faster than that of orally administered cutamesine or a pharmaceutically-acceptable salt thereof. A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can produce a therapeutic onset of action at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, or at least about 6 hours faster than that of orally administered cutamesine.

[0183] A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can reduce side effects of cutamesine. A sublingual formulation of cutamesine or a pharmaceutically-acceptable salt thereof can reduce the total drug load necessary to result in a therapeutic effect. In some embodiments, a lower sublingual dosage formulation of cutamesine or a pharmaceutically-acceptable salt thereof can deliver similar effects compared to a higher oral dosage formulation of cutamesine or a pharmaceutically-acceptable salt thereof or enhanced effects compared to a higher oral dosage formulation.Treatment of Subjects with Neurodegenerative Disease

[0184] The invention discloses methods for treating a subject afflicted a neurodegenerative disease.

[0185] A neurodegenerative disease can be a progressive disease in which cells of the central nervous system stop working and / or die. Non-limiting examples of neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, prion disease, Amyotrophic lateral sclerosis (ALS), motor neuron disease, Huntington’s disease, dementia, spinal muscular atrophy, and spinocerebellar ataxia.

[0186] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative disease that results in the progressive loss of motor neurons that control predominantly voluntary muscles. Different types of ALS can be classified by the types ofmotor neurons that are affected - upper motor neurons (e.g., in the motor cortex of the brain) or lower motor neurons (e.g., neurons of the spinal cord).

[0187] Dementia is a disorder characterized by the loss of cognitive function (e.g., thinking, remembering, reasoning) to such an extent that interferes with daily life. One form of dementia is Alzheimer’s disease. Alzheimer’s disease is a neurodegenerative disease that interferes with cognitive and functional impairment (e.g., memory impairment).

[0188] Parkinson’s disease is a neurodegenerative disorder characterized by unintended or uncontrollable movements such as shaking, stiffness, or difficulty with balance and coordination. Parkinson’s disease can be caused by falling dopamine levels, which can be caused by nerve cell damage.

[0189] In some embodiments, the invention provides cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof for use in treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease). In some embodiments, the invention provides cutamesine, smilagenin, a pharmaceutically- acceptable salt thereof, or a combination thereof for use in the manufacture of a medicament for the treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease).

[0190] Treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease) can result in reducing muscle weakness by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

[0191] Treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease) can result in reducing motor neuron loss by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

[0192] The subject can be a human. Treatment can include treating a human in a clinical trial. A treatment can comprise administering to a subject a pharmaceutical composition comprising one or more of cutamesine, smilagenin, a pharmaceutically-acceptable salt thereof, or a combination thereof, as described throughout the disclosure.

[0193] In some embodiments, the invention provides cutamesine, smilagenin, apharmaceutically-acceptable salt thereof, or a combination thereof, for use in treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease). In some embodiments, the invention provides a cutamesine composition, a smilagenin composition, a pharmaceutically-acceptable salt thereof, or a combination thereof for use in the manufacture of a medicament for the treatment of a neurodegenerative disease (e.g., ALS, dementia, Parkinson’s disease, Alzheimer’s disease).

[0194] Non-limiting examples of possible subjects for administration include the following. Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rats, mice, and guinea pigs. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, or infants.EMBODIMENTS

[0195] Embodiment 1. A combination comprising a therapeutically-effective amount of cutamesine or a pharmaceutically-acceptable salt thereof and a therapeutically-effective amount of smilagenin.

[0196] Embodiment 2. The combination of embodiment 1, wherein the combination is in a unit dosage form that contains the cutamesine or the pharmaceutically-acceptable salt thereof and the smilagenin.

[0197] Embodiment 3. The combination of embodiment 1, wherein the cutamesine is the pharmaceutically-acceptable salt, and the pharmaceutically-acceptable salt is cutamesine dihydrochloride and the smilagenin.

[0198] Embodiment 4. The combination of embodiment 1, wherein the combination comprises a first unit dosage form and a second unit dosage form, wherein the first unit dosage form contains the cutamesine or the pharmaceutically-acceptable salt thereof and the second unit dosage form contains the smilagenin.

[0199] Embodiment 5. The combination of embodiment 1, wherein the combination comprises a first unit dosage form and a second unit dosage form, wherein the first unit dosage form contains the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt is cutamesine dihydrochloride and the second unit dosage form contains the smilagenin.

[0200] Embodiment 6. A pharmaceutical composition comprising: a) cutamesine or a pharmaceutically acceptable salt thereof, and b) a sapogenin.

[0201] Embodiment 7. The pharmaceutical composition of embodiment 6, wherein thesapogenin is smilagenin.

[0202] Embodiment 8. The pharmaceutical composition of embodiment 6, wherein the composition further comprises a pharmaceutically-acceptable excipient.

[0203] Embodiment 9. The pharmaceutical composition of embodiment 6, wherein the composition further comprises a glutamate salt.

[0204] Embodiment 10. The pharmaceutical composition of embodiment 6, wherein the cutamesine or the pharmaceutically acceptable salt thereof and the sapogenin are present in a mass ratio between 1:200 and 1:10 cutamesine: sapogenin.

[0205] Embodiment 11. The pharmaceutical composition of embodiment 10, wherein the cutamesine or the pharmaceutically acceptable salt thereof and the sapogenin are present in a mass ratio of about 3:180 cutamesine: sapogenin.

[0206] Embodiment 12. The pharmaceutical composition of embodiment 6, wherein cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a concentration of at least 1 pM.

[0207] Embodiment 13. The pharmaceutical composition of embodiment 6, wherein cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a concentration of about 1 pM to about 300 nM.

[0208] Embodiment 14. The pharmaceutical composition of embodiment 6, wherein the sapogenin is present in the pharmaceutical composition in a concentration of at least 1 pM.

[0209] Embodiment 15. The pharmaceutical composition of embodiment 6, wherein the sapogenin is present in the pharmaceutical composition in a concentration of about 1 pM to about 300 nM.

[0210] Embodiment 16. The pharmaceutical composition of embodiment 15, wherein the pharmaceutical composition is suitable for sublingual administration.

[0211] Embodiment 17. The pharmaceutical composition of embodiment 6, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

[0212] Embodiment 18. A pharmaceutical composition comprising cutamesine or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is a sublingual dosage form.

[0213] Embodiment 19. The pharmaceutical composition of embodiment 18, wherein the composition further comprises a pharmaceutically-acceptable excipient.

[0214] Embodiment 20. The pharmaceutical composition of embodiment 18, wherein the pharmaceutical composition further comprises lactose.

[0215] Embodiment 21. The pharmaceutical composition of embodiment 18, wherein the pharmaceutical composition further comprises mannitol starch.

[0216] Embodiment 22. The pharmaceutical composition of embodiment 18, wherein the pharmaceutical composition further comprises sodium starch glycolate.

[0217] Embodiment 23. The pharmaceutical composition of embodiment 18, wherein the pharmaceutical composition further comprises magnesium stearate.

[0218] Embodiment 24. The pharmaceutical composition of embodiment 18, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

[0219] Embodiment 25. The pharmaceutical composition of embodiment 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

[0220] Embodiment 26. The pharmaceutical composition of embodiment 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

[0221] Embodiment 27. The pharmaceutical composition of embodiment 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

[0222] Embodiment 28. The pharmaceutical composition of embodiment 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 3 mg.

[0223] Embodiment 29. The pharmaceutical composition of embodiment 18, further comprising a binder, a superdisintegrant, and a lubricant.

[0224] Embodiment 30. The pharmaceutical composition of embodiment 18, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

[0225] Embodiment 31. The pharmaceutical composition of embodiment 18, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in an amount of about 0.5 mg to about 10 mg.

[0226] Embodiment 32. The pharmaceutical composition of embodiment 18, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

[0227] Embodiment 33. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is a tablet.

[0228] Embodiment 34. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

[0229] Embodiment 35. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mm and a thickness from about 2.7 mm to about 2.9 mm.

[0230] Embodiment 36. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which the sublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is less than sixty seconds.

[0231] Embodiment 37. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which the sublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is less than forty seconds.

[0232] Embodiment 38. The pharmaceutical composition of embodiment 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which the sublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is 33 ± 5 seconds.

[0233] Embodiment 39. A pharmaceutical composition comprising cutamesine or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the solid dosage form is subjected to a shake test in which the solid dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the solid dosage form in the water, then a time needed for disintegration of the solid dosage form into fine particles is less than sixty seconds.

[0234] Embodiment 40. The pharmaceutical composition of embodiment 39, wherein the time needed for disintegration of the solid dosage form into fine particles is less than forty seconds.

[0235] Embodiment 41. The pharmaceutical composition of embodiment 39, whereinthe time needed for disintegration of the solid dosage form into fine particles is 33 ± 5 seconds.

[0236] Embodiment 42. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable excipient.

[0237] Embodiment 43. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition further comprises mannitol starch.

[0238] Embodiment 44. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition further comprises lactose.

[0239] Embodiment 45. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition further comprises sodium starch glycolate.

[0240] Embodiment 46. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition further comprises magnesium stearate.

[0241] Embodiment 47. The pharmaceutical composition of embodiment 39, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

[0242] Embodiment 48. The pharmaceutical composition of embodiment 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

[0243] Embodiment 49. The pharmaceutical composition of embodiment 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

[0244] Embodiment 50. The pharmaceutical composition of embodiment 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

[0245] Embodiment 51. The pharmaceutical composition of embodiment 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 3 mg.

[0246] Embodiment 52. The pharmaceutical composition of embodiment 39, further comprising a binder, a superdisintegrant, and a lubricant.

[0247] Embodiment 53. The pharmaceutical composition of embodiment 39, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

[0248] Embodiment 54. The pharmaceutical composition of embodiment 39, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in anamount of about 0.5 mg to about 10 mg.

[0249] Embodiment 55. The pharmaceutical composition of embodiment 39, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

[0250] Embodiment 56. The pharmaceutical composition of embodiment 39, wherein the sublingual dosage form is a tablet.

[0251] Embodiment 57. The pharmaceutical composition of embodiment 39, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

[0252] Embodiment 58. The pharmaceutical composition of embodiment 39, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mm and a thickness from about 2.7 mm to about 2.9 mm.

[0253] Embodiment 59. A pharmaceutical composition comprising cutamesine or a pharmaceutically-acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the solid dosage form is subjected to a dissolution study in which the solid dosage form is placed in a medium of 2 mL of distilled water at 25 °C in a 15 mL glass funnel for sixty seconds, at which point the medium is drawn by vacuum through a 0.45 pm membrane into a collection tube, after which the medium is analyzed by HPLC, then a percent dissolution of cutamesine or the pharmaceutically acceptable salt thereof of about 80 % to about 95 % is obtained.

