CN117794531A

CN117794531A - Therapeutically effective oral administration of 2-arylbenzimidazoles

Info

Publication number: CN117794531A
Application number: CN202280053507.6A
Authority: CN
Inventors: J·O·汉内斯塔德; S·A·史密斯; S·K·卡科卡尔
Original assignee: Tranquist Therapy Inc
Current assignee: Tranquist Therapy Inc
Priority date: 2021-06-17
Filing date: 2022-06-17
Publication date: 2024-03-29

Abstract

Methods for reducing neuroinflammation and/or treating a neurodegenerative disease in a subject are provided. The method comprises orally administering to a subject suffering from a neuroinflammatory and/or neurodegenerative disease a compound comprising formula (I) (TQS-168) or a pharmaceutically acceptable thereofPharmaceutical compositions of the subject salts in amounts that provide defined plasma and brain exposure of TQS-168 and/or active metabolites after administration.

Description

Therapeutically effective oral administration of 2-arylbenzimidazoles

Cross Reference to Related Applications

The present application claims the benefit and priority of U.S. provisional application number 63/211,636 filed on month 17 of 2021 and U.S. provisional application number 63/300,551 filed on month 1 of 2022, each of which is incorporated by reference herein in its entirety for all purposes.

1. Background of the invention

The 2-arylbenzimidazole compound TQS-168-2- (4-tert-butylphenyl) -1H-benzimidazole, previously known as ZLN-005, is an activator of Ppargc1 alpha (PGC-1 alpha) gene expression. Zhang et al Diabetes 62:1297-1307 (2013). TQS-168 has been demonstrated to inhibit bone marrow-mediated inflammation and reduce disease severity in a mouse model of neurodegenerative diseases, including Parkinson's disease, alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS), when orally administered to mice at 25-50 mg/kg. See U.S. patent No. 10,272,070. TQS-168 has also been demonstrated to inhibit microglial metabolic dysfunction in aged mice, to inhibit inflammatory cytokine production in microglial cells in aged mice, to inhibit systemic inflammation in aged mice, and to alleviate behavioral dysfunction in aged mice when administered orally at 25mg/kg to mice. See U.S. patent No. 10,653,669.TQS-168 and structurally related 2-arylbenzimidazoles have also been shown to be effective in the treatment of systemic immune activation. See WO 2021/2626617.

TQS-168 is highly insoluble. In animal model experiments reported in U.S. Pat. Nos. 10,272,070 and 10,653,669, TQS-168 was prepared as an oral suspension and administered to experimental animals by oral gavage. The plasma and brain concentrations of the compounds after administration were not reported and no Pharmacokinetic (PK) information was provided.

To establish an effective oral dosing regimen suitable for a human patient, it is necessary to define the plasma and brain concentrations and total exposure of TQS-168 that provide pharmacodynamic benefits.

2. Summary of the invention

We have now demonstrated that TQS-168 induces PGC-1α protein expression in murine bone marrow cell line BV2 in vitro at a concentration ranging from 0.7 μM (175.21 ng/mL) to 20 μM (5006 ng/mL), and that TQS-168 inhibits LPS-induced secretion of pro-inflammatory cytokines by BV2 cells and human primary bone marrow cells in vitro at a concentration ranging from 0.3 μM (75.09 ng/mL) to 20 μM (5006 ng/mL).

These in vitro experiments predicted that plasma free drug and brain concentrations of TQS-168 in the range of 0.3-20. Mu.M (75.09-5006 ng/mL) inhibited bone marrow-mediated neuroinflammation.

When administered orally at 25-50mg/kg, TQS-168 was previously demonstrated to inhibit bone marrow-mediated inflammation and reduce disease severity in murine models of neurodegenerative diseases, including Parkinson's disease, alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS). U.S. patent No. 10,272,070. We have now measured the plasma, liver and brain concentrations of TQS-168 after a single oral dose of 25mg/kg administered to mice, which was previously found to provide therapeutic effects. At this previously determined effective dose, TQS-168's mean plasma C _max 93.4ng/ml or 0.37. Mu.M, and mean brain C _max Higher, 542.0ng/ml or 2.16. Mu.M. These concentrations are in a range of concentrations that have been demonstrated to induce PGC-1 alpha protein expression and reduce LPS-mediated inflammatory cytokine release in vitro. These in vivo data demonstrate that plasma concentrations of TQS-168 in the range of 0.3. Mu.M-20. Mu.M inhibit bone marrow-mediated neuroinflammation. Evidence of accumulation in the brain suggests that plasma concentrations of TQS-168 below 0.37. Mu.M may also be effective in treating neuroinflammation.

We have also demonstrated that TQS-621, the major phase 1 metabolite of TQS-168, effectively inhibits LPS-induced IL-6 and TNFα secretion by primary human PBMC. These data demonstrate that at least some of the therapeutic effects observed after oral administration of TQS-168 may be attributable to the activity of metabolite TQS-621.

We have also performed phase 1 human clinical trials and measured plasma concentrations of TQS-168 and the active metabolite TQS-621 using three different formulations and demonstrated that pharmacodynamically relevant plasma concentrations could be achieved with oral suspensions of several solid formulations of API.

Thus, in a first aspect, there is provided a method for reducing neuroinflammation and/or treating a neurodegenerative disease in a subject. The method comprises the following steps:

Orally administering to a subject suffering from a neuroinflammatory and/or neurodegenerative disease at least one dose of a pharmaceutical composition comprising a compound of formula (I) (TQS-168) or a pharmaceutically acceptable salt thereof,

in an amount that provides an average peak concentration of TQS-168 in plasma of at least 750ng/mL after administration (C _max )。

In various embodiments, TQS-168 or a salt thereof is administered in an amount that provides a mean plasma C of TQS-168 of at least 1000ng/mL, at least 1250ng/mL, at least 1500ng/mL, or at least 1750ng/mL after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of at least 3000 ng-hr/ml, at least 4000 ng-hr/ml, at least 5000 ng-hr/ml, at least 5500 ng-hr/ml, at least 6000 ng-hr/ml, or at least 7,000 ng-hr/ml after administration _0-t . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of about 6000 ng-hr/ml after administration _0-t 。

In various embodiments, plasma C to TQS-168 _max Time (T) _max ) No more than 2 hours, no more than 90 minutes, or no more than 75 minutes. In a specific embodiment, the TQS-168 plasma T _max Is about 60 minutes.

In a second aspect, methods for reducing neuroinflammation and/or treating a neurodegenerative disease in a human subject are provided. The method comprises the following steps:

Orally administering a pharmaceutical composition comprising a compound of formula (I) (TQS-168) or a pharmaceutically acceptable salt thereof to a subject suffering from neuroinflammatory and/or neurodegenerative disorders,

in an amount which provides a mean peak plasma concentration (C) of the compound of formula (II) (TQS-621) of at least 1000ng/mL after administration _max )

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides plasma C of TQS-621 of 200-2750ng/mL, 300-2200ng/mL, or 400-1800ng/mL after administration _max 。

In another aspect, methods for reducing neuroinflammation and/or treating a neurodegenerative disease in a subject are provided. The method comprises the following steps:

in an amount that provides after administration

(a) An average peak concentration of TQS-168 in plasma of at least 750ng/mL (C _max ) A kind of electronic device

(b) No more than 75 minutes of C in plasma to TQS-168 _max Average time (T) _max ) The method comprises the steps of carrying out a first treatment on the surface of the And

(c) Average peak concentration (C) of at least 1000ng/mL of compound of formula (II) (TQS-621) in plasma _max )

And

(d) No more than 4 hours of C in plasma to TQS-621 _max Average time (T) _max )。

In some embodiments of the methods herein, TQS-168 or a salt thereof is administered at a daily oral dosage of 200-800mg, 300-700mg, 400-600mg, or 400-500 mg. In particular embodiments, TQS-168 or a salt thereof is administered at a daily oral dosage of 400mg or 450 mg.

In various embodiments of the methods described herein, TQS-168 or a salt thereof is administered in a liquid suspension. In certain embodiments, TQS-168 or a salt thereof is administered in a liquid solution.

In certain embodiments, TQS-168 or a salt thereof is administered in a solid dosage form. In a specific solid form embodiment, TQS-168 or a salt thereof is crystalline. In particular solid form embodiments, TQS-168 or a salt thereof is amorphous, and in particular amorphous embodiments, is a spray dried dispersion or hot melt extrudate. In certain embodiments, the solid dosage form is a pouch, capsule or tablet.

In various embodiments, the subject has a neurodegenerative disease selected from the group consisting of motor neuron disease, amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, vascular dementia, frontotemporal lobar degeneration (frontotemporal dementia), dementia with lewy bodies, parkinson's disease, huntington's disease, demyelinating disease, and Multiple Sclerosis (MS). In specific embodiments, the subject has a motor neuron disease. In particular embodiments, the subject has ALS. In specific embodiments, the subject has alzheimer's disease.

In some embodiments, the subject is at least 40 years old and has no previously diagnosed neurodegenerative disease. In specific embodiments, the subject is at least 60 years old or at least 65 years old.

3. Brief description of the drawings

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings where:

FIG. 1 is a Western blot showing induction of PGC-1 alpha protein expression in BV2 murine microglial cell line after in vitro incubation with TQS-168 at 20. Mu.M.

FIG. 2 is a Western blot showing increased PGC-1α protein expression dose response in BV2 cells contacted with TQS-168 in vitro.

FIG. 3 is a bar graph quantifying protein expression levels measured from a scan of Western blots shown in FIG. 2. "PD" indicates PD169316, a p38 MAPK inhibitor. "Rosi" refers to rosiglitazone (AVANDIA), a PPARgamma agonist.

FIGS. 4a-4f show in vitro secretion of cytokines by BV2 cells after LPS activation in the presence of two positive controls, one negative control and TQS-168 at 4 different concentrations. These figures show dose-responsive inhibition of LPS-stimulated TNF alpha and IL-6 cytokine release at various concentrations of TQS-168. Cytokine secretion was measured using a Cytokine Bead Array (CBA) Fluorescence Activated Cell Sorting (FACS) assay.

FIGS. 5a-5b show inhibition of TNFα production by LPS-stimulated in vitro BV2 bone marrow cells by treatment with 5 μM (FIG. 5 a) and 20 μM (FIG. 5 b) TQS-168, respectively.

FIGS. 6a-6d show the dose response of TQS-168 mediated inhibition of the release of the pro-inflammatory cytokine TNFα from LPS stimulated microglial BV2 cells at 24 hours as measured using ELISA. FIGS. 6a and 6c show absolute (FIG. 6 a) and relative (FIG. 6 c) inhibition of TNFα secretion in BV2 cells stimulated with 0.3ng/mL LPS. FIGS. 6b and 6d show absolute (FIG. 6 b) and relative (FIG. 6 d) inhibition of TNFα secretion in BV2 cells stimulated with 1ng/mL LPS.

FIG. 7 shows inhibition of LPS-induced TNF alpha secretion in human PBMC cells treated with various concentrations of TQS-168 in vitro.

FIGS. 8a-8c show the mean plasma (FIG. 8 a), liver (FIG. 8 b) and brain (FIG. 8 c) concentrations of TQS-168 at various time points following a single oral dose of 25mg/kg TQS-168 in mice.

FIGS. 9a-9c show the plasma concentrations of TQS-168 at various time points following a single oral dose of 50mg/kg in wild type mice.

FIG. 10 shows the plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in three individual mice.

FIG. 11 shows the mean plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in three mice.

FIG. 12a shows the plasma concentrations of TQS-168 at various time points following a single oral dose of 50, 150 or 500mg/kg in rats. FIG. 12b shows the average brain concentration of TQS-168 at various time points following a single oral dose of 500mg/kg in rats.

FIG. 13 shows the dose-dependent C of TQS-168 after oral dosing of 50mg/kg, 150mg/kg and 500mg/kg in rats _max (ng/mL) value.

FIG. 14 shows the dose-dependent AUC (ng. Times. Min/mL) of TQS-168 following oral dosing of 50mg/kg, 150mg/kg and 500mg/kg in rats.