[0254] Embodiment 60. The pharmaceutical composition of embodiment 59, wherein a percent dissolution of cutamesine or the pharmaceutically acceptable salt thereof of 92.3 ± 2.1 % is obtained for a form containing 1 mg of cutamesine or the pharmaceutically acceptable salt thereof and 87.9 ± 5.7 % for a form containing 3 mg of cutamesine or the pharmaceutically acceptable salt thereof.

[0255] Embodiment 61. The pharmaceutical composition of embodiment 59, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable excipient.

[0256] Embodiment 62. The pharmaceutical composition of embodiment 59, wherein the pharmaceutical composition further comprises lactose.

[0257] Embodiment 63. The pharmaceutical composition of embodiment 59, wherein the pharmaceutical composition further comprises mannitol starch.

[0258] Embodiment 64. The pharmaceutical composition of embodiment 59, wherein the pharmaceutical composition further comprises sodium starch glycolate.

[0259] Embodiment 65. The pharmaceutical composition of embodiment 59, whereinthe pharmaceutical composition further comprises magnesium stearate.

[0260] Embodiment 66. The pharmaceutical composition of embodiment 59, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

[0261] Embodiment 67. The pharmaceutical composition of embodiment 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

[0262] Embodiment 68. The pharmaceutical composition of embodiment 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

[0263] Embodiment 69. The pharmaceutical composition of embodiment 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

[0264] Embodiment 70. The pharmaceutical composition of embodiment 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 3 mg.

[0265] Embodiment 71. The pharmaceutical composition of embodiment 59, further comprising a binder, a superdisintegrant, and a lubricant.

[0266] Embodiment 72. The pharmaceutical composition of embodiment 59, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

[0267] Embodiment 73. The pharmaceutical composition of embodiment 59, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in an amount of about 0.5 mg to about 10 mg.

[0268] Embodiment 74. The pharmaceutical composition of embodiment 59, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

[0269] Embodiment 75. The pharmaceutical composition of embodiment 59, wherein the sublingual dosage form is a tablet.

[0270] Embodiment 76. The pharmaceutical composition of embodiment 59, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

[0271] Embodiment 77. The pharmaceutical composition of embodiment 59, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mmand a thickness from about 2.7 mm to about 2.9 mm.

[0272] Embodiment 78. A method for treating a condition, the method comprising sublingually administering to a subject in need thereof a therapeutically-effective amount of cutamesine or a pharmaceutically-acceptable salt thereof.

[0273] Embodiment 79. A method for treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of the therapeutic combination of any one of embodiments 1-77.

[0274] Embodiment 80. The method of any one of embodiments 78-79, wherein the condition is a neurodegenerative disease.

[0275] Embodiment 81. The method of any one of embodiments 78-80, wherein the condition is a central nervous system disease.

[0276] Embodiment 82. The method of any one of embodiments 78-80, wherein the condition is a central nervous system disease.

[0277] Embodiment 83. The method of any one of embodiments 78-80, wherein the condition is amyotrophic lateral sclerosis (ALS).

[0278] Embodiment 84. The method of any one of embodiments 78-80, wherein the condition is dementia.

[0279] Embodiment 85. The method of any one of embodiments 78-80, wherein the condition is Alzheimer’s Disease.

[0280] Embodiment 86. The method of any one of embodiments 78-80, wherein the condition is Parkinson’s Disease.

[0281] Embodiment 87. The method of any one of embodiments 78-86, wherein the therapeutically-effective amount is about 0.5 mg to about 10 mg.

[0282] Embodiment 88. The method of any one of embodiments 78-86, wherein the therapeutically-effective amount is about 0.5 mg to about 5 mg.

[0283] Embodiment 89. The method of any one of embodiments 78-86, wherein the therapeutically-effective amount is about 1 mg.

[0284] Embodiment 90. The method of any one of embodiments 78-86, wherein the therapeutically-effective amount is about 3 mg.

[0285] Embodiment 91. The method of any one of embodiments 78-90, further comprising administering to the subject a therapeutically-effective amount of a sapogenin.

[0286] Embodiment 92. The method of embodiment 91, wherein the sapogenin is smilagenin.EXAMPLESEXAMPLE 1: General Methods

[0287] Differentiation of iPSCs into Motor Neurons

[0288] Differentiation of iPSCs into motor neurons (MNs) was performed as shown in FIG. 1. iPSCs were plated into Matrigel (Coming)-coated T25 flasks in a neural medium composed of DMEM / F12 (Gibco) and Neurobasal medium (Life Technologies) at a 1: 1 ratio, 0.5x B27 (Life Technologies), 0.5x N2 (Life Technologies), lx antibiotic-antimycotic (Gibco) and 100 pM ascorbic acid (AA, Sigma- Aldrich), enriched with 3 pM CHIR99021 (Selleckchem), 2 pM SB431542 (Selleckchem) and 2 pM DMH1 (Selleckchem), and fully changed every other day for 6 days. At day 6, cells were split and replated onto 10 pg / ml Poly-L-Omithine (PLO, Sigma-Aldrich) / 5 pg / ml laminin (Sigma- Aldrich) coated flasks at a ratio 1 :3 to 1 :6 in a media composed of the neural medium described previously, supplemented with 1 pM CHIR99021, 2 pM SB431542, 2 pM DMH1, 0.1 pM retinoic acid (RA, Sigma-Aldrich) and 0.5 pM purmorphamine (Sigma- Aldrich), with medium fully changed every other day for 6 days. At day 12, cells were split and replated onto PLO / laminin coated flasks at a ratio 1 :3 to 1 :6 in a media composed of the neural medium described previously, supplemented with 3 pM CHIR99021, 2 pM SB431542, 2 pM DMH1, 0.1 pM RA, 0.5 pM purmorphamine and 0.5 mM valproic acid (VP A, Sigma-Aldrich), with medium fully changed every other day for 6 days. After 6 days, motor neuron progenitor cells (MNPCs) were cryopreserved for later use or passaged and maintained in the MNPCs expansion medium. For every step, the corresponding culture medium was supplemented with 10 pM ROCK inhibitor Y-27632 (Selleckchem) for the first 24 hours to improve survival.

[0289] Glutamate and Viability Assay

[0290] As shown in FIG. 2, MNPCs from the different lines were plated in opaque white with clear flat bottom tissue culture-treated 96 well plates coated with PLO / Laminin at 17.5 k cells per wells in motor neuron final differentiation medium composed of the neural medium supplemented with 0.5 pM RA, 0.1 pM purmorphamine, 0.1 pM Compound E (StemCell Technologies), 10 ng / ml insulin like growth factor-1 (IGF-l,Peprotech), brain-derived neurotrophic factor (BDNF, Peprotech) and ciliary neurotrophic factor (CNTF, Peprotech) supplemented with lOpM ROCK inhibitor Y-27632 (Selleckchem). After 24 hours, the media was fully changed for motor neuron final differentiation medium without ROCK inhibitor, and the MNs were cultured for 4 weeks. Breathe-Easy sealing membranes (Sigma- Aldrich) were applied onto the plates to minimize evaporation.

[0291] Alternatively, to decrease cell clumping, MNPCs were first passaged in priming medium (basic neural medium supplemented with 0.5 pM RA and 0.1 pM purmorphamine) for 6 days with medium changed every other day, before plating in final differentiation medium.

[0292] For the 3414 cell line, due to the high number of contaminating cells in the MN culture, a treatment with 1 pM cytosine arabinoside (Ara C, Sigma) for 4 hours was performed at 6 days post-plating.

[0293] At day 27 post-plating, 10 pl of the medium per well was removed and replaced by lOpl of media supplemented with a solution of 10X glutamate (Sigma) and CTZ (Tocris) and test compounds. The final concentration of glutamate was 0.1 mM and concentration of CTZ depends on the cell line (10 pM for 3414 (C90rf72), 25 pM forTD17 (sALS), and 50 pM for A4V (SOD1-A4V).

[0294] After 24 hours, the viability of MNs was determined with the ATP-based luminescence assay Cell-Titer Gio (Promega) according to the manufacturer’s instructions and the luminescence readings were acquired using a SpectraMax® ID3 Microplate Reader (Molecular Devices). For each treatment, the percentage of viability was determined by normalizing the raw luminescence values to those of untreated wells for each cell line.EXAMPLE 2: Cutamesine and Smilagenin Combination in Spinal Cord Motor Neurons

[0295] In this experiment, the protective effects of cutamesine and smilagenin were evaluated on WT or SOD1 tg primary spinal motor neurons and on co-cultures of spinal explants and myoblasts, injured with glutamate.

[0296] Genotyping of SOD1 Tg Embryos

[0297] Pregnant female rats SOD1-G93A (Sprague Dawley, Taconic), of 14 days of gestation, were used. On the day of the dissection (from pregnant females at 14 days of gestation), a piece of each embryo brain (~3 mm3) was placed in a 2 mL tube of free DNase with a new scalpel. The DNA was extracted with the SYBR® Green Extract-N-Amp™ tissue PCR kit (Sigma Aldrich). 120 pL of extraction solution was put on each piece of embryo heads. Then the heads were incubated for 10 minutes at room temperature. At the end of this incubation period, the heads were incubated for 5 minutes at 95 °C. Immediately after this last incubation, 100 pL of neutralizing solution was added. Each DNA extract was diluted at 1 / 40 and stored at +4 °C until use.

[0298] SOD1-G93A gene was determined using genomic fragment with human SOD1 primers (5'-CATCAGCCCTAATCCATCTGA-3' (SEQ ID NO: 1);5'-CGCGACTAACAATCAAAGTGA-3' (SEQ ID NO: 2)). The SOD1 primers were dilutedat 3 pM in sterile, ultrapure water. A mix for PCR was prepared with ultrapure water (4 pL per sample), primer at 3pM (2 pL per sample), and Master Mix (10 pL per sample). In a PCR 96-well plate, 16 pL of PCR mix was added in each well. 4 pL of each diluted DNA was added according to a plan deposit.

[0299] The RT-PCR was run using the CFX96™ Bio-Rad RT-PCR system, using the following program: Initial denaturation (95 °C, 20 sec) / 45 cycles (95 °C, 10 sec; 65 °C, 10 sec; 72 °C, 30 sec) / Melt curve (95 °C, 15 sec; 64 °C, 1 min; 90 °C, 30 sec; 60 °C 15 sec). The amplification plots and melt curves were analyzed with the Bio-Rad software. The results for each sample were compared to negative control (ultrapure water) and to the positive control (DNA from Tg embryos).

[0300] Primary Culture of Spinal Cord Motor Neurons

[0301] Rat spinal cord motor neurons (MNs) were cultured. Briefly, pregnant female rats of 14 days gestation (Sprague Dawley; Taconic) were euthanized using a deep anesthesia with CO2 chamber and a cervical dislocation. Then fetuses were removed from the uterus and immediately placed in ice-cold L15 Leibovitz medium with a 2% penicillin (10,000 U / ml) and streptomycin (10 mg / ml) solution (PS) and 1% bovine serum albumin (BSA).

[0302] Spinal cords were treated for 20 minutes at 37 °C with a trypsin-EDTA solution at a final concentration of 0.05 % trypsin and 0.02 % EDTA. The dissociation was stopped by addition of Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g / liter of glucose, containing DNAse I grade II (final concentration 0.5 mg / mL) and 10 % fetal calf serum (FCS). Cells were mechanically dissociated by three forced passages through the tip of a 10- ml pipette. Cells were spun by centrifuge at 515 x g for 10 minutes at 4 °C. The supernatant was discarded, and the pellet was resuspended in a defined culture medium consisting of Neurobasal medium with a 2 % solution of B27 supplement, 2 mmol / liter of L-glutamine, 2 % of PS solution, and 10 ng / mL of brain-derived neurotrophic factor (BDNF). Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion test. The cells were seeded at a density of 20,000 per well in 96-well plates precoated with poly-L-lysine and were cultured at 37 °C in an air (95%)-CO2 (5%) incubator. The medium was changed every 2 days. The wells of the first lines and columns were not used for culture (to avoid any edge effect) and were filled with sterile water.

[0303] Primary Spinal Cord Explant and Myoblast Co-Culture

[0304] The human muscle cell line was established from dissociated cells (22,000 cells per wells), plated in gelatin-coated 0.1% in water on 48 well-plates, and grown in a proliferation medium consisting of mix of 62 % of MEM medium and 25 % of Ml 99 mediumsupplemented with glutamine 2 mM, human insulin 10 pg / ml, Human recombinant Epidermal growth factor lOng / ml (EGF), human recombinant Fibroblast growth factor basic 2ng / ml (bFGF), fetal calf serum 10% (FCS) and 2% of Penicillin 10.000 U / ml and Streptomycin 10.000 pg / ml (PS). The medium was changed every 2 days.

[0305] Five days after the start of culture, immediately after satellite cell fusion, whole transverse slices of 13-day-old rat Wistar embryos (Janvier Labs, France) spinal cords with 4 dorsal root ganglia (DRG) attached were placed on the muscle monolayer (one explant per well in the central area). DRG was necessary to achieve a good ratio of innervation.Innervated cultures were maintained in a mixed (67% / 25%) medium composed of MEM and medium 199, supplemented with 5 % FCS, insulin 5 pg / ml, glutamine 2 mM and 2% PS. After 24 hours of co-culture, neurites were observed growing out of the spinal cord explants. These neurites made contacts with myotubes and induced the first contractions after ~ 8 days of co-culture. Quickly thereafter, innervated muscle fibers located in the proximity to the spinal cord explants were virtually continuously contracting. Innervated fibers were morphologically and spatially distinct from the non-innervated ones and could easily be distinguished from them. The plates were maintained at 37 °C in a humidified incubator, in an atmosphere of air (95%)-CC>2 (5%).

[0306] Test Compounds and Glutamate Exposure (Primary Culture of Spinal Neurons)

[0307] On day 13 of culture, primary motor neurons were pre-treated with the test compounds, for 1 hour. The test compounds were applied alone or combined to test potential synergy. Riluzole was used as a reference compound.

[0308] On day 13 of culture, and after the 1 hour of pre-incubation, glutamate was added to a final concentration of 5 pM diluted in control medium still in presence of the compounds for 20 minutes. After 20 minutes, glutamate was washed out and fresh culture medium was added for an additional 24 hours.

[0309] Test Compound and Glutamate Exposure (Co-Culture of Spinal Explant and Myoblasts)

[0310] On day 27 of culture, the co-culture was pre-treated with the test compounds for 1 hour or six hours prior to the application of glutamate. The test compounds were applied alone or combined to test potential synergy. Riluzole was used as a reference compound. Then glutamate was applied (60 pM, 20 minutes) on the co-cultures still in presence of the test compounds.

[0311] After 20 minutes of injury, the supernatant was removed with fresh medium, with test compound or riluzole, for an additional 48 hours.

[0312] Organization of Culture Plates

[0313] Test compounds were tested on cultures in 96-well plates (6 wells per condition).Conditions evaluated are shown in Tables 1-2. (FIGs. 3A-3H (cutamesine) and FIGs. 4A- 4H (smilagenin)).Table 1Table 2

[0314] Effective concentrations of each compound were determined and used in combination experiments (Tables 3-5) (FIGs. 22A-22I, FIGs. 23A-23H, FIGs. 24A-24H, and FIGs.38A-38C).Table 3Table 5

[0315] To determine the optimal ratio of the combination of cutamesine to smilagenin, assessments were made of motor neuron survival, neurite outgrowth, and TDP-43 pathology in rodent primary motor neuron culture in which cell stress and death were induced by glutamate (FIG. 221). The lowest efficacious dose was the 1:10 ratio of cutamesine dihydrochloride: smilagenin on accumulated phosphorylated TDP-43 in the cytosol.

[0316] Effective combination concentrations of each compound are determined and used (Table 6)Table 6

[0317] The compounds are tested on cultures (5 culture wells per conditions, 48 well-plate). Test compounds and riluzole are pre-incubated for 1 hour and 6 hours before the glutamate application. The most efficient concentration of each compound is tested in co-cultures.

[0318] Immunostaining on Primary Spinal Neurons

[0319] 24 hours after intoxication, the supernatants are discarded, and cells are fixed by a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20 °C. After permeabilization with 0.1% of saponin, cells are incubated for 2 hours with 1) a mouse monoclonal antibody anti microtubule-associated-protein 2 (MAP 2) at dilution of 1 / 400 in PBS containing 1% FCS and 0.1% of saponin (this antibody is revealed with Alexa Fluor 488 goat anti-mouse IgG at the dilution 1 / 400 in PBS containing 1 % FCS, 0.1 % saponin, for 1 hour at room temperature) and 2) a) a rabbit polyclonal antibody anti-nuclear TAR DNA- binding protein 43 (TDP-43) at dilution of 1 / 100 in PBS containing 1 % FCS and 0.1 % of saponin (the antibody TDP-43 is revealed with Alexa Fluor 568 goat anti-rabbit at a dilution of 1 / 400 in PBS containing 1 % FCS, 0.1 % saponin, for 1 hour at room temperature) or b) a rabbit polyclonal antibody anti-phosphorylatedTDP-43 at dilution of 1 / 200 in PBS containing 1 % FCS and 0.1% of saponin (the antibody phosphoTDP-43 is revealed with Alexa Fluor 568 goat anti-rabbit at a dilution of 1 / 400 in PBS containing 1 % FCS, 0.1 % saponin, for 1 hour at room temperature).

[0320] Nuclei are counterstained with the fluorescent dye Hoechst (Sigma Aldrich), a marker of cell number. For each condition, 30 pictures representative of all the well area per well are taken using ImageXpress® with 20x magnification, using the same acquisition parameters. From the images, analyses are directly performed by MetaXpress®. Analysis of neuron survival is measured. Analysis of neurite network is measured. Analysis of cytoplasmic TDP- 43 in MAP2 positive neurons is measured. Analysis of phosphorylated TDP-43 in MAP2 positive neurons is measured.

[0321] Immunostaining of Co-Cultures of Spinal Explants and Myoblasts

[0322] After 48 hours of intoxication, cells are incubated with 500 nM of a-bungarotoxin coupled with Alexa 488 during 15 minutes in the culture medium at 37 °C to detect motor endplates. After 2 washings in PBS, cells are fixed by a solution of 4 % of paraformaldehyde in PBS, pH = 7.3 for 20 minutes at room temperature. The cells are washed 2 times in PBS. A solution of PBS containing 0.1% of saponin and 1% FCS for 15 minutes at room temperature is used to permeabilize cells and block non-specific sites. Then co-cultures are incubated with a rabbit monoclonal anti-neurofilament 200 KD antibody (NF) at the dilution of 1 / 500 in PBS containing 1% FCS, 0.1% saponin, for 2 hours at room temperature. Antibody against NFstain the axon of motor neuron is used to evaluate the global neurite network. These antibodies are revealed with Alexa Fluor 568 goat anti-rabbit IgG at the dilution 1 / 400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature and an anti-mouse 488. Nuclei are counterstained with the fluorescent dye Hoechst (sigma 1 / 1000). For each condition, 20 pictures representative of all the well area per well are taken using ImageXpress® with lOx magnification using the same acquisition parameters. From the images, analyses are directly performed by MetaXpress®. Total neurite length is measured. The number of NMJs is measured. The mean size of NMJs is measured.

[0323] All values are expressed as mean + / - SEM. Statistical analysis is performed with GraphPad Prism using a one-way ANOVA, followed by a Dunnett’s test or a Fisher’s LSD test. p<0.05 is considered significant.EXAMPLE 3: Sublingual Formulation of Cutamesine, General Methods

[0324] Preparation of Calibration and Qualification Solutions

[0325] To prepare the water TFA 0.01% solution, 100 pL of trifluoroacetic acid were dissolved in 1000 mL of HPLC water.

[0326] To prepare the diluting solvent, an equal volume mixture of water / acetonitrile was prepared. 500 mL of acetonitrile were dissolved in 500 mL of HPLC water.

[0327] To prepare the cutamesine dihydrochloride stock solution, a stock solution of cutamesine at 1.0 mg / mL was prepared. Cutamesine dihydrochloride was massed (corresponding to 10 mg of cutamesine base) and dissolved in the diluting solvent and the volume was made up to 10 mL with the same solvent.

[0328] To prepare the cutamesine dihydrochloride standard solution, six standard solutions of cutamesine dihydrochloride were prepared in concentrations between 10 pg / mL and 200 pg / mL by diluting the stock solution in the dilution solvent according to Table 7.Table 7: Cutamesine Standard Solutions

[0329] Reconstituted solutions of cutamesine dihydrochloride were prepared to evaluate the effect of excipients on the HPLC analyses. 6 reconstituted solutions from 10 pg / mL to 200 pg / mL were prepared by diluting the stock solutions of cutamesine and placebo in theappropriate proportions reported in Table 8.Table 8: Cutamesine Reconstituted Solutions

[0330] To prepare the placebo stock solution, two placebo tablets were suspended in about 8 mL of diluting solvent. The mixture was stirred in an ultrasonic bath for about 5 minutes. The volume was made up to 10 mL with the same solvent. The solution was spun by centrifuge at8000 rpm for 10 minutes and fdtered through a 0.45 pm PVDF membrane.