FIG. 15 shows the plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in three rats.

FIG. 16 shows the mean plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in rats.

FIG. 17 shows the plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in three dogs.

FIG. 18 shows the mean plasma concentrations of TQS-168 at various time points following a single Intravenous (IV) dose of 0.5mg/kg in dogs.

FIG. 19A shows the mean plasma concentration of TQS-168 following an oral dose of TQS-168 of 45mg/kg in mice. FIG. 19B shows the average brain concentration of TQS-168 following an oral dose of TQS-168 of 45mg/kg in mice.

FIG. 20 shows phase 1 metabolites from liver metabolism of TQS-168 following oral administration.

FIG. 21A shows the absolute inhibition of LPS-stimulated IL-6 secretion by the TQS-168 metabolite TQS-621 from previously frozen PBMC obtained from the first healthy human volunteer donor. FIG. 21B shows the relative inhibition of IL-6 inhibition expressed as a percent activity.

FIG. 22A shows the absolute inhibition of LPS-stimulated IL-6 secretion by TQS-168 metabolite TQS-621 from previously frozen PBMC obtained from a second healthy human donor. FIG. 22B shows the relative inhibition of IL-6 inhibition expressed as a percent activity.

FIG. 23A shows the absolute inhibition of LPS-stimulated TNFα secretion by previously frozen PBMC of the first donor by TQS-168 and metabolite TQS-621. FIG. 23B shows the relative inhibition of IL-6 inhibition expressed as a percent activity.

FIG. 24A shows the absolute inhibition of LPS-stimulated TNF alpha secretion by TQS-168 and metabolite TQS-621 from previously frozen PBMC obtained from the second donor. Fig. 24B shows the relative inhibition of tnfα inhibition expressed as a percent activity.

FIGS. 25A-25C graphically depict the change in plasma concentration of TQS-168 over time following a single oral dose of TQS-168 in mice given 50mg/mL TQS-168 in different formulations.

FIGS. 26A-26C graphically depict the change in plasma concentration of TQS-621 over time after a single oral dose in mice given 50mg/mL TQS-168 in varying amounts.

FIGS. 27A-B graphically depict the change in plasma concentration of TQS-168 over time in humans after a single oral dose of 60mg, 180mg or 540mg of a TQS-168 methylcellulose powder suspension formulation. Fig. 27A is a linear diagram. Fig. 27B is a logarithmic graph.

FIGS. 28A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time in humans following a single oral dose of 60mg, 180mg or 540mg of a TQS-168 methylcellulose powder suspension formulation. Fig. 28A is a linear diagram. Fig. 28B is a logarithmic graph.

FIGS. 29A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time in humans following a single oral dose of 60mg of an TQS-168 methylcellulose powder suspension oral formulation. Fig. 29A is a linear diagram. Fig. 29B is a logarithmic graph.

FIGS. 30A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time in humans following a single oral dose of 180mg of TQS-168 methylcellulose powder suspension oral formulation. Fig. 30A is a linear diagram. Fig. 30B is a logarithmic graph.

FIGS. 31A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time in humans following a single oral dose of 540mg of an TQS-168 methylcellulose powder suspension oral formulation. Fig. 31A is a linear diagram. Fig. 31B is a logarithmic graph.

FIGS. 32A-B graphically depict the change in plasma concentration of TQS-168 over time after a single dose of: for oral suspensions in the fasted state, 60mg, 180mg or 540mg of TQS-168 Methylcellulose (MC) powder; for oral suspensions in the fed state, 90mg of Spray Dried Dispersion (SDD) powder; 180mg of SDD powder for oral suspension in the fasted state; or 180mg Hot Melt Extrudate (HME) powder in a fasted state. Fig. 32A is a linear diagram. Fig. 32B is a logarithmic graph.

FIGS. 33A-B graphically depict the plasma concentration of metabolite TQS-621 as a function of time after a single dose of: for oral suspensions in the fasted state, 60mg, 180mg or 540mg of TQS-168 Methylcellulose (MC) powder; for oral suspensions in the fed state, 90mg of Spray Dried Dispersion (SDD) powder; 180mg of SDD powder for oral suspension in the fasted state; or 180mg Hot Melt Extrudate (HME) powder in a fasted state. Fig. 33A is a linear diagram. Fig. 33B is a logarithmic graph.

FIGS. 34A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time after a single dose of 90mg of a TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation) in a human in the fed state. Fig. 34A is a linear diagram. Fig. 34B is a logarithmic graph.

Fig. 35A-B graphically depicts the change in plasma concentration of TQS-168 over time after a single dose of 90mg Spray Dried Dispersion (SDD) powder suspension (oral formulation) in humans in fed and fasted states. Fig. 35A is a linear diagram. Fig. 35B is a logarithmic graph.

FIGS. 36A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time after a single dose of 90mg Spray Dried Dispersion (SDD) powder suspension (oral formulation) in humans in fed and fasted states. Fig. 35A is a linear diagram. Fig. 35B is a logarithmic graph.

FIGS. 37A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time following a single oral dose of 90mg of a TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation) in a person in a fasted state. Fig. 37A is a linear diagram. Fig. 37B is a logarithmic graph.

FIGS. 38A-B graphically depict the change in plasma concentration of TQS-168 over time after a single dose of 90mg, 180mg or 270mg of a TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation) in a person in a fasted state. Fig. 38A is a linear diagram. Fig. 38B is a logarithmic graph.

FIGS. 39A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time after a single dose of 90mg, 180mg or 270mg of a TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation) in a person in a fasted state. Fig. 39A shows a linear diagram. Fig. 39B shows a logarithmic graph.

Figures 40A-B plot the plasma concentration of TQS-168 over time (day 1) after a single dose of 90mg or 120mg Spray Dried Dispersion (SDD) powder suspension (oral formulation) in humans in fed and fasted states, respectively. Fig. 40A is a linear diagram. Fig. 40B is a logarithmic graph.

Figures 41A-B plot the change in plasma concentration of TQS-168 over time after a single daily dose of 90mg or 120mg spray-dried dispersion (SDD) powder suspension (oral formulation) in humans in fed and fasted states, respectively, for 7 consecutive days. Fig. 41A is a linear diagram. Fig. 41B is a logarithmic graph.

Fig. 42A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time after a single dose of 90mg or 120mg of TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation), respectively, in humans in fed and fasted states. Fig. 42A is a linear diagram. Fig. 42B is a logarithmic graph.

FIGS. 43A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time after a single dose of 90mg or 120mg of a TQS-621 spray-dried dispersion powder suspension (oral formulation), respectively, in fed and fasted humans, for 7 consecutive days. Fig. 43A is a linear diagram. Fig. 43B is a logarithmic graph.

Fig. 44A-B graphically depicts the change in plasma concentration of TQS-168 over time after a single dose of TQS-168 Methylcellulose (MC) powder suspension formulation, spray-dried dispersion (SDD) powder suspension formulation, or Hot Melt Extrudate (HME) powder suspension formulation in humans at different doses. Fig. 44A is a linear diagram. Fig. 44B is a logarithmic graph.

FIGS. 45A-B graphically depict the change in plasma concentration of TQS-621 over time after a single dose of a different dose of a TQS-168 Methylcellulose (MC) powder suspension formulation, a Spray Dried Dispersion (SDD) powder suspension formulation, or a Hot Melt Extrudate (HME) powder suspension formulation. Fig. 45A is a linear diagram. Fig. 45B is a logarithmic graph.

FIGS. 46A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time after a single dose of 180mg of a TQS-168 spray-dried dispersion (SDD) powder suspension (oral formulation) in a person in a fasted state. Fig. 46A is a linear diagram. Fig. 46B is a logarithmic graph.

FIGS. 47A-B graphically depict the change in plasma concentrations of TQS-168 and metabolite TQS-621 over time after a single dose of 180mg of a TQS-168 Hot Melt Extrudate (HME) powder suspension (oral formulation) in a person in a fasted state. Fig. 34A is a linear diagram. Fig. 34B is a logarithmic graph.

FIGS. 48A-B graphically depict the change in plasma concentration of TQS-621 over time after a single dose of a TQS-168 Methylcellulose (MC) powder suspension formulation, a Spray Dried Dispersion (SDD) powder suspension formulation, or a Hot Melt Extrudate (HME) powder suspension formulation in humans in a fasted state at different doses. Fig. 48A is a linear diagram. Fig. 48B is a logarithmic graph.

Fig. 49A-B graphically depicts the change in plasma concentration of TQS-168 over time following a single continuous daily dose of 120mg Spray Dried Dispersion (SDD) powder suspension (oral formulation) in a person in a fasted state. Fig. 49A is a linear diagram. Fig. 49B is a logarithmic graph.

FIGS. 50A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time following successive single daily doses of a spray-dried dispersion (SDD) powder suspension (oral formulation) of TQS-168 in humans in a fasted state. Fig. 50A is a linear diagram. Fig. 50B is a logarithmic graph.

FIGS. 51A-B graphically depict the change in plasma concentration of TQS-168 over time (day 1) after a single dose of 90mg, 120mg or 300mg of spray-dried dispersion (SDD) powder suspension (oral formulation) in a person in a fed/fasted state. Fig. 51A is a linear diagram. Fig. 51B is a logarithmic graph.

Fig. 52A-B graphically depict the change in plasma concentration of TQS-168 over time after a single daily dose of 90mg, 120mg, or 300mg spray-dried dispersion (SDD) powder suspension (oral formulation), respectively, in a person in a fed/fasted state for 7 days. Fig. 52A is a linear diagram. Fig. 52B is a logarithmic graph.

Figures 53A-B plot the change in plasma concentration of metabolite TQS-621 over time (day 1) after a single dose of 90mg, 120mg or 300mg of spray-dried dispersion (SDD) powder suspension (oral formulation) in humans in the fed/fasted state. Fig. 53A is a linear diagram. Fig. 53B is a logarithmic graph.

FIGS. 54A-B graphically depict the change in plasma concentration of metabolite TQS-621 over time (day 1) after a single dose of a spray-dried dispersion (SDD) powder suspension (oral formulation) of TQS-168 90mg, 120mg or 300mg for 7 consecutive days in a person in a fed/fasted state. Fig. 54A is a linear diagram. Fig. 54B is a logarithmic graph.

Fig. 55A-B graphically depicts the change in plasma concentration of TQS-168 over time after a single dose of TQS-168 Methylcellulose (MC) powder suspension formulation, spray-dried dispersion (SDD) powder suspension formulation, or Hot Melt Extrudate (HME) powder suspension formulation at different doses. Fig. 55A is a linear diagram. Fig. 55B is a logarithmic graph.

4. Detailed description of the invention

4.1. Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms "individual," "host," and "subject" are used interchangeably and refer to an animal to be treated, including, but not limited to, humans and non-human primates; rodents, including rats and mice; a bovine animal; equine animals; sheep; a feline; and canine animals. "mammal" means one or more members of any mammalian species. Non-human animal models, i.e., mammals, non-human primates, mice, rabbits, etc., can be used for experimental research.

"patient" refers to a human subject, including healthy human donors.

The terms "treatment", "treatment" and grammatical variants thereof are used in the broadest sense as understood in the clinical arts. Thus, these terms do not require cure or complete remission of the disease and encompass obtaining any clinically desirable pharmacological and/or physiological effect. Unless otherwise indicated, "treatment" and "treatment" do not encompass prophylaxis.

The phrase "therapeutically effective amount" refers to an amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect treatment of the disease, condition, or disorder. The "therapeutically effective amount" may vary depending on the compound, the disease and its severity, the age, weight, etc., of the subject to be treated.

The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a subject. Examples of pharmaceutically acceptable salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, fluoroheptanoate (fluhydrocaptanoate), glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate (palmoate), pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluene sulfonate, and undecanoate.

Other examples of pharmaceutically acceptable salts include salts with suitable cations such as N ⁺ 、NH ₄ ⁺ And NW ₄ ⁺ (wherein W may be C ₁ -C ₈ Alkyl group), and the like. For therapeutic use, salts of the compounds of the present disclosure may be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also be used, for example, to prepare or purify pharmaceutically acceptable compounds.

The compounds that are basic in nature, which are included in the compositions and methods of the present invention, are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, sucrate, formate, benzoate, glutamate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate)) salts.

Compounds that are acidic in nature, comprised in the compositions and methods of the present invention, are capable of forming base salts with a variety of pharmacologically acceptable cations. Examples of such salts include alkali or alkaline earth metal salts, and in particular calcium, magnesium, sodium, lithium, zinc, potassium and iron salts.

Compounds comprising basic or acidic moieties included in the compositions and methods of the invention may also form pharmaceutically acceptable salts with various amino acids. The compounds of the present disclosure may contain both acidic and basic groups; for example 1 amino group and 1 carboxylic acid group. In such cases, the compound may be present as an acid addition salt, a zwitterionic or a basic salt.

The range is as follows: throughout this disclosure, various aspects of the invention are presented in a range format. Ranges include the recited endpoints. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual values within the range. For example, descriptions of ranges such as 1 to 6 should be considered to have specifically disclosed sub-ranges within the range such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers, e.g., 1, 2, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

In this disclosure, "comprises/comprising," "contains," "has," "includes" and language variants thereof have the meanings assigned to them in the united states patent laws, allowing additional components beyond those explicitly recited.

The term "or" as used herein is to be understood as inclusive unless specifically stated or apparent from the context.

The terms "a," "an," and "the" as used herein are to be construed as singular or plural unless specifically stated or apparent from the context. That is, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As an example, "an element" means one element or more than one element.

Unless specifically stated or otherwise apparent from the context, the term "about" as used herein is to be understood to be within normal tolerances in the art, e.g., within 2 standard deviations of the mean, and is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% and still more preferably ±0.1% from the value. Where percentages are provided with respect to the amount of components or materials in the composition, the percentages are to be understood as percentages by weight unless otherwise indicated or understood from the context.

It should be understood that the order of steps or order of performing certain actions is not important so long as the present disclosure remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

The terms "pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" and "pharmaceutically acceptable adjuvant" are used interchangeably and refer to an excipient, diluent, carrier or adjuvant that is useful in the preparation of pharmaceutical compositions that are generally safe, non-toxic and biologically or otherwise undesirable, and include excipients, diluents, carriers and adjuvants that are acceptable for veterinary use as well as for human pharmaceutical use. The phrase "pharmaceutically acceptable excipient" includes one or more than one such excipient, diluent, carrier and/or adjuvant.

The terms "sustained release", "delayed release" and "controlled release" as used herein refer to the delayed or prolonged release of a therapeutic agent or API of a pharmaceutical formulation. These terms may further refer to compositions that provide a delayed or prolonged duration of action, such as Pharmacokinetic (PK) parameters of pharmaceutical compositions comprising a therapeutically effective amount of an active pharmaceutical ingredient as described herein.

In general, reference to or description of an element (such as hydrogen or H) is intended to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Thus, contains a radioisotope such as tritium, ¹⁴ C、 ³² P and ³⁵ the compounds of S are within the scope of the present technology. Procedures for inserting such markers into compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

Unless a specific stereochemistry is specifically indicated, all chiral, diastereomeric, and racemic forms of a compound are intended. Thus, the compounds described herein include optical isomers that are enriched or resolved at any or all asymmetric atoms, as will be apparent from the description. Racemic mixtures of the R-and S-enantiomers, as well as enantiomerically enriched stereoisomers comprising the R-and S-enantiomers, as well as individual optical isomers, may be separated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are within the scope of the present technology.

The compounds described herein may exist as solvates, particularly hydrates, and unless otherwise indicated, means all such solvates and hydrates. Hydrates may form during the manufacture of a compound or a composition comprising a compound, or hydrates may form over time due to the hygroscopic nature of a compound. The compounds of the present technology may also exist as organic solvates, including DMF, ether, alcohol solvates, and the like. The identification and preparation of any particular solvate is within the skill of one of ordinary skill in synthetic organic or pharmaceutical chemistry.

As described herein, the present disclosure is directed to various embodiments of the compounds, compositions, and methods of the present disclosure. The various embodiments described are intended to provide a variety of illustrative examples and should not be construed as alternative types of description. Rather, it should be noted that descriptions of the various embodiments provided herein may have overlapping ranges. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present technology.

4.2. Summary of experimental observations

We have demonstrated that TQS-168 induces PGC-1 alpha gene and protein expression in murine bone marrow cell line BV2 in vitro at concentrations ranging from 0.7. Mu.M to 20. Mu.M, and that TQS-168 inhibits LPS-induced secretion of pro-inflammatory cytokines by BV2 cells and human primary bone marrow cells in vitro at concentrations ranging from 0.3. Mu.M to 20. Mu.M.

These in vitro experiments predicted that plasma and brain concentrations of TQS-168 in the range of 0.3-20. Mu.M inhibited bone marrow-mediated neuroinflammation.

When administered orally at 25-50mg/kg, TQS-168 was previously demonstrated to inhibit bone marrow-mediated inflammation and reduce disease severity in murine models of neurodegenerative diseases, including Parkinson's disease, alzheimer's disease, and Amyotrophic Lateral Sclerosis (ALS). U.S. patent No. 10,272,070. We have measured the plasma, liver and brain concentrations of TQS-168 after a single oral dose of 25mg/kg was administered to mice, which was previously found to provide therapeutic effects. At this previously determined effective dose, TQS-168's mean plasma C _max 93.4ng/ml or 0.37. Mu.M, and brain C _max Higher, 542.0ng/ml or 2.16. Mu.M, in a concentration range that was demonstrated to induce PGC-1 alpha protein expression and reduce LPS-mediated inflammatory cytokine release in vitro. These in vivo data demonstrate that plasma concentrations of TQS-168 in the range of 0.3. Mu.M-20. Mu.M inhibit bone marrow-mediated neuroinflammation. Evidence of preferential accumulation in the brain suggests that plasma concentrations of TQS-168 below 0.37. Mu.M may also be effective in treating neuroinflammation.

We have also demonstrated that TQS-621, the major phase 1 metabolite of TQS-168, effectively inhibits LPS-induced IL-6 and TNFα secretion by primary human PBMC. These data demonstrate that at least some of the therapeutic effects observed after oral administration of TQS-168 can be attributed to the activity of metabolite TQS-621.

The data demonstrate that oral administration of TQS-168 in solution provides a higher TQS-168 and TQS-621C than seen with two different suspension formulations _max And total drug exposure.

4.3. Methods of reducing neuroinflammation and/or treating neurodegenerative disorders

4.3.1. Pharmacokinetic dosage regimen

Thus, in a first aspect, there is provided a method for reducing neuroinflammation and/or treating a neurodegenerative disease in a subject. The method comprising orally administering to a subject suffering from a neuroinflammatory and/or neurodegenerative disease a pharmaceutical composition comprising a compound of formula (I) (TQS-168) (MW 250.3) or a pharmaceutically acceptable salt thereof,

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in an amount that provides (a) an average peak blood or plasma TQS-168 concentration (C) of at least 50nM (12.515 ng/mL) after administration _max ). In certain embodiments, the amount provides (a) an average peak blood or plasma TQS-168 concentration (C) of at least 50nM (12.515 ng/mL) _max ) And (b) no more than 360 minutes of TQS-168 in blood or plasma to C _max Average time (T) _max ). In some embodiments, C is measured in plasma _max And T _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides at least 100nM (25.03 ng/mL), 150nM (37.545 ng/mL), 200nM (50.06 ng/mL), 250nM (62.575 ng/mL), 300nM (75.09 ng/mL), 350nM (87.605 ng/mL), 400nM (100.12 ng/mL), 450nM (112.635 ng/mL), 500nM (125.15 ng/mL), 550nM (137.665 ng/mL), 600nM (150.18 ng/mL), 650nM (162.695 ng/mL), 700nM (175.21 ng/mL), 750nM (187.725 nM), 800nM (200.24 ng/mL), 850nM (212.755 ng/mL), 900nM (225.27 ng/mL), 950nM (237.785 nM), 1 μM (250.3 ng/mL), 2 μM (500.6 ng/mL), 2.5 μM (625.75 mL), 3 μM (125.9 ng/mL), 3 μM (37 μM), 5 μM (37 μM), 37 μM (5 μM (37.5 μM), 37 μM (37 μM), 5 μM (37 μM (37.5 μM), 37 μM (37.5 μM) 10. Mu.M (2503 ng/mL), 10.5. Mu.M (2628.15 ng/mL), 11. Mu.M (2753.3 ng/mL), 11.5. Mu.M (2878.45 ng/mL), 12. Mu.M (3003.6 ng/mL), 12.5. Mu.M (3128.75 ng/mL), 13. Mu.M (3253.9 ng/mL), 13.5. Mu.M (3379.05 ng/mL), 14. Mu.M (3504.2 ng/mL), 14.5. Mu.M (3629.35 ng/mL), 15. Mu.M (3754.5 ng/mL), 15.5. Mu.M (3879.65 ng/mL), 16. Mu.M (4004.8 ng/mL), 16.5. Mu.M (4129.95 ng/mL), 1 7. Mu.M (4255.1 ng/mL), 17.5. Mu.M (4380.25 ng/mL), 18. Mu.M (4505.4 ng/mL), 18.5. Mu.M (4630.55 ng/mL), 19. Mu.M (4755.7 ng/mL), 19.5. Mu.M (4880.85 ng/mL), 20. Mu.M (5006 ng/mL), 20.5. Mu.M (5131.15 ng/mL), 21. Mu.M (5256.3 ng/mL), 21.5. Mu.M (5381.45 ng/mL), 22. Mu.M (5506.6 ng/mL), 22.5. Mu.M (5631.75 ng/mL), 23. Mu.M (5756.9 ng/mL), 23.5. Mu.M (5882.05 ng/mL), 24. Mu.M (6007.2 ng/mL), 24.5. Mu.M (6132.35 ng/mL), or 25. Mu.M (6275.5 ng/mL) TQS-168 mean blood or plasma C _max 。

In certain embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of at least 3.5. Mu.M (876.05 ng/mL), 4. Mu.M (1001.2 ng/mL), 4.5. Mu.M (1126.35 ng/mL), 5. Mu.M (1151.5 ng/mL), 5.5. Mu.M (1376.65 ng/mL), 6. Mu.M (1501.8 ng/mL), 6.5. Mu.M (1626.95 ng/mL), 7. Mu.M (1752.1 ng/mL), 7.5. Mu.M (18778.25 ng/mL), or 8. Mu.M (2002.4 ng/mL) after administration _max 。

In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of at least 4 μM (1001.2 ng/mL), 4.5 μM (1126.35 ng/mL), 5 μM (1151.5 ng/mL), or 5.5 μM (1376.65 ng/mL) after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of 2 μM (500.6 ng/mL) to 8 μM (2002.4 ng/mL), 2.5 μM (625.75 ng/mL) to 7.5 μM (1877.25 ng/mL), 3 μM (750.9 ng/mL) to 7 μM (1752.1 ng/mL), 3.5 μM (876.05 ng/mL) to 6.5 μM (1626.95 ng/mL), or 4 μM (1001.2 ng/mL) to 6 μM (1501.8 ng/mL) after administration _max . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of 4 μM (1001.2 ng/mL) to 5 μM (1151.5 ng/mL) after administration _max 。

In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of about 4.5 μM (1126.35 ng/mL) after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or of TQS-168 of at least 700ng/mL, 750ng/mL, 800ng/mL, 850ng/mL, 900ng/mL, 950ng/mL, 1000ng/mL, 1500ng/mL, or 2000ng/mL after administrationPlasma C _max . In certain embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of at least 900ng/mL, 950ng/mL, 1000ng/mL, 1500ng/mL, or 2000ng/mL of TQS-168 after administration _max . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of at least 1000ng/mL, 1100ng/mL, 1200ng/mL, 1300ng/mL, 1400ng/mL, 1500ng/mL, 1600ng/mL, 1700ng/mL, 1800ng/mL, 1900ng/mL, or 2000ng/mL after administration _max . In certain embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of 900ng/mL to 1300ng/mL, or 1000ng/mL to 1200ng/mL, after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-168 of about 1100ng/mL after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered, the amount providing at least 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M, 5.5. Mu.M, 6. Mu.M, 6.5. Mu.M, 7. Mu.M, 7.5. Mu.M, 8. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M, 3.5. Mu.M, 2.M average brain C of TQS-168 of 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM, 17 μM, 17.5 μM, 18 μM, 18.5 μM, 19 μM, 19.5 μM, 20 μM, 20.5 μM, 21 μM, 21.5 μM, 22 μM, 22.5 μM, 23 μM, 23.5 μM, 24 μM, 24.5 μM, or 25 μM _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides a brain/plasma ratio of TQS-168 of 0.5-10 after administration. In certain embodiments, TQS-168 is administered in an amount that provides a brain/plasma ratio of TQS-168 of 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5 after administration. In certain embodiments, the TQS-168 is administered in an amount that provides a brain/plasma ratio of TQS-168 of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0 after administration.