[0331] High Performance Liquid Chromatography (HPLC) Assay

[0332] A HPLC assay method was developed and optimized to determine specificity, linearity, accuracy, and repeatability of cutamesine solutions with concentrations ranging from 10 to 200 mg / L.

[0333] Cutamesine dihydrochloride standards were injected. Calibration graph plots peak area in mAU min versus concentrations in pg / mL. Regression coefficient was calculated from the straight-line equation. Concentration of cutamesine dihydrochloride was determined from the peak area.

[0334] Concentration of standard solutions was calculated using the following formula: 100where m is the mass of cutamesine in the stock solution in mg, P is the purity of the cutamesine standard in decimal (e.g. 0.95), V is the volume of the stock solution sample in mL, 368.5124 is the molar mass of cutamesine in g / mol, 441.4343 is the molar mass of cutamesine dihydrochloride in g / mol, and Va is the final dilution volume of standard solution in mL (e.g., 5 mL).

[0335] The concentration of cutamesine in the test solutions was calculated as follows:where Acutamesine is the area of cutamesine peak in mAU min, a is the slope of calibration curve.

[0336] Chromatographic parameters were stated using a standard solution of cutamesine at100 pg / L. Chromatogram and characteristic values of the cutamesine peak at 230 nm areshown in FIG. 5. The 2D and 3D UV spectra of the cutamesine peak are shown in FIGs. 6A- 6B. The spectra were recorded from 200 to 500 nm. Specificity was verified by comparing the chromatogram of a cutamesine standard solution (FIG. 7A) to a chromatogram of a cutamesine standard solution in the presence of excipients (FIG. 7B). No interfering peaks coming from excipients were observed.

[0337] A linearity study was carried out with standard solutions and reconstituted standard solutions (formulated cutamesine) from 10 to 200 mg / L. Each solution was prepared by successive dilution from a single stock solution. Regression lines were calculated, excluding the origin (FIGs. 8A-8B). The response was linear from 10 to 200 mg / L.

[0338] Accuracy was demonstrated for standard and reconstituted standard solutions. The recovery of each individual sample was determined by comparing the values of the reconstituted standard solutions with the linear regression curve of the standard solutions. Results are found in Table 9. Individual recoveries varied between 98.12% and 99.66% and the mean recovery (99.12%) was within 98%-102%.Table 9: Accuracy

[0339] Repeatability of responses was assessed by six injections of 200 mg / L standard solution. The relative standard deviation of the cutamesine peak areas was calculated. Results are found in Table 10. The %RSD was less than 2.0%.Table 10: Repeatability

[0340] Stability of standard solutions was tested by storing different concentrations of cutamesine standard solutions at room temperature protected from light, and injected at 1, 3, and 7 days. Percent recoveries were calculated versus the initial concentration. Results are shown in Table 11. Standard solutions were found to be stable at least after seven days of storage at room temperature and in the absence of light.Table 11: Stability

[0341] The HPLC method was qualified for the analysis of cutamesine solutions. The HPLC method demonstrated specificity and repeatability. Linearity and accuracy were confirmed with cutamesine solutions from 10 mg / L to 200 mg / L in both the absence and the presence of excipients. Stability of the standards was verified over a period of seven days at room temperature.EXAMPLE 4: Liquid Formulation of Cutamesine

[0342] A forced degradation study of an aqueous solution of cutamesine at 1.0 mg / mL was performed to evaluate the possibility of considering a liquid formulation of cutamesine.

[0343] An aqueous solution of SA4503 was prepared at 1.1 mg / mL. This concentration was chosen so that the final concentration of the solution would be 1.0 mg / mL after the addition of the acidic, basic, and oxidizing reagents used during the forced degradation study.

[0344] In a 25 mL volumetric flask, 34.6 mg cutamesine was dissolved in pure water and the volume was made up to 25 mL with the same solvent. The stress conditions of the degradation study are shown in FIG. 9A, where cutamesine levels are expressed as the percent recovery versus the control. Degradation was monitored by measuring the cutamesine concentration using the HPLC assay method described in Example 3. A summary of the degradation results is found in FIG. 9L.

[0345] For the unstressed control sample, the cutamesine solution was aliquoted in 1.0 mL volumes into four 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of pure water was added to each vial as to attain a concentration of 1.0 mg / mL. Each aliquotwas homogenized and stored at room temperature (20°C-23°C) away from light.

[0346] For the acid hydrolysis reaction, the cutamesine solution was aliquoted in 1.0 mL volumes into three 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of concentrated hydrochloric acid (37%) was added to each vial. Each aliquot was homogenized and stored at room temperature (20°C-23 °C) away from light. No degradation was observed after 7 days. Cutamesine dihydrochloride appeared stable under acidic conditions (FIGs. 9B-9C).

[0347] For the basic hydrolysis reaction, the cutamesine solution was aliquoted in 1.0 mL volumes into three 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of concentrated sodium hydroxide solution (10 N) was added to each vial. Each aliquot was homogenized and stored at room temperature (20°C - 23 °C) away from light. The addition of NaOH IN to the solutions led to precipitation of cutamesine (FIGs. 9D-9E). The remaining amount of cutamesine corresponded to solubility in basic form (e.g., 0.220 mg / mL).

[0348] For the oxidation reaction, the cutamesine solution was aliquoted in 1.0 mL volumes into three 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of a 30% hydrogen peroxide solution was added to each vial. Each aliquot was homogenized and stored at room temperature (20 °C - 23 °C) away from light. Cutamesine was found not to be stable under strong oxidizing conditions; after 7 days, only 80% was recovered (FIGs. 9F- 9G)

[0349] For the photolysis reaction, the cutamesine solution was aliquoted in 1.0 mL volumes into three 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of pure water was added to each vial. Each aliquot was homogenized and exposed to daylight at room temperature (20 °C - 23 °C). Under photolytic conditions, cutamesine slightly degraded (FIGs. 9H-9I). After 7 days, the amount of cutamesine was found to be 97.17% of the initial concentration. Degradation peaks are visible at T=7 days. Some of these peaks have the same retention times of those observed under oxidizing conditions.

[0350] For the thermolysis reaction, the cutamesine solution was aliquoted in 1.0 mL volumes into three 1.5 mL clear glass vials (one vial for each analysis time point). 0.1 mL of pure water was added to each vial. Each aliquot was homogenized and stored in an oven at 70 °C away from light. No degradation peaks were observed. Cutamesine was found to be stable at 70 °C for 7 days (FIGs. 9J-9K).

[0351] An aqueous solution of cutamesine at 1.0 mg / mL was found to be stable in strong acid medium (IN HC1) for 7 days at room temperature and away from daylight. An aqueoussolution of cutamesine at 1.0 mg / mL was also found to be stable at 70 °C for 7 days. An aqueous solution of cutamesine at 1.0 mg / mL was found to precipitate and be slightly soluble in an alkaline medium (IN NaOH). An aqueous solution of cutamesine at 1.0 mg / mL was found to be unstable in a strong oxidizing medium (H2O2 3%) at room temperature. Finally, an aqueous solution of cutamesine at 1.0 mg / mL was found to degrade slightly under daylight after 7 days at room temperature.

[0352] These results show that when cutamesine is protected from light and oxidation, the liquid form of cutamesine is a viable formulation.EXAMPLE 5: Sublingual Formulation of Cutamesine

[0353] Tablets of two different strengths of cutamesine were prepared - 1 mg and 3 mg. Cutamesine and excipients were massed according to Table 12.Table 12: Preparation of Sublingual Tablets

[0354] Powders were combined and homogenized using geometric dilution. Then the magnesium stearate was added, and the resulting powder was again homogenized before the press step.

[0355] Ten tablets from each strength and ten placebo tablets were randomly reserved for thickness and diameter uniformity study. Measurements were carried out with a Digital Vernier.

[0356] A compatibility study of cutamesine and the excipient mixture was performed usingDifferential Thermal Analysis (DTA). Approximately 8-13 mg of cutamesine, placebo, and formulation were placed in a 100 pL platinum cup. Samples were heated from 25 °C to 300 °C at a rate of 10 °C / minute under airflow. Indium, aluminum, and zinc were used as standards for temperature calibration.

[0357] A group of 20 tablets was randomly taken from each formulation and accurately massed. Each tablet mass was compared with the average of the masses.

[0358] 10 cutamesine tablets were randomly taken and analyzed by HPLC. Each tablet was powdered, and the active ingredient was extracted with water in a 5 mL volumetric flask. The contents of the flasks were spun by centrifuge at 8000 G for 10 minutes and then filtered through a 0.45 pm PVDF membrane. After appropriate dilution, the solution was analyzed by HPLC according to the qualified method. The drug content was expressed as a percentage of the claimed label and should be 100 ± 15%.

[0359] Shaking tests were performed to evaluate the disintegration time for active tablets and placebo tablets. One tablet was placed in a 20 mL glass beaker containing 2 mL of pure water at 37 ± 0.5 °C. The beaker was shaken gently back and forth to disperse the tablet. The time for which the tablet disintegrate into particles was recorded. Tests were performed in triplicate.

[0360] In vitro dissolution tests were performed using a method specifically designed for fast-disintegrating sublingual tablet dosage forms (FIG. 10). This method simulates the sublingual cavity in contrast to the official compendial method, which uses large volumes of dissolution medium with constant agitation. A 0.45-pm PVDF filter membrane was prewetted with 50 pL of distilled water and placed between the 15 mL glass funnel and the fritted glass base, which were clamped and inserted into the Buchner flask. A 10 mL disposable plastic collection tube was placed at the outlet tip of the clamped unit to collect the filtrate. The Buchner flask was connected to a vacuum line controlled by automatic shut-off, quick-disconnect coupling inserts (on / off switches).

[0361] 2 mL of distilled water at 25 °C was added to the 15 mL glass funnel as dissolution medium. The tablet was placed undisturbed in the dissolution medium. Time points ranging from 30 seconds to 120 seconds (stopwatch) were previously tested to assess the dissolution rate of a representative formulation of cutamesine tablets. Based on these results, the 60 second time point was selected for the subsequent experiments. At each time point, full vacuum was applied by opening the on / off switch, causing the total volume of dissolution medium to be withdrawn immediately through a 0.45 pm filter membrane into the collection tube and terminating any further dissolution. The membrane prevented the passage ofundissolved particles and was replaced with a new membrane for each dissolution analysis. The cutamesine content of each sample was measured by HPLC as described above. To obtain the percentage of drug released, cutamesine content (mg) in the filtrate was compared with the mean content uniformity of ten individual cutamesine tablets. The reusable parts in contact with the cutamesine, including the glass funnel and fritted glass base, were thoroughly cleaned before each test.