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of TQS-168 measured in plasma after administration of at least 2000 ng.hr/ml, 2500 ng.hr/ml, 3000 ng.hr/ml, 3500 ng.hr/ml, 4000 ng.hr/ml, 4500 ng.hr/ml, 5000 ng.hr/ml, 5500 ng.hr/ml, 6000 ng.hr/ml, 6500 ng.hr/ml, 7000 ng.hr/ml, 7500 ng.hr/ml, 8000 ng.hr/ml, 8500 ng.hr/ml, or 9000 ng.hr/ml _0-t . In certain embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of TQS-168 in plasma of at least 4000 ng.hr/ml, 4500 ng.hr/ml, 5000 ng.hr/ml, 5500 ng.hr/ml, 6000 ng.hr/ml, 6500 ng.hr/ml, or 7000 ng.hr/ml after administration _0-t 。

In certain embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of TQS-168 in plasma of 4000 ng-hr/ml to 8000 ng-hr/ml after administration _0-t . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of TQS-168 in plasma of 5000 ng-hr/ml to 7000 ng-hr/ml after administration _0-t . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an AUC of TQS-168 in plasma of about 6000 ng-hr/ml after administration _0-t 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average T of TQS-168 in blood or plasma of no more than 360 minutes after administration _max . In certain embodiments, TQS-168 or a salt thereof is administered in a formulation that provides an average T in blood or plasma of no more than 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 225, 200, or 180 minutes _max . In certain embodiments, TQS-168 or a salt thereof is administered in a formulation that provides an average T in blood or plasma of no more than 90 minutes, 60 minutes, or 45 minutes _max 。

In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides no more than 12 after administrationAverage T of TQS-168 in blood or plasma for 0 min, 90 min or 60 min _max . In particular embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average T of about 60 minutes after administration _max 。

In another aspect, methods for treating neuroinflammation and/or treating a neurodegenerative disease in a subject are provided. The method comprises orally administering to a subject suffering from a neuroinflammatory and/or neurodegenerative disease a pharmaceutical composition comprising TQS-168 or a pharmaceutically acceptable salt thereof in an amount which, after administration, provides (a) a mean peak plasma concentration (C) of the compound of formula (II) (TQS-621) (MW 266.3) of at least 50nM _max )

And (b) no more than 360 minutes of C in plasma to TQS-621 _max Average time (T) _max )。

In some embodiments, TQS-168 or a salt thereof is administered, the amount providing at least 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M, 5.5. Mu.M, 6. Mu.M, 6.5. Mu.M, 7. Mu.M, 7.5. Mu.M, 8. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M, 10. Mu.M, 1. Mu.M, 2.5. Mu.M, 3.5. Mu.M, 4.5. Mu.M, 7. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M average blood or plasma C of TQS-621 of 10.5. Mu.M, 11. Mu.M, 11.5. Mu.M, 12. Mu.M, 12.5. Mu.M, 13.5. Mu.M, 14. Mu.M, 14.5. Mu.M, 15. Mu.M, 15.5. Mu.M, 16. Mu.M, 16.5. Mu.M, 17. Mu.M, 17.5. Mu.M, 18. Mu.M, 18.5. Mu.M, 19. Mu.M, 19.5. Mu.M, 20. Mu.M, 20.5. Mu.M, 21. Mu.M, 21.5. Mu.M, 22. Mu.M, 22.5. Mu.M, 23.5. Mu.M, 24. Mu.M, 24.5. Mu.M or 25. Mu.M _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-621 of at least 75ng/mL, 100ng/mL, 125ng/mL, 150ng/mL, 175ng/mL, 200ng/mL, 225ng/mL, 250ng/mL, 300ng/mL, 350ng/mL, 400ng/mL, 450ng/mL, 500ng/mL, 550ng/mL, or 600ng/mL after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average blood or plasma C of TQS-621 of 100-700ng/mL, 200-600ng/mL, or 300-500ng/mL after administration _max 。

In some embodiments, TQS-168 or a salt thereof is administered, the amount providing at least 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M, 5.5. Mu.M, 6. Mu.M, 6.5. Mu.M, 7. Mu.M, 7.5. Mu.M, 8. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M, 3.5. Mu.M, 2.M average brain C of TQS-621 of 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM, 17 μM, 17.5 μM, 18 μM, 18.5 μM, 19 μM, 19.5 μM, 20 μM, 20.5 μM, 21 μM, 21.5 μM, 22 μM, 22.5 μM, 23 μM, 23.5 μM, 24 μM, 24.5 μM, or 25 μM _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides a brain/plasma ratio of TQS-621 of 0.5-10 after administration. In certain embodiments, TQS-168 is administered in an amount that provides a brain/plasma ratio of TQS-168 of 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5 after administration. In certain embodiments, the TQS-168 is administered in an amount that provides a brain/plasma ratio of TQS-168 of at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0 after administration.

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides an average T of TQS-621 in blood or plasma of no more than 360 minutes after administration _max . In certain embodiments, TQS-168 or a salt thereof is administered in a formulation that provides an average T in blood or plasma of no more than 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 225, 200, or 180 minutes _max . In certain embodiments, the process comprisesTQS-168 or a salt thereof is administered in a formulation that provides an average T in blood or plasma of no more than 90 minutes, 60 minutes or 45 minutes _max 。

In another aspect, a method of treating a neuroinflammatory and/or neurodegenerative disease in a subject is provided. The method comprises orally administering to a subject suffering from a neuroinflammatory and/or neurodegenerative disease a pharmaceutical composition comprising TQS-168 or a pharmaceutically acceptable salt thereof in an amount that provides (a) an average peak concentration of TQS-168 in plasma of at least 50nM (C _max ) And (b) no more than 360 minutes of C in plasma to TQS-168 _max Average time (T) _max ) The method comprises the steps of carrying out a first treatment on the surface of the And (C) an average peak concentration (C) of at least 50nM in plasma (TQS-621) _max ) And (d) no more than 360 minutes of C in plasma to TQS-621 _max Average time (T) _max )。

In some embodiments, TQS-168 or a salt thereof is administered, the amount providing at least 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M, 5.5. Mu.M, 6. Mu.M, 6.5. Mu.M, 7. Mu.M, 7.5. Mu.M, 8. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M, 10. Mu.M, 1. Mu.M, 2.5. Mu.M, 3.5. Mu.M, 4.5. Mu.M, 7. Mu.M, 8.5. Mu.M, 9. Mu.M, 9.5. Mu.M average blood or plasma C of TQS-168 of 10.5. Mu.M, 11. Mu.M, 11.5. Mu.M, 12. Mu.M, 12.5. Mu.M, 13.5. Mu.M, 14. Mu.M, 14.5. Mu.M, 15. Mu.M, 15.5. Mu.M, 16. Mu.M, 16.5. Mu.M, 17. Mu.M, 17.5. Mu.M, 18. Mu.M, 18.5. Mu.M, 19. Mu.M, 19.5. Mu.M, 20. Mu.M, 20.5. Mu.M, 21. Mu.M, 21.5. Mu.M, 22. Mu.M, 22.5. Mu.M, 23.5. Mu.M, 24. Mu.M, 24.5. Mu.M or 25. Mu.M _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides at least 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 2.5 μM, 3.5 μM, 6 μM, 6.5 μM, 7 μM, 10 μM, 11 μM, or a salt thereof after administration, Average brain C of TQS-168 of 14.5 μM, 15 μM, 15.5 μM, 16 μM, 16.5 μM, 17 μM, 17.5 μM, 18 μM, 18.5 μM, 19 μM, 19.5 μM, 20 μM, 20.5 μM, 21 μM, 21.5 μM, 22 μM, 22.5 μM, 23 μM, 23.5 μM, 24 μM, 24.5 μM or 25 μM _max 。

In some embodiments, TQS-168 or a salt thereof is administered in an amount that provides at least 50nM, 100nM, 150nM, 200nM, 250nM, 300nM, 350nM, 400nM, 450nM, 500nM, 550nM, 600nM, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, 10 μM, 10.5 μM, 11 μM, 11.5 μM, 12 μM, 12.5 μM, 13 μM, 13.5 μM, 14 μM, 14.5 μM, 15 μM, 5 μM after administrationAverage brain C of TQS-621 of M, 15.5. Mu.M, 16. Mu.M, 16.5. Mu.M, 17. Mu.M, 17.5. Mu.M, 18. Mu.M, 19. Mu.M, 19.5. Mu.M, 20. Mu.M, 20.5. Mu.M, 21. Mu.M, 21.5. Mu.M, 22. Mu.M, 22.5. Mu.M, 23. Mu.M, 23.5. Mu.M, 24. Mu.M, 24.5. Mu.M or 25. Mu.M _max 。

Inhibition of peripheral inflammation may provide benefits in the treatment of neuroinflammation. For example, fingolimod, now approved for the treatment of relapsing-remitting Multiple Sclerosis (MS), alleviates MS pathology by reducing lymphocyte outflow from lymph nodes. Thus, in some embodiments, TQS-168 is administered in an amount that provides optimal concentrations of TQS-168 and metabolite TQS-621 in the peripheral and central compartments.

In various embodiments, TQS-168 is administered in an amount that provides an optimal concentration ratio for one or more of the following:

TQS-168 plasma TQS-168 brain

TQS-621 plasma TQS-621 brain

TQS-168 plasma TQS-621 plasma

TQS-168 brain TQS-621 brain.

4.3.2. Oral dosage

In various embodiments, the daily oral dosage of TQS-168 is at least 0.5mg/kg. In various embodiments, the oral dosage of TQS-168 is at least 1mg/kg. In certain embodiments, the dose is at least 2mg/kg, at least 3mg/kg, at least 4mg/kg, at least 5mg/kg, at least 6mg/kg, at least 7mg/kg, at least 8mg/kg, at least 9mg/kg, or at least 10mg/kg.

In various embodiments, the daily oral dosage of TQS-168 is at least 10mg/kg. In certain embodiments, the dose is at least 15mg/kg, at least 20mg/kg, at least 25mg/kg, 30mg/kg, at least 35mg/kg, at least 40mg/kg, at least 45mg/kg, at least 50mg/kg, at least 55mg/kg, at least 60mg/kg, at least 65mg/kg, at least 70mg/kg, at least 75mg/kg, at least 80mg/kg, at least 85mg/kg, at least 90mg/kg, at least 95mg/kg, at least 100mg/kg, at least 150mg/kg, at least 175mg/kg, or at least 200mg/kg. In certain embodiments, the dose is 250mg/kg, 300mg/kg, 350mg/kg, 400mg/kg, 450mg/kg, 500mg/kg, 600mg/kg, 650mg/kg, 700mg/kg, 750mg/kg, 800mg/kg, 850mg/kg, 900mg/kg, 950mg/kg, or 1000mg/kg. In certain embodiments, the oral dose is 0.5mg/kg to 100mg/kg per day. In certain embodiments, the oral dose is 2mg/kg to 100mg/kg per day. In certain embodiments, the oral dose is 25mg/kg to 1000mg/kg per day.

In various embodiments, the oral daily dose of TQS-168 is 25mg/kg. In certain embodiments, the dose is at least 25mg/kg. In certain embodiments, the dose is at least 50mg/kg, at least 100mg/kg, at least 150mg/kg, at least 175mg/kg, or at least 200mg/kg. In certain embodiments, the dose is 250mg/kg, 500mg/kg, 750mg/kg, or 1000mg/kg. In certain embodiments, the oral dose is 25mg/kg to 1,000mg/kg per day.

In various embodiments, the daily oral dosage is 10-5000mg. In certain embodiments, the dose is 10mg, 15mg, 20mg, 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, or 1000mg. In certain embodiments, the dose is 1500mg, 2000mg, 2500mg, 3000mg, 3500mg, 4000mg, 4500mg, or 5000mg.

In various embodiments, the daily dose is 25-2000mg. In certain embodiments, the dose is 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 900mg, 925mg, 950mg, 975mg, or 1000mg.

In certain embodiments, the daily oral dosage is 200-800mg. In specific embodiments, the daily oral dosage is 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg or 500mg. In specific embodiments, the dose is 400mg or 450mg. In certain embodiments, the daily oral dose is 400mg or 450mg in a spray-dried dispersion formulation.

4.3.3. Oral dosage form

In some embodiments, TQS-168 or a salt thereof is administered in a suspension. In other embodiments, TQS-168 or a salt thereof is administered in solution. In some embodiments, TQS-168 or a salt thereof is administered in a solid dosage form. In a specific embodiment, the solid dosage form is a capsule. In a specific embodiment, the solid dosage form is a tablet. In particular embodiments, TQS-168 is in crystalline or amorphous form. In a specific embodiment, TQS-168 is in an amorphous form.

4.3.4. Patient(s)

In various embodiments, the subject has neuroinflammation. In certain embodiments, the subject is not diagnosed with a neurodegenerative disease. In specific embodiments, the subject is not diagnosed with a neurodegenerative disease and is at least 40, 45, 50, 55, 60, 65, 70, or 75 years old. In particular embodiments, the subject is not diagnosed with a neurodegenerative disease, but has one or more signs or symptoms of cognitive impairment. In specific embodiments, the subject has Mild Cognitive Impairment (MCI).

In various embodiments, the subject has a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is selected from the group consisting of motor neuron disease, amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, vascular dementia, frontotemporal lobar degeneration (frontotemporal dementia), dementia with lewy bodies, parkinson's disease, huntington's disease, demyelinating diseases, and Multiple Sclerosis (MS).

In specific embodiments, the subject has a motor neuron disease. In particular embodiments, the subject has ALS. In specific embodiments, the subject has alzheimer's disease. In specific embodiments, the subject has vascular dementia. In specific embodiments, the subject has frontotemporal dementia (FTD). In specific embodiments, the subject has dementia with lewy bodies (lewy body disease). In specific embodiments, the subject has parkinson's disease. In specific embodiments, the subject has huntington's disease. In yet other embodiments, the subject has a demyelinating disease. In one embodiment, the subject has MS.

4.3.5. Other embodiments

Other embodiments are provided in the numbered clauses below.

1. A method of treating a neuroinflammatory and/or neurodegenerative disease in a subject, the method comprising:

in an amount that provides after administration

(a) Average peak concentration of TQS-168 in plasma of at least 50nM (C _max ) A kind of electronic device

(b) No more than 360 minutes of C in plasma to TQS-168 _max Average time (T) _max )。

2. The method of clause 1, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 100nM, 250nM or 500nM _max 。

3. The method of clause 2, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 750nM _max 。

4. The method of clause 3, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 1. Mu.M _max 。

5. The method of clause 4, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 5. Mu.M _max 。

6. The method of clause 5, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 7.5. Mu.M _max 。

7. The method of clause 6, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-168C of at least 10. Mu.M _max 。

8. The method of any of clauses 1-7, wherein the TQS-168 or a salt thereof is administered in a formulation providing

(b) Mean plasma TQS-168T for no more than 275 minutes _max 。

9. The method of clause 8, wherein the TQS-168 or a salt thereof is administered in a formulation that provides

(b) Mean plasma TQS-168T for no more than 250 minutes _max 。

10. The method of clause 9, wherein the TQS-168 or a salt thereof is administered in a formulation providing

(b) Mean plasma TQS-168T for no more than 225 minutes _max 。

11. The method of clause 10, wherein the TQS-168 or a salt thereof is administered in a formulation that provides

(b) Average plasma TQS-168T of no more than 180, 90, 60 or 45 minutes _max 。

12. A method of treating a neuroinflammatory and/or neurodegenerative disease in a subject, the method comprising:

in an amount that provides after administration

(a) Average peak plasma concentration (C) of at least 50nM of compound of formula (II) (TQS-621) _max )

And

(b) No more than 360 minutes of C in plasma to TQS-621 _max Average time (T) _max )。

13. The method of clause 12, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 100nM, 250nM or 500nM _max 。

14. The method of clause 13, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 750nM _max 。

15. The method of clause 14, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 1. Mu.M _max 。

16. The method of clause 15, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 5. Mu.M _max 。

17. The method of clause 16, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 7.5. Mu.M _max 。

18. The method of clause 17, wherein the TQS-168 or salt thereof is administered in an amount that provides after administration

(a) Mean plasma TQS-621C of at least 10. Mu.M _max 。

19. The method of any of clauses 12-18, wherein the TQS-168 or a salt thereof is administered in a formulation providing

(b) Mean plasma TQS-621T for no more than 275 minutes _max 。

20. The method of clause 19, wherein the TQS-168 or a salt thereof is administered in a formulation that provides

(b) Mean plasma T of TQS-168 for no more than 250 minutes _max 。

21. The method of clause 20, wherein the TQS-168 or a salt thereof is administered in a formulation that provides

(b) Mean plasma T of TQS-168 for no more than 225 minutes _max 。

22. The method of clause 21, wherein the TQS-168 or a salt thereof is administered in a formulation that provides

(b) Mean plasma T of TQS-168 of no more than 180, 90, 60 or 45 minutes _max 。

23. A method of treating a neuroinflammatory and/or neurodegenerative disease in a subject, the method comprising:

in an amount that provides after administration

(b) No more than 360 minutes of C in plasma to TQS-168 _max Average time (T) _max ) The method comprises the steps of carrying out a first treatment on the surface of the And

(c) Average peak concentration (C) of compound of formula (II) (TQS-621) in plasma of at least 50nM _max )

And

(d) No more than 360 minutes of C in plasma to TQS-621 _max Average time (T) _max )。

24. The method of any of clauses 1-23, wherein the TQS-168 or a salt thereof is administered in a suspension.

25. The method of any of clauses 1-23, wherein the TQS-168 or a salt thereof is administered in solution.

26. The method of any of clauses 1-23, wherein the TQS-168 or a salt thereof is administered in a solid dosage form.

27. The method of clause 26, wherein the solid dosage form is a capsule.

28. The method of clause 26, wherein the solid dosage form is a tablet.

29. The method of any of clauses 1-24 or 26-28, wherein the TQS-168 is in crystalline or amorphous form.

30. The method of any of clauses 1-24 or 26-28, wherein the TQS-168 is in an amorphous form.

31. The method of any of clauses 1-30, wherein the subject has a neurodegenerative disease selected from the group consisting of motor neuron disease, amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, vascular dementia, frontotemporal lobar degeneration (frontotemporal dementia), dementia with lewy bodies, parkinson's disease, huntington's disease, demyelinating disease, and Multiple Sclerosis (MS).

32. The method of clause 31, wherein the subject has a motor neuron disease.

33. The method of clause 31, wherein the subject has ALS.

34. The method of clause 31, wherein the subject has alzheimer's disease.

35. The method of clause 31, wherein the subject has vascular dementia.

36. The method of clause 31, wherein the subject has frontotemporal dementia (FTD).

37. The method of clause 31, wherein the subject has dementia with lewy bodies (lewy body disease).

38. The method of clause 31, wherein the subject has parkinson's disease.

39. The method of clause 31, wherein the subject has huntington's disease.

40. The method of clause 31, wherein the subject has a demyelinating disease.

41. The method of clause 31, wherein the subject has MS.

42. The method of any one of clauses 1 to 41, wherein the dose of TQS-168 is at least 0.5mg/kg.

43. The method of clause 42, wherein the dose of TQS-168 is at least 2mg/kg.

44. The method of clause 43, wherein the dose of TQS-168 is at least 4mg/kg.

45. The method of clause 44, wherein the dose of TQS-168 is at least 8mg/kg.

46. The method of clause 45, wherein the dose of TQS-168 is at least 12mg/kg.

47. The method of clause 46, wherein the dose of TQS-168 is at least 14mg/kg.

48. The method of clause 47, wherein the dose of TQS-168 is at least 16mg/kg.

49. The method of clause 48, wherein the dose is 2mg/kg.

50. The method of clause 49, wherein the dose is 4mg/kg.

51. The method of clause 50, wherein the dose is 8mg/kg.

5. Experimental examples

The following are examples of specific embodiments for practicing the invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. Such techniques are well explained in the literature.

5.1. Example 1-TQS-168 in vitro induction of PGC-1 alpha protein expression in murine bone marrow cell lines at 20. Mu.M

Frozen BV2 murine microglia were thawed and propagated in complete medium (RPMI, 10% heat-inactivated FBS,1% L-glutamine, 1% Pen-Strep) until the growth rate reached the log phase.

For protein expression analysis, DMSO (1:1000 final dilution) or 20. Mu.M TQS-168 in DMSO (1:1000 final dilution) was added to the cell culture. After 24 hours of stimulation, the supernatant was discarded, and Cell lysis buffer (Cell Signal) was added to the adherent cells to extract proteins. The total protein of all samples was quantified and normalized by BCA assay.

Two replicates of untreated cultures and three replicates of TQS-168 treated cultures were studied, each replicate containing lysates from 5,000,000 BV2 bone marrow cells.

Pgc1α expression was detected by western blotting using anti-PGC-1α antibodies (SC 13067, santa Cruz Biotechnology,1:500 dilution). The use of anti-beta-actin antibodies (SC 8432, santa Cruz Biotechnology,1:2000 dilution) to quantify beta-actin, a housekeeping gene, is not clear whether its expression level is affected by TQS-168. Representative western blots are shown in figure 1.

As shown, TQS-168 induced PGC-1 alpha protein expression in murine BV2 microglia in vitro at 20. Mu.M.

5.2. Example 2-TQS-168 PGC-1 alpha protein expression in murine bone marrow cell lines was induced in vitro at a concentration of 0.7 to 20. Mu.M

For protein expression analysis, DMSO (1:1000 final dilution) or TQS-168 in DMSO (1:1000 final dilution) at 20. Mu.M, 6.8. Mu.M, 2.2. Mu.M and 0.7. Mu.M was added to BV2 cell cultures. After 24 hours of stimulation, the supernatant was discarded, and Cell lysis buffer (Cell Signal) was added to the adherent cells to extract proteins. The total protein of all samples was quantified and normalized by BCA assay. PGC-1 a was then detected by western blotting using anti-PGC-1 a antibodies (SC 13067, santa Cruz Biotechnology,1:500 dilution). Beta-actin was detected using anti-beta-actin antibodies (SC 8432, santa Cruz Biotechnology,1:2000 dilution). The results are shown in fig. 2. All procedures were performed according to standard molecular biology protocols for protein expression studies.

Fig. 3 is a bar graph quantifying protein expression levels measured from a scan of western blots shown in fig. 2. PD refers to PD169316, a p38 MAPK inhibitor.

FIGS. 2 and 3 demonstrate that TQS-168 induces PGC-1 alpha protein expression in murine BV2 cells in vitro at concentrations ranging from 0.7. Mu.M to 20. Mu.M.

5.3. Example 3-TQS-168 inhibits inflammatory cytokine secretion (CBA assay) of LPS-stimulated BV2 cells in vitro at concentrations ranging from 1. Mu.M to 20. Mu.M

Lipopolysaccharide (LPS) is a natural ligand of the TLR4/CD14 complex, which is highly expressed on bone marrow cells.