[0362] Direct compression was used to prepare fast-disintegrating sublingual tablets of cutamesine. Sodium starch glycolate was used as the superdisintegrant, mannitol starch as the filler and binder, and magnesium stearate as the lubricant, in the proportions given in Table 13Table 13: Composition of Cutamesine Tablets

[0363] Characteristics of the tablets are shown in Table 14. FIG. 11 shows images of the tablets.Table 14: Diameter and Thickness of Tablets

[0364] Twenty tablets were randomly taken and massed to determine mass variation. Masses and variation are shown in FIG. 12.

[0365] The uniformity of drug content was tested on 10 tablets for each formulation (FIG. 13). The drug content is expressed in mg of cutamesine per tablet and as a percentage of the label claim (e.g., 1.0 mg and 3.0 mg). Each individual content of both formulations did not exceed ± 15%. The average percentage of drug content for cutamesine 1 mg was 104.0 ± 2.7%. The average percentage of drug content for cutamesine 3 mg was 99.5 ± 1.2%. Results showed uniform drug distribution in all tablets.

[0366] The shake test is a rapid tablet disintegration (or dispersion) test adapted to the specificities of sublingual tablets. The sample tablet was placed in a beaker containing a volume of water equivalent to the limited volume of saliva present in the sublingual cavity (approximately 1.5 mL). The beaker was manually shaken gently back and forth to simulate tumbling and tongue pressure in the oral cavity. Disintegration times of the tablets into fine particles are given in Table 15. Active and placebo tablets disintegrated into fine particles in about 30 seconds.Table 15: Disintegration Time of Cutamesine Tablets

[0367] The in vitro method was designed to evaluate the dissolution of fast-disintegrating sublingual tablets. The method has the advantage of testing dissolution in media volumes as small as 2 mL, which corresponds to the volume of saliva secreted in 2 minutes. The recommended time for sublingual tablets to remain under the tongue is 120 seconds. Results are shown in Table 16. Amounts of cutamesine released after 60 seconds were 92.3 ± 2.1% and 87.9 ± 5.7% for the 1 mg and the 3 mg tablets, respectively.Table 16: In Vitro Dissolution of Cutamesine Tablets

[0368] To test thermal stability, cutamesine, formulated cutamesine, and excipients were sampled and analyzed by differential thermal analyses (DTA), which were carried out under air. Temperature was increased from room temperature to 300 °C at 10 °C per minute (FIG. 14A). Until 225 °C, the mass loss profde was similar for cutamesine and excipients, while that of the formulated powder exhibited a larger loss. This loss was due to the preparation of the powder mixture. The preparation required prolonged trituration under normal atmosphere to ensure homogeneity of concentration (geometric dilution) in view of the small quantity of active ingredient in the tablet. The formulated cutamesine sample lost 2.72% of mass until 200 °C. This loss was mainly attributed to the humidity absorbed by the powder during its preparation.

[0369] As shown in FIG. 14B, the behavior of cutamesine was typical of a meltingdecomposition sequence. The melting point was estimated at 224.75 °C and the maximum of the decomposition peak was reached at 269.91 °C. The melting point of mannitol (highest- mass excipient in the formulation) is observed at 168.10 °C. Dissolution of cutamesine in the liquid matrix of the mannitol following melting is possible.

[0370] 1 mg cutamesine tablets were submitted to thermal degradation at 70 °C for 48 hours. The forced degradation assay is described previously in Example 4. Results are shown in FIGs. 15A-15B. The chromatogram was essentially identical to that of the cutamesine standard. All detected impurities were present in the standard at the same proportions.EXAMPLE 6: Bioavailability of Cutamesine Sublingual Tablets

[0371] Drugs administered via the sublingual route are directly absorbed into blood circulation and avoid the hepatic first-pass metabolism. Cutamesine transport through the oral mucosal epithelial barrier was evaluated in vitro using cell culture inserts (e.g., Transwell® cell culture inserts) (FIG. 16). Bioavailability of cutamesine was studied on SCC4 cell monolayers as an alternative sublingual barrier model. SCC4 cell lines are cells of oral epithelium extracted from human tongue squamous cell carcinoma. Cells were prepared according to the preparation method shown in FIG. 17.

[0372] A stock solution of cutamesine in HBSS buffer was prepared (FIG. 18A). 12.60 mg of cutamesine dihydrochloride was added to a 10 mL volumetric flask. 8 mL HBSS buffersolution was added, and the suspension was shaken until complete dissolution of cutamesine. The volume was completed to the gauge with HBSS. The solution was filter sterilized on 0.2 mm PVDF membrane under a PSM laminar flow hood.

[0373] A stock solution of cutamesine tablet in HBSS was prepared (FIG. 18B). One 3 mg cutamesine tablet and two 1 mg cutamesine tablets were added to a 5 mL volumetric flask. 4 mL HBSS buffer solution was added, and the suspension was shaken and sonicated to homogenize the suspension. The volume was completed to the gauge with HBSS. The solution was filter sterilized on 0.2 mm PVDF membrane under a PSM laminar flow hood. The concentration was checked by HPLC and adjusted at 1 g / L with HBSS buffer solution.

[0374] A placebo stock solution was prepared. Three placebo tablets were added to a 5 mL volumetric flask. 4 mL HBSS buffer solution was added, and the suspension was shaken and sonicated to homogenize the suspension. The volume was completed to the gauge with HBSS. The solution was filter sterilized on 0.2 mm PVDF membrane under a PSM laminar flow hood.

[0375] For cell monolayer treatment, the culture media was removed from each insert and well. The apical compartment was rinsed with 500 pL HBSS lx. The basolateral compartment was rinsed with 1500 pL HBSS lx. The two compartments were emptied and 500 pL HBSS lx was added to the apical compartment and 1500 pL HBSS lx was added to the basolateral compartment. Cell monolater resistance was measured (R at t=0), and the two compartments were emptied. 1500 pL HBSS lx was added to each basolateral compartment and 500 pL of a sample was added to a well. The concentration of cutamesine was followed by HPLC in each compartment at t = 0, 15, 30, 60, and 180 minutes. After 180 minutes, the solutions of each compartment were individually collected. 500 pL HBSS lx was added to the apical compartment and 1500 pL HBSS lx was added to the basolateral compartment. The resistance of the cell monolayers was measured (R at t=l 80 minutes). Plate plans are shown in FIGs. 19A-19B.

[0376] Transepithelial / transendothelial electrical resistance (TEER) is accepted as a quantitative technique to measure the integrity of tight junction dynamics in cell culture models of epithelial monolayers. Cell monolayer resistances were measured before and after the bioavailability tests. Results are shown in FIG. 20A-20B. R is the resistance to ion flux between the apical and basolateral sides of the cell monolayer and therefore the integrity of the intercellular tight junctions. TEER values represent the resistance per surface unit corrected by subtraction of the background (“Control without cell monolayer”).

[0377] FIGs. 21A-21D show kinetic analytical results of both apical and basolateralcompartments during the transepithelial transfer of cutamesine. Apparent permeability (Papp) was used to determine the degree of permeability to rank different drug substances and to comprehend transportation pathways. The Papp is calculated using the equation:where dQ / dt is the slope of the cumulative fraction absorbed versus time in seconds, A is the area of the fdter in cm2, and Co is the initial concentration in the apical chamber. Drug permeability according to Papp is summarized in Table 17. Calculated Papp varied between 3.7810'6to 5.91*10'6cm / s, indicating that cutamesine is characterized by standard permeability in the Apparent Drug Permeability Index.Table 17: Apparent Drug Permeability Index

[0378] The kinetic transfer was not linear, particularly during the initial times. From 15 to 180 minutes, the mean transfer rate from the apical to the basolateral chamber was V = 0.09 pg / min cm2At the initial times, no difference in basolateral concentrations was observed between cutamesine alone and formulated cutamesine. However, at high concentrations, the excipients appeared to ameliorate the transfer in the case of the highest concentration (FIG.21D)

[0379] In conclusion, SCC-4 cell lines (oral epithelium extracted from human tongue squamous cell carcinoma) were suitable for modeling the sublingual bioavailability of cutamesine. Excipients had no deleterious effect on the cell monolayer. The transfer kinetics of cutamesine corresponded to what is expected of a standard transfer (0.09 pg / min cm2).EXAMPLE 7 : Administration of Cutamesine / Smilagenin in Mice

[0380] Superoxide dismutase (SOD1) mouse models of ALS were used to study synergistic effects of cutamesine and smilagenin. Mice were treated with cutamesine, smilagenin, a combination of cutamesine and smilagenin, or only vehicle (control) (n=8 per group, 4 male and 4 female). Mice were treated daily from day 70 to day 105. FIG. 39A illustrates a schematic of the experimental procedure.

[0381] Neuroscores were provided at day 105, at which some mice were symptomatic (FIG. 39B). However, no mice were at endstage (stage 7-8) as of day 105.

[0382] Also at day 105, lumbar spinal cord tissue was collected from the SOD1-G93A mice for each of the four conditions (cutamesine, smilagenin, a combination of cutamesine andsmilagenin, and only vehicle (control)). RNA was isolated from the tissue samples and bulk RNA sequencing occurred. RNAseq analysis was performed using R. RNA sequencing reads were aligned to the hg38 reference genome. Read counts were exported and subjected to filtering to remove low-expressed genes. All expression values were log-normalized and library size-normalized.

[0383] Transcriptomic analysis was performed on the lumbar spinal tissue and differentially expressed genes were determined (FIGs. 39C-39E). Approximately 30 differentially expressed genes were found when comparing cutamesine vs. vehicle (FIG. 39C). Approximately 300 differentially expressed genes were found when comparing smilagenin vs. vehicle (FIG. 39D). Approximately 3,000 differentially expressed genes were found comparing smilagenin and cutamesine vs. vehicle (FIG. 39E).EXAMPLE 8: Administration of Cutamesine / Smilagenin in Humans

[0384] A subject is diagnosed with ALS. The subject is prescribed a sublingual cutamesine tablet or a solid dose formulation of cutamesine and a solid dose formulation of smilagenin as described herein for ALS therapy. The subject begins a course of therapy for ALS by taking a unit dosage form on a prescribed schedule.

[0385] Forty-five patients are randomized to receive six-months of a double-blind, placebo- controlled treatment. Six patients are given 90 mg smilagenin and 3 mg cutamesine. Six patients are given 90 mg smilagenin and 6 mg cutamesine. Six patients are given 180 mg smilagenin and 3 mg cutamesine. Six patients are given 180 mg smilagenin and 6 mg cutamesine. Six patients are given a placebo control. After the first treatment course, two doses are determined. In the second six-month treatment course, five patients receive the first dose, five patients receive the second dose, and five patients receive the placebo control (FIG. 40)

[0386] The treatments are reviewed for safety as a primary endpoint with additional secondary endpoints of PK, neurofilament and other biomarkers, ALSFRS-R, and PRO / CRO. EXAMPLE 9: Synthesis of Cutamesine and Salts Thereof

[0387] Preparation of tert-butyl 4-(3-phenylpropyl)piperazine-l-carboxylate (Intermediate 1)

[0388] 1-Boc-piperazine (125 g, 0.67 mol, 1.0 eq.) (Boc represents a / c / 7-buty 1 oxy carbonylprotecting group) and K2CO3 (185.52 g, 1.34 mol, 2.0 eq.) were added to a nitrogen-purged, 3 L Chemglass reactor equipped with an overhead agitator. 1212.5 mL isopropanol (IP A) was added to the reactor and the mixture was stirred with the overhead agitator. (3- bromopropyl)benzene (146.98 g, 0.74 mol, 1.1 eq.) was added to the mixture and 125 mL IP A was injected to rinse down any solid substance on the wall of the reactor to the mixture. The resulting mixture was stirred at 22 ± 3 °C for at least 30 minutes. The temperature was raised to 72 ± 3 °C and the resulting mixture was agitated at 72 ± 3 °C for 20-24 hours. Then the mixture was cooled down to 50 °C and a sample was taken to measure the reaction conversion by high performance liquid chromatography (HPLC). After the desired conversion was achieved, the reacted mixture was cooled down to 22 ± 3 °C. The mixture was filtered with a filter funnel with 1 part Celite (wet with IP A) connected to a receiver and vacuum line. 535 mL IPA was used in the filtration to rinse any residue from the reactor to the filter funnel. Filtration provided a solution containing / c / 7-butyl 4-(3- phenylpropyl)piperazine-l -carboxylate (Intermediate 1) with a yield greater than 70%. Intermediate 1 solution was used directly in the next step without further purification.

[0389] Deprotection of Intermediate 1 to form l-(3-phenylpropyl)piperazine dihydrochloride (Intermediate 2)

[0390] Intermediate 1 was deprotected to remove the Boc protecting group by reacting with HC1 in IPA (HC14PA, HC1 concentration 5.0-6.0 M). In a clean and dry reactor purged with N2, HC14PA (675 mL) was added and heated to 45 ± 3 °C over 25 minutes under agitation. The Intermediate 1 solution was added to the reactor while maintaining the temperature at 45 ± 3 °C. 133.75 mL IPA was used to rinse down any residual Intermediate 1 solution to the reactor. The mixture was stirred at 45 ± 3 °C for at least 3-4 hours. After the reaction was complete (confirmed by HPLC), the reacted mixture was cooled down to 22 ± 3 °C over 20- 30 minutes and then stirred for at least 16-18 hours at 22 ± 3 °C. The reacted mixture (slurry) was run through a filter funnel connected to a receiver and vacuum line. 401.25 mL IPA was used to rinse down any residue from the reactor to the filter funnel. Additional 535 mL IPA was used to wash the solid phase with sufficient mixing. The mixture was filtered and the solid phase was collected and dried with suction and N2 for at least 18 hours to produce l-(3- phenylpropyl)piperazine dihydrochloride (Intermediate 2) as a solid. The solid was tested forresidual piperazine (<3% w / w), IPA (<5000 ppm), and water content (<3% w / w). Intermediate 2 was produced at 145.13 g (yield 78%).JH NMR (400 MHz, DMSO-d6) 5 12.01 (s, 1H), 10.09 (s, 2H), 7.33-7.14 (m, 5H), 3.86-3.24 (m, 8H), 3.22-3.02 (m, 2H), 2.62 (t, J=7.8 Hz, 2H), 2.13-1.88 (m, 2H). 13C NMR (101 MHz, DMSO-d6) 8 140.55, 128.46, 128.30, 126.16, 55.27, 47.63, 40.15, 39.94, 39.73, 39.63, 39.52, 39.31, 39.10, 38.90, 31.95, 24.73.

[0391] Preparation of 3,4-dimethoxyphenylethyle tosylate (Intermediate 3)

[0392] 2-(3,4-dimethoxyphenyl)ethanol (90.38 g, 0.50 mol, 1.0 eq.) and 542.50 mL 2- methyltetrahydrofuran (2-MeTHF) were added to a nitrogen-purged, 3 L Chemglass reactor equipped with an overhead agitator. The mixture was stirred at room temperature.Tetramethylethylenediamine (TMEDA, 86.48 g, 1.50 mol, 1.5 eq.) was added to the mixture and the mixture was cooled to 0 ± 3 °C. A solution of -toluene sulfonyl chloride (TsCl, 141.84 g, 1.50 mol, 1.5 eq.) in 2-MeTHF (271.25 mL) was added to the mixture while the temperature was maintained at 0 ± 3 °C (not higher than 5 °C). 90 mL 2-MeTHF was used to rinse the TsCl container to transfer TsCl to the reactor. The mixture was stirred at 0 ± 3 °C for 2-3 hours. A sample was taken for reaction conversion by HPLC.

[0393] After the desired conversion was achieved, the reacted mixture was cooled down to - 20 °C and maintained at -20 °C for at least 16 hours. The temperature was then raised to 10 ± 3 °C. 2 M NaOH (488.70 g total solution of NaOH in water, 1.8 eq. NaOH) was added to the mixture to quench the reaction. The temperature was maintained below 25 °C during the quench. The temperature was then adjusted to 22 ± 3 °C and the mixture was stirred for 2-3 hours. A sample of the upper organic phase was taken for reaction conversion by HPLC.

[0394] After the desired conversion was achieved, the mixture was held to allow layers to separate for at least 30 minutes (a lower aqueous layer and an upper organic layer). The lower aqueous layer was drained. The organic layer was washed with HC1 solution, water, and K2CO3 solution. The temperature of the organic layer was adjusted to 10 ± 3 °C. 2 M HC1 (488.70 g total solution of HC1 in water) was added to the organic layer over at least 20minutes (with temperature not higher than 25 °C). The mixture was stirred at 22 ± 3 °C for 30 minutes and then held without stirring to allow layers to separate for at least 30 minutes. The lower aqueous layer was drained. The organic layer was washed with water. 452.50 mL water was added to the organic layer and the mixture was stirred at 22 ± 3 °C for 30 minutes. After that, the mixture was held to allow layers to separate for at least 30 minutes. The lower aqueous layer was drained and the organic layer was washed with K2CO3 solution. 452.50 g 1 M K2CO3 solution was added and the mixture was stirred at 22 ± 3 °C for 30 minutes. Then the mixture was held to allow layers to separate for at least 30 minutes. The lower aqueous layer was drained and the organic layer was transferred to a polyethylene (PE) container. 180 mL 2-MeTHF was used to rinse down the organic layer to the PE container. The organic layer containing 3,4-dimethoxyphenylethyle tosylate (Intermediate 3) with a purity of at least 90% was used directly in the next step without further purification.

[0395] Preparation of l-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine

[0396] K2CO3 (311.59 g, 5 eq.) and Intermediate 3 solution (166.85 g of Intermediate 3, 1 eq.) were added to a nitrogen-purged, 3 L Chemglass reactor equipped with an overhead agitator. The mixture was stirred. 151.25 mL 2-MeTHF was used to rinse any residual Intermediate 3 from the PE container to the reactor. Intermediate 2 (125 g, 1 eq.) was dissolved in water in another PE container and added to the Intermediate 3 mixture solution.125 mL water was used to rinse any residual Intermediate 2 from the other PE container to the reactor. The mixture was stirred at 22 ± 3 °C for 30 minutes and then at 72 ± 3 °C for 20- 24 hours. The temperature was cooled down to 50 °C and a sample was taken for conversion measurement by HPLC. After the reaction was complete (confirmed by HPLC), the mixture was cooled down to 22 ± 3 °C and the upper organic layer was washed sequentially with 756.25 mL 1 M NaOH (lx) and water (2x). After removing the aqueous layer, 500 mL 2-MeTHF was added and the mixture was distilled under atmospheric pressure to about 750 mL. The distillation was repeated once to obtain 750 mL solution. Water content of the solution was less than 0.15% w / w. The solution was fdtered using a fdter funnel with 0.5 part Celitel (wet with 2-MeTHF) connected to a receiver and vacuum line. 605 mL 2-MeTHF was used to rinse down any residue substance from the reactor to the fdter funnel. Filtration provided a solution containing cutamesine.

[0397] Preparation of l-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (cutamesine dihydrochloride salt)

[0398] The cutamesine solution (1.0 eq. cutamesine) was added to a clean and dry reactor. 151.25 mL 2-MeTHF was used to rinse any residual cutamesine from the container to the reactor. HC1 solution (135.27 g HC1, 3 eq.) mixed with 605 mL 2-MeTHF was added to the reactor over 2-3 hours under stirring. 151.25 mL 2-MeTHF was used to rinse the HC1 solution from the container to the reactor. The resulting mixture was stirred at 22 ± 3 °C for 16-18 hours. The mixture was fdtered with a fdter funnel connected to a receiver and vacuum line (302.50 mL 2-MeTHF) to rinse down any residual substance from the reactor to the fdter funnel. The solid phase was rinsed with 453.75 mL 2-MeTHF twice and dried with suction / N2 for at least 18-20 hours to produce cutamesine HC1 salt (135.35 g, 68% yield). The dried cutamesine HC1 salt was tested for 2-MeTHF (<5000 ppm), TMEDA (<1500 ppm) and water content (<0.5% w / w).