LPS was used to induce BV-2 cells to secrete cytokines. Cells were incubated with TQS-168 at various concentrations to assess whether TQS-168 could inhibit LPS-promoted release of various cytokines by cell cultures in vitro. Cytokine secretion was measured by cell measurement bead array (CBA) Fluorescence Activated Cell Sorting (FACS).

Briefly, one vial of BV2 frozen stock (1 million cells/mL complete medium) was thawed to 10mL of complete medium per vial, for a total of 4 vials. The vials were then centrifuged at 1800rpm for 3 minutes to wash off the frozen medium. Four vials were then combined into 25mL of complete medium. TQS-168 was prepared in DMSO.

Cells were incubated for 24 hours in medium (negative control), LPS (positive control), DMSO+LPS (positive control, additionally controlling the presence of DMSO in a TQS-168 stock solution) or LPS+TQS-168 at final concentrations of 1. Mu.M, 5. Mu.M, 10. Mu.M and 20. Mu.M.

After incubating the well plates for 24 hours, they were centrifuged at 1800rpm for 5 minutes. Next, 150. Mu.l per well was transferred to a new set of plates, of which 50. Mu.l was used for CBA. Plates were then centrifuged with cells, followed by the addition of 100. Mu.L of DAPI (50 ml PBS+10. Mu.L DAPI stock at 1:5000 dilution). The plates were then incubated in the dark for 5 minutes, followed by addition of 100. Mu.L of PBS and centrifugation at 1800rpm for 5 minutes. Finally, the plates were resuspended in 200 μl PBS and tested.

Multiplexed cell measurement bead array (CBA) assays were performed according to standard techniques to detect the presence of secreted murine TNFα, IL-6, IFNγ, IL-12, monocyte chemotactic protein 1 (MCP-1) and IL-10 in cell culture media.

Conclusion(s)

As shown in fig. 4, BV2 cells treated with 100ng/ml LPS showed strong release of tnfα and IL-6 pro-inflammatory cytokines without significant induction of secretion of INF- γ, IL-10, IL-12 and MCP-1. TQS-168 was able to reduce LPS-induced secretion of TNFα and IL-6 by BV2 cells at concentrations ranging from 1. Mu.M to 20. Mu.M. tQS-168 was concentration dependent for the reduction of TNF alpha (FIG. 4 a) and IL-6 (FIG. 4 b).

5.4. Example 4-TQS-168 in vitro inhibition of LPS-induced TNF alpha secretion by BV2 bone marrow cells at 5. Mu.M and 20. Mu.M (CBA assay)

A total of 4-11 replicates of BV2 cultures under different conditions were studied. Frozen BV2 microglial cell lines were thawed and propagated in complete medium (RPMI, 10% heat-inactivated FBS,1% L-glutamine, 1% Pen-Strep) until the growth rate reached the log phase.

For TNFalpha stimulation, cells were stimulated with 100ng/mL LPS for 24 hours. DMSO (1:1000 final dilution) or TQS-168 in DMSO (1:1000 final dilution) at 5. Mu.M or 20. Mu.M was added to the cell culture. After 24 hours of stimulation, supernatants were collected for cytokine analysis using CBA assay (BD Biosciences) according to the manufacturer's protocol. Tnfα expression was normalized to DMSO-treated conditions.

Statistical analysis using ANOVA had a significant threshold at p-value < 0.05.

Conclusion(s)

As shown in FIGS. 5a and 5b, TQS-168 inhibited TNF alpha production by LPS-stimulated BV-2 bone marrow cells at 5. Mu.M and 20. Mu.M, respectively, as compared to the control.

1.1.5.5. Example 5-TQS-168 inhibits TNF alpha Release (ELISA) of LPS-stimulated BV2 cells in vitro at concentrations of 0.3. Mu.M to 10. Mu.M

TNFa ELISA procedure

First, 100. Mu.L of supernatant was removed from the cell plate and transferred to a dilution plate, which was then centrifuged at 216Xg for 10 minutes to remove microparticles. The dilution plate is immediately assayed, or an aliquot is taken and stored at less than or equal to-20 ℃; avoiding repeated freeze-thaw cycles.

Next, a standard curve was prepared as follows: first 900ul of calibrator dilution RD5K was pipetted into a 700pg/mL tube, then 200 ul of the appropriate calibrator dilutions were pipetted into the remaining tubes. Stock solutions were used to create dilutions. The resulting tube was then thoroughly mixed. Mouse TNFα standard (700 pg/mL) was used as the high standard, and calibrator diluent RD5T was used as the zero standard at 0 pg/mL.

Assay dilutions RD1-63 (50 uL) were then added to the center of each well and mixed prior to and during use. Then, 50ul of standard, control or sample was added to the center of each well and covered with tape. The plates were then mixed for 1 minute and incubated for 2 hours at room temperature.

Each well was then aspirated and washed by filling each well five times with wash buffer (400 ul). After the last wash, the remaining wash buffer is removed by aspiration or decantation. TNFαIL-6 conjugate (100 ul) was then added to each well, covered with tape, incubated for 2 hours at room temperature, and washed and/or aspirated five times.

Then a substrate solution (100 uL) was added to each well, incubated at room temperature in the dark for 30 minutes, then 100uL of stop solution was added and mixed. The optical density of each well was determined within 30 minutes using a microplate reader set to 450 nm.

Measurement setup and parameters and data processing

Plates were read at 450nM and 570nM on a Spectrostar Nano machine with built-in MARS data analysis.

Optimizing parameters

Titration of LPS stimulation was performed to optimize assay dynamic range.

For TNFα ELISA, BV-2 cells were very responsive to low concentrations of LPS. Concentration ranges of 0.1ng/mL to 1,000ng/mL were tested. After 22 hours of stimulation, 0.3ng/ml LPS produced sufficient TNF alpha release from these cells (8-10 fold above background) without saturating the linear range of the ELISA detection system. If higher LPS concentrations are used to stimulate BV-2 cells, it is recommended that the sample solution be kept within the linear range of the detection system. Current tnfα protocols use 10,000 cells/well in 96-well plates. Cell count titration can optimize the S/B ratio. Miniaturization from 96 wells to 384 wells is also possible by this assay.

Conclusion(s)

FIGS. 6a-6d show a decrease in the dose response of TQS-168 mediated microglial BV2 cells to the release of the pro-inflammatory cytokine TNFα measured at 24 hours using ELISA. FIGS. 6a and 6c show absolute (FIG. 6 a) and relative (FIG. 6 c) inhibition of TNFα secretion by BV2 cells stimulated with 0.3ng/mL LPS. FIGS. 6b and 6d show the absolute (FIG. 6 b) and relative (FIG. 6 d) inhibition of TNFα secretion by BV2 cells stimulated with 1ng/mL LPS.

In BV-2 cells treated with 0.3ng/mL LPS, administration of TQS-168 inhibited TNFα production in a concentration-dependent manner, with up to about 25% inhibition observed for cells administered 10. Mu.M TQS-168.

Similarly, in BV-2 cells treated with 1ng/mL LPS, administration of TQS-168 inhibited TNFα production in a concentration-dependent manner, with up to about 35% inhibition observed for cells administered 10. Mu.M TQS-168.

5.6. Example 6-TQS-168 inhibition of LPS-induced TNF- α production by primary human bone marrow cells

Research method

Peripheral Blood Mononuclear Cells (PBMCs) from 4 different healthy volunteers were used in this study. Fresh blood samples were collected at the Stanford blood center and treated with Ficoll gradient for PBMC isolation. PBMC samples were stored in liquid nitrogen at-80 ℃ for subsequent analysis of TNF- α production.

For TNF- α stimulation, frozen PBMC samples were thawed and allowed to stand at 37℃and cells were stimulated with 100ng/ml LPS for 24 hours. DMSO (1:1000 final dilution) or TQS-168 in DMSO (1:1000 final dilution) at various concentrations was added to cell cultures of LPS-stimulated PBMC to evaluate the effect of T-168 on TNF- α production by human primary bone marrow cells. After 24 hours of stimulation, supernatant samples from different conditions were collected and TNF-a concentrations in the supernatants were analyzed using a cell measurement bead array (CBA) assay according to the protocol of BD Biosciences. TNF- α was quantified by median fluorescence intensity reading (MFI). The samples were analyzed directly after staining with LSRII flow cytometer.

As shown in FIG. 7, in human PMBC cells treated with 100ng/mL LPS, administration of TQS-168 at 5 μM and 20 μM resulted in statistically significant inhibition of TNFα production compared to untreated.

5.7. EXAMPLE 7 TQS-168 tissue concentration after a single oral dose in mice

Research method

In the first experiment, a single dose of TQS-168 was administered by oral gavage at 25mg/kg to a total of 3-4 male C57BL6/J mice. TQS-168 was prepared as a suspension for oral gavage with 0.5% methylcellulose in PBS.

Tissues were collected at various time points after dosing and processed for analysis of TQS-168 concentration by LC-MS. Tissues analyzed included plasma, brain and liver. Prior to brain and liver collection, animals were thoroughly perfused with 20mL ice-cold PBS to remove contaminating blood. The data are shown in Table 1 (TQS-168 concentration, ng/mL).

Conclusion(s)

Mean plasma C _max 93.4ng/ml or 0.37. Mu.M. Mean brain C _max 542.0ng/ml or 2.16. Mu.M. The mean concentrations (ng/ml) of TQS-168 for plasma (FIG. 8 a), liver homogenate (FIG. 8 b) and brain homogenate (FIG. 8 c) are plotted in FIG. 8.

In a second experiment, mice were given 50mg/kg TQS-168 by oral gavage. For group 1, TQS-168 was prepared as a suspension at a concentration of 5.0mg/mL with 0.5% methylcellulose in PBS. For group 2, TQS-168 was prepared as a solution in 10% polyethylene glycol (PEG) 400|30% Kleptose (Roquette) |60% water at a concentration of 5.0 mg/mL.

Plasma exposure (ng/mL) for group 1 mice is presented in Table 2, and plasma exposure (ng/mL) for group 2 mice is presented in Table 3.

FIGS. 9a-9c show the plasma concentration of TQS-168 in group 1 mice and group 2 mice as a function of time.

Conclusion(s)

After a single oral dose of 50mg/kg, this dose is twice that of the dose that has been previously demonstrated to inhibit bone marrow-mediated inflammation and reduce disease severity in animal models of neurodegenerative diseases including parkinson's disease, alzheimer's disease and Amyotrophic Lateral Sclerosis (ALS), C in plasma _max 2137ng/mL or 8.54. Mu.M to C _max Time (T) _max ) 50 minutes.

5.8. EXAMPLE 8 TQS-168 plasma and brain concentrations following a single oral dose in mice

A single oral dose of 45mg/kg TQS-168 was administered to mice. TQS-168 was prepared as a solution in 10% polyethylene glycol (PEG) 400|30% Kleptose (Roquette) |60% water at a concentration of 5.0 mg/mL.

The concentrations in plasma and brain were measured at different time points and various pharmacokinetic parameters were calculated as shown in tables 4 and 5 below and in fig. 19A and 19B.

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Brain/plasma C of TQS-168 _max The ratio was 1.842. The brain/plasma AUC ratio of TQS-168 was 1.814.

5.9. EXAMPLE 9 metabolite TQS-621 inhibits LPS-stimulated human PBMC Release of pro-inflammatory cytokines

The major metabolites of liver metabolism from TQS-168 have been described after oral administration. See Sun et al, quick Commun. Mass Spectrom.32:480-488 (2018), incorporated herein by reference. We synthesized the phase 1 metabolites from liver metabolism of TQS-168 shown in FIG. 20 and tested their ability to inhibit LPS-stimulated proinflammatory cytokine secretion in vitro.

Briefly, 20,000 human PBMCs were aliquoted into each well. LPS was added at 1ng/mL and the cells were incubated for 24 hours in the presence of LPS and TQS-168 or one of its metabolites. The readings were human IL-6ELISA and human TNFα ELISA. For the assay, the supernatant was diluted in culture medium. The supernatant dilution of IL-6ELISA was 1:8, and the supernatant dilution of human TNFα ELISA was 1:2. The results are shown in fig. 21A and 21B.