[0399] Recrystallization of cutamesine HCl salt

[0400] A sample (e.g., 150 g) of cutamesine HCl salt was added to a clean and dry reactor. 2250 mL ethanol was added to the reactor and mixture was stirred. 112.50 mL water was added to the reactor and the temperature of the mixture was raised to 78 ± 3 °C over 1 hour. The mixture was stirred at 78 ± 3 °C for 1 hour. The temperature of the mixture was lowered to 22 ± 3 °C over 1-2 hours and the mixture was stirred at 22 ± 3 °C for 16 hours. The temperature of the mixture was then lowered to 2 ± 3 °C over 1 hour and the mixture was stirred at 2 ± 3 °C for 4-5 hours. The mixture was then separated by fdter funnel and ethanol- water (297 mL ethanol and 2.25 mL water) was used to rinse any residual substance from the reactor and further to rinse the solid phase (445.5 mL ethanol and 3.38 mL water). The solid(recrystallized cutamesine dihydrochloride salt) was collected and dried with suction / N2 for at least 18-20 hours. The recrystallized cutamesine dihydrochloride salt was tested for ethanol (<5000 ppm), 2-MeTHF (< 5000 ppm), IP A (<5000 ppm), piperazine (<1500 ppm), and water content (<0.5% w / w). 135 g cutamesine dihydrochloride salt was collected (90% yield). *H NMR (400 MHz, D2O) 8 7.43-7.33 (m, 2H), 7.32-7.30 (m, 1H), 7.31-7.23 (m, 2H), 6.97 (d, J=8.3 Hz, 1H), 6.95 (d, J=1.9 Hz, 1H), 6.89 (dd, J=8.2, 1.9 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.80-3.53 (m, 8H), 3.52 (dd, J=9.2, 6.9 Hz, 2H), 3.32-3.21 (m, 2H), 3.10-2.93 (m, 2H), 2.72 (t, J=7.4 Hz, 2H), 2.17-1.95 (m, 2H).13C NMR (101 MHz, D2O) 8 148.30, 147.31, 140.19, 128.82, 128.52, 128.49, 126.60, 121.41, 112.25, 112.12, 57.66, 56.36, 55.69, 55.67, 48.71, 48.51, 31.52, 29.17, 24.94.

[0401] XRD Analysis

[0402] The cutamesine dihydrochloride salt was observed to have two polymorphic forms (Form A and Form B). Two samples of polymorphic form A (Form A-l and Form A-2) and Form B were characterized by X-ray diffraction (XRD) using an X-ray wavelength of 1.5406 A. Forms A-l and A-2 were observed to have an X-ray diffraction pattern comprising peaks at 4.9°, 9.8°, 14.7°, and 19.6° ± 0.2 20 as measured by X-ray diffraction using an X-ray wavelength of 1.5406 A. Form B was observed to have an X-ray diffraction pattern comprising peaks at 4.5°, 12.3°, and 18.5° ± 0.2 20 as measured by X-ray diffraction using an X-ray wavelength of 1.5406 A. The estimated crystallinity for each polymorph was computed using DIFFRAC.EVA V5 software (V5). Form A-l and Form B were observed to have estimated relative crystallinity values of 87%. FIG. 25 illustrates the X-ray diffraction spectra of Form A-l, Form A-2, and Form B using an X-ray wavelength of 1.5406 A.

[0403] Thermal analysis: TA Instruments DSC 25 was used for differential scanning calorimetry (DSC) analysis of Form A-l and Mettler Toledo DSC 3 was used for DSC analysis of Form B. 5-8 mg material was weighed into an aluminum pan, sealed, and poked prior to analysis. The temperature was increased from room temperature to 400 °C at a rate of 10 °C / minute. TA Instruments TGA 550 was used for thermogravimetric analysis (TGA) of Form A and B. 5-9 mg material was placed in an aluminum pan and sealed. The temperature was increased from room temperature to 600 °C at a rate of 10 °C / minute.

[0404] Form A-l was observed to have a glass transition with a peak with an onset temperature of 168 °C, indicating the presence of an amorphous content within the test sample. An endothermic event was observed to appear with an onset temperature of 194 °C. The event could be attributed to the melting of an undetermined / unknown solid form. An endothermic event was observed to occur at 271 °C, corresponding to the melting of Form A-1, followed by a peak starting at 332°C, corresponding to sample decomposition. Form A-l was observed to show sample decomposition at elevated temperatures greater than 250 °C via TGA. FIG. 26 illustrates the differential scanning calorimetry analysis of Form A-l. FIG. 27 illustrates the TGA graph of Form A-l.

[0405] Form B was observed at have an endothermic event at 270 °C, corresponding to the melting point of Form B. Sample decomposition was observed after 300 °C. TGA analysis showed sample decomposition occurring at elevated temperatures of approximately 264 °C. FIG. 28 illustrates the differential scanning calorimetry analysis of Form B. FIG. 29 illustrates the TGA graph of Form B.

[0406] TABLE 18 summarizes the results of the DCS and TGA analysis for Form A-l and Form B.Table 18: DSC and TGA analysis of Form A-l and Form B

[0407] Dynamic Vapor Sorption (DVS) Analysis

[0408] The hygroscopicity of Form A-l and Form B was measured using dynamic vapor sorption (DVS) analysis. An approximately 6 mg sample of Form A-l or Form B was placed in a flat-bottom pan in a DVS chamber. Over the course of the analysis, the relative humidity (RH) was increased in stages of 10%, beginning at 0% (RH) and ending at 90% RH. At each stage of the analysis, the RH was held until the mass was stable for a maximum of 6 hours, and an image of the sample taken. After 90% RH was reached, the humidity was decreased in 10% RH stages until 0% RH was reached. The process was repeated for a total of two cycles.

[0409] During the first period corresponding to the initial drying period, the mass of Form A- 1 decreased. The decrease was attributable to the removal of surface moisture. As the % RH increased, Form A-l was observed to gradually gain mass. At 60% RH, sample sorption wasmeasured to be -0.2%, while at 80% RH, the sample was measured to gain 0.4% water molecules, and at 90% RH, the sample was measured to gain 0.6% water molecules. As the relative pressure was reduced and the RH decreased, Form A-l desorbed water molecules in a similar fashion to the sorption cycle. Form A-l exhibited a similar sample sorption and desorption pattern in the second cycle as the first. Form A-l was observed to be slightly hygroscopic due to the 0.4% gain of water molecules at 80% RH. FIG. 30 illustrates the DVS spectrum across the two cycles. FIG. 31 illustrates reversible isotherm plots for Form A-l across the two DVS cycles, indicating a physisorption process in which water gets adsorbed and desorbed in a similar fashion. FIG. 32 illustrates images of Form A-l taken during the DVS analysis at 0% RH of the first cycle, 90% RH of the first cycle, and 0% RH of the 1stcycle during desorption. Form A-l was observed to be in the form of shiny white particles, which were unchanged following the adsorption and desorption cycle. Form A-l was analyzed by powder X-ray diffraction (BXRD) before and after DVS. FIG. 33 illustrates the X-ray diffraction spectra using X-ray wavelength of 1.5406 A. No change in the peak 20 angles was observed after the DVS analysis. The absence of a change indicates no change in the solid form of Form A-l upon exposure to different relative humidity conditions.

[0410] Following an initial 2-hr drying period to remove surface moisture from Form B, the sample was subjected to the adsorption and desorption cycles as previously described. As the %RH was increased, the sample was observed to gain mass gradually. At 60% RH and above, sample sorption was measured to be -0.8%. At 80% RH, the sample of Form B was measured to gain 1.5% water molecules and gain 2.2% water molecules at 90% RH. During the desorption phase, as the relative pressure was decreased, the sample was measured to desorb water molecules, retaining 0.8% at 0% RH. The sorption and desorption cycles in the second DVS cycle were observed to be identical with the sample retaining 0.8% water at 0% RH. The retained moisture corresponded to surface moisture not part of the crystal structure. Form B was observed to be slightly hygroscopic due to the 1.5% gain of water molecules at 80% RH. FIG. 34 illustrates the DVS spectrum for Form B across two cycles. FIG. 35 illustrates isotherm plots for Form B. The isotherm plots for the first cycle were observed to be irreversible for the first cycle as the sample retained 0.8% of water molecules inside the crystal lattice. The second DVS cycle was observed to have fully reversible sorption and desorption plots that were identical to the first. FIG. 36 illustrates live images of Form B taken during the DVS analysis at 0% RH of the first cycle, 90% RH of the first cycle, and 0% RH of the 1stcycle during desorption. Form B was observed to be in the form of a white powder, which was unchanged following adsorption and desorption. Form B was analyzed bypowder X-ray diffraction (BXRD) before and after DVS. FIG. 37 illustrates the X-ray diffraction spectra using X-ray wavelength of 1.5406 A. No change in the peak 20 angles was observed after the DVS analysis. This observation indicated no change in the solid form of Form B upon exposure to different relative humidity conditions.

[0411] Form A-l and Form B of cutamesine were observed to be two different solid crystalline forms. Differential scanning calorimetry indicated that Form A-l is slightly more thermodynamically stable than Form B is, with a difference of 1 °C in the measured melting points observed between the two forms.EXAMPLE 10: Evaluation of cutamesine and smilagenin in spinal explant and neuromuscular junctions.

[0412] In this experiment, the protective effects of cutamesine and smilagenin were evaluated on wildtype or primary spinal motor neurons and on co-cultures of spinal explants and myoblasts, injured with glutamate.

[0413] Spinal explants from El 3.5 rat embryos were prepared and maintained in culture with cultured human myotubes according to the procedure described in EXAMPLE 2. The spinal explant and myotube co-culture was maintained in culture for 28 days. After 28 days of culture, the co-culture was pre-treated with cutamesine, smilagenin, or cutamesine and smilagenin for one or six hours prior to glutamate injury. Glutamate (60 pM) was applied to the co-cultures for 20 minutes in the presence of cutamesine, smilagenin, or cutamesine and smilagenin. After 20 minutes, the supernatant was removed, and replaced with fresh medium containing cutamesine, smilagenin, or cutamesine and smilagenin for an additional 48 hours. After 48 hours, the co-cultures were evaluated for neurite length, the number of neuromuscular junctions (NMJs), the NMJ area, the neurite network, and the neurite network close to NMJs as measures of innervation of muscle fibers. A schematic of the experimental workflow is illustrated in FIG. 41A.

[0414] FIG. 41B illustrates the number of NMJs in the co-culture of spinal cord explant and myoblasts treated glutamate. Co-treatment with cutamesine and smilagenin increased the mean number of NMJs following glutamate treatment to a greater extent than cutamesine or treatment alone. FIG. 41C illustrates the mean area of NMJs in the co-culture treated with glutamate. Co-treatment with cutamesine and smilagenin increased the area of NMJs following glutamate treatment to a greater extent than cutamesine or smilagenin treatment alone. FIG. 4 ID illustrates a measurement of the neurite network in the co-culture treated with glutamate. Treatment with smilagenin, cutamesine, and smilagenin and cutamesine significantly increased the neurite network following glutamate injury. FIG. 41E illustrates ameasurement of the neurite network close to NMJs in the co-culture treated with glutamate. Co-treatment with cutamesine and smilagenin significantly increased the neurite network close to NMJs following treatment with glutamate.EXAMPLE 11: Beneficial Effect in Preclinical Models of the Combination ofCutamesine and Smilagenin

[0415] The multifactorial nature of the pathophysiology of ALS leads to the preference of pleiotropic and combination therapies. Combining compounds engaging multiple disease targets simultaneously provides synergistic effects.