FIG. 21A shows the absolute inhibition of LPS-stimulated IL-6 secretion by the TQS-168 metabolite TQS-621 from PBMC obtained from the first donor. FIG. 21B shows the relative inhibition of IL-6 inhibition expressed as a percent activity. The structure of TQS-621 is shown in formula II below:

FIG. 22A shows the absolute inhibition of LPS-stimulated IL-6 secretion by the TQS-168 metabolite TQS-621 from PBMC obtained from the second donor. FIG. 22B shows the relative inhibition of IL-6 inhibition expressed as a percent activity.

FIG. 23A shows the absolute inhibition of LPS-stimulated TNFα secretion by PBMC of the first donor by TQS-168 and metabolite TQS-621. Fig. 23B shows the relative inhibition of tnfα inhibition expressed as a percent activity. FIG. 24A shows the absolute inhibition of LPS-stimulated TNF alpha secretion by TQS-168 and metabolite TQS-621 on PBMC of the second donor. Fig. 24B shows the relative inhibition of tnfα inhibition expressed as a percent activity.

The data show that stage 1 metabolite TQS-621 is a potent inhibitor of IL-6 and TNFα secretion by LPS stimulated human PBMC, confirming that at least some of the therapeutic effects observed following oral administration of TQS-168 may be attributed to the activity of the active metabolite TQS-621.

5.10. EXAMPLE 10 plasma concentrations of TQS-168 and active metabolite TQS-621 following a single oral dose of TQS-168 in mice

A single dose of TQS-168 of 50mg/kg was administered to C57BL/6 male mice using one of three formulations: group 1-5.0 mg/ml of suspension in PBS solution of 0.5% methylcellulose; group 2-5.0 mg/ml of a solution in PEG400 (Fluka) 10% | Kleptose (Roquette)% | sterile water 60%; group 3-5.0 mg/ml of suspension in Labrafil M1944 CS (Gattefosse)% |Masine CC (Gattefosse) 5% |Miglyol 812N 10% | sterile water 55%.

Plasma concentrations of TQS-168 and TQS-621 were measured over time.

Table 6 presents TQS-168 plasma concentration data for group 1 mice; table 7 presents the TQS-168 plasma concentration data for group 2 mice; table 8 provides the TQS-168 plasma concentration data for group 3 mice.

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Solution formulation group 2 provided a higher TQS-168C than suspension formulations group 1 (1121 ng/mL) (4.5. Mu.M) and group 3 (611 ng/mL) (2.4. Mu.M) _max (1950 ng/mL) (7.9. Mu.M), and higher exposure (AUC) than suspension formulations of groups 1 (8111) and 3 (4153) _inf =11404 hr ng/mL). The results are plotted in fig. 25A (group 1), 25B (group 2) and 25C (group 3).

Table 9 presents the TQS-621 plasma concentration data for group 1 mice; table 10 presents the TQS-621 plasma concentration data for group 2 mice; table 11 provides the TQS-168 plasma concentration data for group 3 mice.

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TQS-621 plasma C of group 2 mice receiving the solution formulation _max 449ng/mL, thereby providing a C of metabolite TQS-621 and parent TQS-168 of 0.152 _max Ratio and AUC of 0.136 _last Ratio.

The results are plotted in fig. 26A (group 1), 26B (group 2) and 26C (group 3).

5.11. EXAMPLE 11 concentration of TQS-621 in the brain following a single oral dose of TQS-168 in mice

TQS-168 was prepared as a 4.5mg/ml solution in PEG 400% I Kleptise 30% I sterile water 60%. A single oral dose of 45mg/kg was administered. TQS-621 concentrations were measured over time in plasma and brain. The data are provided in tables 12 and 13 below.

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5.12. EXAMPLE 12 TQS-168 concentration in plasma and brain after a single oral dose in rats

Research method

Male (n=3) and female (n=3) Sprague-Dawley rats were given a single dose of TQS-168 by oral gavage at 50, 150 or 500mg/kg of TQS-168. TQS-168 was prepared as a suspension with 0.5% methylcellulose in PBS.

Plasma samples were collected at 30, 60, 120, 240 and 1440 minutes after dosing. Brain samples were collected in some animals at 60, 240 and 1440 minutes after a single oral dose of TQS-168 at 500 mg/kg. Samples were processed to analyze TQS-168 concentration by LC-MS.

The data are presented as TQS-168 concentrations (ng/ml) in plasma (FIG. 12 a) and brain homogenates (FIG. 12 b). FIGS. 12a and 12b show that TQS-168 was detected in both plasma and brain tissue of treated rats. In the oral administration of TQS-168The maximum concentration of TQS-168 was detected in plasma and brain 240 minutes later (C _max )。

The C of plasma tissue was calculated using PKSolver (Compute Methods Programs biomed.9, 2010; 99 (3): 306-14.Doi:10.1016/j.cmpb.2010.01.007.2010, 21-2 electronic publication) _max And AUC _0-t The values, base data are plotted in fig. 12 a. The results are shown in FIG. 13 (C) _max ) And 14 (AUC), and these values are summarized in table 14.

Conclusion(s)

TQS-168 was detected in rats after single oral doses of 50, 150 and 500mg/kg, dose-dependent C in both plasma and brain tissue _max And AUC.

5.13. EXAMPLE 13 TQS-168 blood after a single intravenous dose in mice

Pulp concentration

Research method

Three male 7-9 week old male CD-1 mice from Lingchong were treated intravenously with 0.5mg/kg TQS-168. TQS-168 was prepared in a solution of 31.6% DMAC+36.8% ethanol+31.6% propylene glycol. Blood was collected at 0.5, 3, 10, 30, 60, 120, 240, 480 and 720 minutes after a single dose of TQS-168 and then processed. Blood sample is collected into K by saphenous vein puncture ₂ In EDTA tubes, centrifugation was performed at 4600rpm for 5 minutes at 4℃and plasma was collected and stored at below-20℃before analysis of TQS-168 concentration by LC-MS. The data are presented as TQS-168 concentration and average concentration per volume of plasma (ng/mL), as shown in fig. 10 (single mouse) and fig. 11 (average), respectively.

Conclusion(s)

TQS-168 was detected in plasma following an intravenous dose of 0.5mg/kg TQS-168 in mice. The terminal elimination half-life was 0.14 hours (8.4 minutes).

5.14. Example 14-detection of TQS-168 in plasma after a single intravenous dose in rats

Research method

A single intravenous dose of 0.5mg/kg TQS-168 was administered to a total of 3 male, 7-9 week old Sprague Dawley rats from Vital River, in a volume of 0.5mL/kg. TQS-168 was prepared in a solution of 31.6% DMAC+36.8% ethanol+31.6% propylene glycol.

Blood samples were collected at 0.5, 3, 10, 30, 60, 120, 240, 480 and 720 minutes after a single dose of TQS-168 and then processed. Blood sample is collected into K by saphenous vein puncture ₂ In EDTA tubes, centrifugation was performed at 4600rpm for 5 minutes at 4℃and plasma was collected and stored at below-20℃before analysis of TQS-168 concentration by LC-MS. The data are presented as TQS-168 concentration (ng/mL) in plasma and plotted in fig. 15 (single rat) and 16 (average).

Conclusion(s)

TQS-168 was detected in plasma in rats after a single intravenous dose of 0.5mg/kg was administered. Half-life was 0.162 hours (9.72 minutes) and clearance (L/hr/kg) was 9.29.

5.15. Example 15-detection of TQS-168 in plasma after a single intravenous dose in dogs

Research method

A total of 3 male 1-3 year old beagle dogs from Beijing Marshall Biotechnology Co., ltd (Beijing Marshall Biotechnology, ltd.) were treated intravenously with 0.5mL/kg of TQS-168 at 0.5 mg/kg. TQS-168 was prepared in a solution containing 31.6% DMAC, 36.8% ethanol and 31.6% propylene glycol.

Blood samples were collected at 0.5, 3, 10, 30, 60, 120, 240, 360, 480, 720 and 1440 minutes after a single dose of TQS-168 and then processed. Blood sample is collected into K ₂ In EDTA tubes, centrifugation was performed at 4600rpm for 5 minutes at 4℃and plasma was collected and stored at below-20℃before analysis of TQS-168 concentration by LC-MS. The data are presented as TQS-168 concentration per unit volume of plasma (ng/mL) and are shown in FIGS. 17 and 18.

5.16. Example 16-part 1 SAD 1 phase trial-double blind randomization study of subjects receiving a single ascending dose of TQS-168 or placebo.

5.16.1. Test design

A double blind, randomized, placebo controlled clinical study was performed to characterize and compare the Pharmacokinetic (PK) profile of TQS-168 and its metabolite TQS-621 following a single ascending dose of TQS-168, presented in 3 different formulations or placebo in healthy subjects.

The subject: at age 18 to 55 years, 18.0 to 32.0kg/m was measured at screening ² Is performed in healthy male subjects with Body Mass Index (BMI) on the randomized, double-blind, placebo-controlled phase 1 single increment dose [ SAD ]]And (5) testing. The subjects all had a weight of at least 55kg at the time of screening. The key criteria for exclusion were the subjects: has the evidence of current SARS-CoV-2 infection, has the clinical manifestations of significant cardiovascular disease, kidney disease, liver disease, skin disease, chronic respiratory disease or gastrointestinal disease, or >Aspartic acid Aminotransferase (AST) or alanine Aminotransferase (ALT) at 1.5 times the Upper Limit of Normal (ULN). Subjects were recruited at one of the study centers in the uk. Each subject provided written informed consent.

And (3) test design: the assays were performed in a plurality of cohorts, with at least 7 subjects per cohort. In cohorts 1-3 of part 1, subjects received a single oral dose of TQS-168 Methylcellulose (MC) suspension, or spray-dried dispersion (SDD) suspension, or Hot Melt Extrudate (HME) suspension, or placebo, in a fasted state. Subjects were assigned to the study treatment group at a rate of 6 TQS-168:2 placebo per cohort. Subjects in cohort 1 received protocol a,60mg TQS-168MC. Subjects in cohort 2 received regimen B,180mg TQS-168. Cohort 3 was divided into three separate phases, with subjects being recipients of a single regimen. Subjects in cohort 3 stage 1 received regimen C,540mg TQS-168MC suspension on day 1. The same subjects in cohort 3 phase 2 received regimen D,180mg TQS-168SDD suspension on day 1, and in cohort 1 phase 3 phase 2, the same subjects received regimen E,180mg TQS-168HME suspension on day 3. See table 15.

IMP: a research pharmaceutical product; N/A: is not applicable.

For group 1-3 stage 1, the screening stage was up to 4 weeks. After confirmation of eligibility, the subjects in each cohort were randomly assigned to receive either active agent (TQS-168) or placebo treatment. Note that this is the first time TQS-168 was administered in humans and thus the sentinel dosing design was followed. Each group is divided into a sentinel group and a main group. The sentinel group consisted of the first two subjects of each cohort. They received dosing prior to the rest of the subjects (main group). Only after active review of safety data for the 24h sentinel group after dosing will the main group of subjects in the group receive dosing. The randomized schedule was constructed such that one subject dosed on the first day received a TQS-168 suspension and one subject received placebo. According to the protocol, all treatments were received once daily after an overnight fast (No. 10 hours fast), except for the cohort in which food impact was evaluated. For cohort 1-3 stage 1, subjects were admitted in the morning one day (day-1) prior to dosing and left on site until they were discharged 48 hours after dosing (day 3). For cohort 3, phase 2, subjects were admitted to the hospital on the morning one day prior to dosing (day-1) and left on site until they were discharged 48 hours after dosing (day 5). According to the regimen, subjects received either in the fasted state after overnight fast (. Gtoreq.10 hours) on the first morning, or in the fed state after eating a high fat breakfast 30 minutes prior to dosing, depending on their prescribed regimen.

After dosing of cohort 1-3 stage 1, and prior to dosing of subjects in cohort 3 stage 2, a transitional stage was observed and safety scrutinized. Examination concludes that it is safe to administer the SDD and HME formulations to a subject at doses similar to the MC formulation. Thus, group 3 phase 2 does not require a sentinel.