[0416] To assess the therapeutic potential of the combination of smilagenin and cutamesine, the combination is tested in preclinical models of ALS, including primary rodent motor neuron cultures, rodent spinal explant and human myotube co-cultures, patient-derived IPSC- motor neurons, and the SOD1-G93A mouse.

[0417] Across all in vitro models tested, the combination of smilagenin and cutamesine protects against motor neuron loss, protects against TDP-43 pathology, and supports neurite length and outgrowth. In addition, SOD1-G93A mice treated with the combination of smilagenin and cutamesine have a slowed disease progression. A transcriptomic profde from the lumbar spinal cord of treated SOD1-G93A mice is made to show differentially expressed genes.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A combination comprising a therapeutically-effective amount of cutamesine or a pharmaceutically-acceptable salt thereof and a therapeutically-effective amount of smilagenin.

2. The combination of claim 1, wherein the combination is in a unit dosage form that contains the cutamesine or the pharmaceutically-acceptable salt thereof and the smilagenin.

3. The combination of claim 1, wherein the cutamesine is the pharmaceutically- acceptable salt, and the pharmaceutically-acceptable salt is cutamesine dihydrochloride.

4. The combination of claim 1, wherein the combination comprises a first unit dosage form and a second unit dosage form, wherein the first unit dosage form contains the cutamesine or the pharmaceutically-acceptable salt thereof and the second unit dosage form contains the smilagenin.

5. The combination of claim 1, wherein the combination comprises a first unit dosage form and a second unit dosage form, wherein the first unit dosage form contains the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt is cutamesine dihydrochloride and the second unit dosage form contains the smilagenin.

6. A pharmaceutical composition comprising: a) cutamesine or a pharmaceutically acceptable salt thereof, and b) a sapogenin.

7. The pharmaceutical composition of claim 6, wherein the sapogenin is smilagenin.

8. The pharmaceutical composition of claim 6, wherein the composition further comprises a pharmaceutically-acceptable excipient.

9. The pharmaceutical composition of claim 6, wherein the composition further comprises a glutamate salt.

10. The pharmaceutical composition of claim 6, wherein the cutamesine or the pharmaceutically acceptable salt thereof and the sapogenin are present in a mass ratio between 1:200 and 1:10 cutamesine: sapogenin.

11. The pharmaceutical composition of claim 10, wherein the cutamesine or the pharmaceutically acceptable salt thereof and the sapogenin are present in a mass ratio of about 3:180 cutamesine: sapogenin.

12. The pharmaceutical composition of claim 6, wherein cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in aconcentration of at least 1 pM.

13. The pharmaceutical composition of claim 6, wherein cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in a concentration of about 1 pM to about 300 nM.

14. The pharmaceutical composition of claim 6, wherein the sapogenin is present in the pharmaceutical composition in a concentration of at least 1 pM.

15. The pharmaceutical composition of claim 6, wherein the sapogenin is present in the pharmaceutical composition in a concentration of about 1 pM to about 300 nM.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is suitable for sublingual administration.

17. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically- acceptable salt of cutamesine is a dihydrochloride salt.

18. A pharmaceutical composition comprising cutamesine or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is a sublingual dosage form.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable excipient.

20. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises lactose.

21. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises mannitol starch.

22. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises sodium starch glycolate.

23. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition further comprises magnesium stearate.

24. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

25. The pharmaceutical composition of claim 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

26. The pharmaceutical composition of claim 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

27. The pharmaceutical composition of claim 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

28. The pharmaceutical composition of claim 18, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 3 mg.

29. The pharmaceutical composition of claim 18, further comprising a binder, a superdisintegrant, and a lubricant.

30. The pharmaceutical composition of claim 18, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

31. The pharmaceutical composition of claim 18, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in an amount of about 0.5 mg to about 10 mg.

32. The pharmaceutical composition of claim 18, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

33. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is a tablet.

34. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

35. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mm and a thickness from about 2.7 mm to about 2.9 mm.

36. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which the sublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is less than sixty seconds.

37. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which thesublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is less than forty seconds.

38. The pharmaceutical composition of claim 18, wherein the sublingual dosage form is solid, wherein if the sublingual dosage form is subjected to a shake test in which the sublingual dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the sublingual dosage form in the water, then a time needed for disintegration of the sublingual dosage form into fine particles is 33 ± 5 seconds.

39. A pharmaceutical composition comprising cutamesine or a pharmaceutically- acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the solid dosage form is subjected to a shake test in which the solid dosage form is placed in a glass beaker containing 2 mL of water and the beaker is manually shaken at 37 ± 0.5 °C to disintegrate the solid dosage form in the water, then a time needed for disintegration of the solid dosage form into fine particles is less than sixty seconds.

40. The pharmaceutical composition of claim 39, wherein the time needed for disintegration of the solid dosage form into fine particles is less than forty seconds.

41. The pharmaceutical composition of claim 39, wherein the time needed for disintegration of the solid dosage form into fine particles is 33 ± 5 seconds.

42. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable excipient.

43. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises mannitol starch.

44. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises lactose.

45. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises sodium starch glycolate.

46. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition further comprises magnesium stearate.

47. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

48. The pharmaceutical composition of claim 39, wherein the cutamesine or thepharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

49. The pharmaceutical composition of claim 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

50. The pharmaceutical composition of claim 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

51. The pharmaceutical composition of claim 39, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 3 mg.

52. The pharmaceutical composition of claim 39, further comprising a binder, a superdisintegrant, and a lubricant.

53. The pharmaceutical composition of claim 39, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

54. The pharmaceutical composition of claim 39, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in an amount of about 0.5 mg to about 10 mg.

55. The pharmaceutical composition of claim 39, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

56. The pharmaceutical composition of claim 39, wherein the sublingual dosage form is a tablet.

57. The pharmaceutical composition of claim 39, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

58. The pharmaceutical composition of claim 39, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mm and a thickness from about 2.7 mm to about 2.9 mm.

59. A pharmaceutical composition comprising cutamesine or a pharmaceutically- acceptable salt thereof, wherein the pharmaceutical composition is a solid dosage form, wherein if the solid dosage form is subjected to a dissolution study in which the solid dosageform is placed in a medium of 2 mL of distilled water at 25 °C in a 15 mL glass funnel for sixty seconds, at which point the medium is drawn by vacuum through a 0.45 pm membrane into a collection tube, after which the medium is analyzed by HPLC, then a percent dissolution of cutamesine or the pharmaceutically acceptable salt thereof of about 80% to about 95% is obtained.

60. The pharmaceutical composition of claim 59, wherein a percent dissolution of cutamesine or the pharmaceutically acceptable salt thereof of 92.3 ± 2.1% is obtained for a form containing 1 mg of cutamesine or the pharmaceutically acceptable salt thereof and 87.9 ± 5.7% for a form containing 3 mg of cutamesine or the pharmaceutically acceptable salt thereof.

61. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises a pharmaceutically-acceptable excipient.

62. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises lactose.

63. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises mannitol starch.

64. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises sodium starch glycolate.

65. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition further comprises magnesium stearate.

66. The pharmaceutical composition of claim 59, wherein the pharmaceutical composition comprises the pharmaceutically-acceptable salt of cutamesine, and the pharmaceutically-acceptable salt of cutamesine is a dihydrochloride salt.

67. The pharmaceutical composition of claim 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 10 mg.

68. The pharmaceutical composition of claim 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 0.5 mg to about 5 mg.

69. The pharmaceutical composition of claim 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in an amount of about 1 mg.

70. The pharmaceutical composition of claim 59, wherein the cutamesine or the pharmaceutically acceptable salt thereof is present in the pharmaceutical composition in anamount of about 3 mg.

71. The pharmaceutical composition of claim 59, further comprising a binder, a superdisintegrant, and a lubricant.

72. The pharmaceutical composition of claim 59, further comprising mannitol starch, sodium starch glycolate, and magnesium stearate.

73. The pharmaceutical composition of claim 59, further comprising: a) mannitol in an amount of about 100 mg to about 150 mg; b) sodium starch glycolate in an amount of about 5 mg to about 15 mg; and c) magnesium stearate in an amount of about 0.5 mg to about 10 mg.

74. The pharmaceutical composition of claim 59, further comprising: a) mannitol in an amount of about 135 mg; b) sodium starch glycolate in an amount of about 9 mg; and c) magnesium stearate in an amount of about 3 mg.

75. The pharmaceutical composition of claim 59, wherein the sublingual dosage form is a tablet.

76. The pharmaceutical composition of claim 59, wherein the sublingual dosage form is a tablet that has a diameter from about 7 mm to about 9 mm and a thickness from about 2.0 mm to about 4.0 mm.

77. The pharmaceutical composition of claim 59, wherein the sublingual dosage form is a tablet that has a diameter from about 8 mm to about 8.2 mm and a thickness from about 2.7 mm to about 2.9 mm.

78. A method for treating a condition, the method comprising sublingually administering to a subject in need thereof a therapeutically-effective amount of cutamesine or a pharmaceutically-acceptable salt thereof.

79. A method for treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of the therapeutic combination of any one of claims 1-77.

80. The method of any one of claims 78-79, wherein the condition is a neurodegenerative disease.

81. The method of any one of claims 78-80, wherein the condition is a central nervous system disease.

82. The method of any one of claims 78-80, wherein the condition is an aging condition.

83. The method of any one of claims 78-80, wherein the condition is amyotrophic lateral sclerosis (ALS).

84. The method of any one of claims 78-80, wherein the condition is dementia.

85. The method of any one of claims 78-80, wherein the condition is Alzheimer’sDisease.

86. The method of any one of claims 78-80, wherein the condition is Parkinson’s Disease.

87. The method of any one of claims 78-86, wherein the therapeutically-effective amount is about 0.5 mg to about 10 mg.

88. The method of any one of claims 78-86, wherein the therapeutically-effective amount is about 0.5 mg to about 5 mg.

89. The method of any one of claims 78-86, wherein the therapeutically-effective amount is about 1 mg.

90. The method of any one of claims 78-86, wherein the therapeutically-effective amount is about 3 mg.

91. The method of any one of claims 78-90, further comprising administering to the subject a therapeutically-effective amount of a sapogenin.

92. The method of claim 91, wherein the sapogenin is smilagenin.

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