In cohorts 4-6, each subject received a single oral dose of TQS-168SDD suspension or placebo in either the fed or fasted state. The subjects of each cohort were randomly assigned to the study treatment group and placebo group at a 5:1 ratio. Cohort 4 received regimen F,90mg TQS-168, which was administered to subjects in a fed state. Cohort 5 received regimen G,90mg TQS-168, which was administered to subjects in a fasted state. Cohort 6 received regimen H,270mg TQS-168, which was administered to subjects in a fasted state. According to the regimen, the subject receives the dose in the morning on the first day, either in the fasted state after overnight fasted (. Gtoreq.10 hours) or in the fed state after a high fat breakfast of 30 minutes prior to dosing. See table 16.

Safety was continuously assessed throughout the trial by monitoring adverse events and concomitant drug use, electrocardiography (ECG), vital signs, laboratory safety assessment and physical examination. Blood samples for pharmacokinetic assessment were collected from each subject on day-1, prior to each dose (.ltoreq.1 hour) and at intervals throughout the study up to 48 hours post dose (where applicable). In cohorts 1-5, no use was expected to exceed C in any individual subject _max AUC of 305ng/mL and 2750ng h/mL _(0-24) Upper exposure limit dose specified prior to the trial of (c). After modification, the upper exposure limit is increased to C _max 1121ng/mL and AUC 5969ng h/mL.

IMP, research drug product; N/A, inapplicable.

For all subjects in cohorts 1-3, safety was continuously assessed throughout the trial by monitoring adverse events and concomitant drug use, electrocardiography (ECG), vital signs, laboratory safety assessment, and physical examination. Blood samples for pharmacokinetic assessment were collected from each subject on day-1, prior to each dose (.ltoreq.1 hour) and at intervals throughout the study up to 48 hours post dose (where applicable).

Evaluation of pharmacokinetics: blood samples for plasma PK analysis were collected at regular time intervals. Venous blood samples were collected from subjects by trained clinical team members. The pre-dosing samples were collected at less than or equal to 1 hour prior to dosing. Samples from 0 to 1 hour post-dose were collected within ±2 minutes of the nominal post-dose sampling time. Samples 1.5 to 12 hours post-dose were collected within + -10 minutes of the nominal post-dose sampling time. Samples from 16 to 48 hours post-dose were collected at a time stamp within ±30 minutes of the nominal post-dose sampling time. Samples were collected in appropriate containers and processed to separate plasma. PK analysis was performed on plasma samples using validated bioanalytical methods.

Statistical analysis: the sample size for the study was selected based on practical considerations and experience from previous studies of similar designs. The number of subjects in each cohort (group) was considered sufficient to evaluate the primary objective of each study. Pharmacokinetic parameters were determined by non-atrioventricular techniques using WinNonlin software version 8.0 or higher (Certara usa.inc., usa). All data are listed and summarized by subject group using descriptive statistics. All statistical analyses were performed using SAS version 9.4 or higher.

tQS-168 Methylcellulose (MC) powder suspension formulations

Methylcellulose (MC) powder suspension formulations of 2- (4-tert-butylphenyl) -1H-benzimidazole (TQS-168; compound of formula I) were prepared by reconstitution as suspensions in methylcellulose vehicle formulations of Table 17 ("vehicle formulations").

Vehicle formulations were prepared as follows: water (1986 g) was heated to 80 ℃ (+5℃), then methylcellulose (10 g) was added and stirred for 30 minutes or more until the methylcellulose was completely dispersed. Sodium dodecyl sulfate (2 g) and 30% simethicone emulsion (2 g) were then added and the mixture was stirred until a translucent, white/off-white, slightly viscous suspension, free form particles formed. The pH of the resulting vehicle formulation was 5.3 (target pH 6.0 +/-3.0).

General procedure for reconstitution of Compounds of formula I in vehicle formulations of Table 17

The required amount of 2- (4-tert-butylphenyl) -1H-benzimidazole (Compound 1; TQS-168) (e.g., 60mg-1000 mg) is weighed into a vial. Vehicle formulations (100 mL) were added to vials containing the compound of formula I to give compound Methylcellulose (MC) powder suspension formulations of formula I.

5.16.3. Methylcellulose (MC) results

A single oral dose of TQS-168 Methylcellulose (MC) powder was administered to healthy male subjects in a fasted state at one of the following doses: 60mg, 180mg or 540mg. Plasma concentrations of TQS-168 and metabolite TQS-621 were measured over time and key pharmacokinetic parameters were determined.

Tables 18 and 19 present the geometric mean of the key pharmacokinetic parameters of TQS-168 and metabolite TQS-621 in subjects following oral administration of TQS-168.

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* Median (min-max)

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TQS-168 single increment dose (SAD) PK profile: group 3 rd orderSegment 1 received 540mg of TQS-168 (scheme C) and provided the highest TQS-168C of 323ng/mL (1.29. Mu.M) _max . Group 2 received 180mg of TQS-168 (scheme B) and provided 53.1ng/mL (0.21. Mu.M) of TQS-168C _max . Group 1 received 60mg of TQS-168 (scheme A) and provided 26.7ng/mL (0.11. Mu.M) of TQS-168C _max . The results are plotted in fig. 27. Group 3 phase 1 also showed a higher peak to peak ratio than group 2 (AUC _0-24 ＝163hr*ng/m,AUC _0-inf =180 hr ng/mL) and group 1 (AUC _0-24 ＝71.2hr*ng/mL,AUC _0-inf Higher exposure (=72.7 hr ng/mL) (AUC _0-24 ＝1440hr*ng/mL,AUC _0-inf ＝1600hr*ng/mL)。

Metabolite TQS-621PK profile: group 3 phase 1 showed a higher metabolite TQS-621C than group 2 (199 ng/mL) (0.75. Mu.M) and group 1 (65.3 ng/mL) (0.25. Mu.M) _max (1110 ng/mL) (4.17. Mu.M). The results are plotted in fig. 28. Similarly, group 3 also showed a higher peak value than group 2 (AUC _0-24 ＝1210hr*ng/mL,AUC _inf =1350 hr ng/mL) and group 1 (AUC _0-24 ＝383hr*ng/mL,AUC _inf Higher metabolite exposure (=410 hr ng/mL) (AUC _0-24 ＝10800hr*ng/m,AUC _inf ＝12700hr*ng/m)。

In the study, the maximum plasma concentration of TQS-168 (C _max ) And TQS-168 exposure (AUC _(0-24) And AUC _(0-inf) ) It appears that the dose increases slightly in sub-proportion to the dose after a single dose of 60 and 180mg TQS-168. However, it should be noted that between the 180mg and 540mg doses of TQS-168, the C of TQS-168 _max 、AUC _(0-24) And AUC _(0-inf) The increase was in a hyperproportional manner, with 6.1-fold, 8.8-fold and 9.5-fold increases for a 3-fold increase in dose, respectively. C when observing the whole dosage range of 60mg-540mg _max 、AUC _(0-24) And AUC _(0-inf) The super-proportions are increased by 12.1 times, 20.2 times and 21.2 times respectively. See fig. 27, 29-31.

With respect to metabolite TQS-621, plasma C after a single dose from 60 to 180mg TQS-168 _max 、AUC _(0-24) And AUC _(0-inf) It appears to increase in proportion to the dose. For doses between 180 and 540mg, C _max 、AUC _(0-24) And AUC _(0-inf) Super-proportional increases, with 5.6, 8.9 and 9.4 fold increases for 3 fold increases in dose, respectively. Furthermore, the dose was increased 9-fold from 60mg to 540mg over the whole dose range, plasma C _max 、AUC _(0-24) And AUC _(0-inf) The super-scale increases by 17.0 times, 28.2 times and 31.0 times, respectively. See fig. 28-31.

After a dose of 60mg, the maximum plasma TQS-168 concentration occurs between 0.5 and 2 hours post-dose, with the median T of TQS-168 _max For 1 hour after administration. The maximum plasma metabolite TQS-621 concentration occurs between 1 and 4 hours post-administration, median T _max For 2.5 hours after administration.

After a dose of 180mg TQS-168, the maximum plasma concentration of TQS-168 occurred between 1 and 4 hours post-administration. The median T at 1 hour post-dose was observed _max . The maximum plasma metabolite TQS-621 concentration occurs between 1 and 4 hours post-administration, median T _max For 1.5 hours after administration.

After 540mg of TQS-168 was administered, the maximum plasma concentration of TQS-168 occurred between 1 and 10 hours post-administration, median T _max For 2.25 hours after administration. Metabolite TQS-621 showed maximum plasma concentration between 2 and 12 hours post-dose, median T _max For 4 hours after administration.

At all doses of TQS-168, the concentration of metabolite TQS-621 exceeded that of TQS-168 after a short lag.

The geometric mean terminal half-life of TQS-168 after the 60mg dose, 180mg dose and 540mg dose was 3.06, 7.36 and 10.1 hours, respectively, and was dose dependent. Geometric mean terminal half-lives of metabolite TQS-621 at doses of 60mg, 180mg and 540mg were 4.89, 9.10 and 11.3 hours, respectively. It should be noted that for all doses of TQS-168, the plasma concentration of TQS-168 was quantifiable starting from 0.5 hours post-dose and was quantifiable until the final sampling time point of 48 hours post-dose. The concentration of TQS-621 was also quantified starting from 0.5 hours post-dose and was still quantified until the final sampling time point of 48 hours post-dose.

5.16.4. Spray-dried dispersion (SDD) formulations and oral suspensions thereof

5.16.4.1 SDD preparation of 2- (4-tert-butylphenyl) -1H-benzimidazole (Compound of formula I)

Preparation of the agent

A spray-dried dispersion (SDD) of 2- (4-tert-butylphenyl) -1H-benzimidazole (compound of formula I; TQS-168) having the composition listed in table 20 was prepared by spray-drying the stock formulation shown in table 21.

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Preparation procedure of 5.16.4.2 spray-dried raw material preparation

The compound of formula I (45.0 g) was slowly added to 2-propanol (1791.1 g) with stirring and placed under a homogenizer (Silverson SL2 homogenizer) with stirring for 5 minutes or more until the compound of formula I was completely dissolved. The reaction mixture was then removed from the homogenizer and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (60.0 g) was slowly added with stirring, placed back under the homogenizer and stirred for 10 minutes or more until the Soluplus was completely dissolved. The reaction mixture was then removed from the homogenizer and slowly added with stirring to amorphous silica @ 244 FP) was returned to the homogenizer and stirred for an additional 15 minutes or more until the amorphous silica was completely dispersed. The resulting suspension is referred to herein as a "stock formulation"

Procedure for the preparation of spray-dried dispersion (SDD) formulations of compounds of formula I5.16.4.3

The spray dryer unit (prosept 4M8 spray dryer) is provided with a compressed air supply. Once the outlet temperature was stable, the feed pump was started and 2-propanol (blank solution) was sprayed through the nozzle as a fine spray into the collection chamber. The spray dryer parameters were adjusted to achieve feed rates within the ranges listed in table 22 below.

The raw material formulation is stirred under a homogenizer at a suitable speed to maintain a uniform dispersion without generating bubbles. The raw formulation was then sprayed as a fine spray through a nozzle into the collection chamber of a spray dryer unit (prosept 4M8 spray dryer, using the parameters set forth in table 3 and set forth with blank solution) where the solvent was rapidly evaporated to produce a solution containing the compound of formula I, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) and silica @244 FP) (SDD formulation of compound 1). Once all of the stock formulation has been sprayed and collected, the stock formulation is replaced with 2-propanol (blank solution) and sprayed through the nozzle of the spray dryer for 5 minutes or more to allow any remaining "stock formulation" to collect within the gas stream. / >

5.16.4.4 Oral suspension of spray-dried dispersion (SDD) formulation of 2- (4-tert-butylphenyl) -1H-benzimidazole (Compound 1)

A spray-dried dispersion (SDD) (e.g., 60-1000 mg) of 2- (4-tert-butylphenyl) -1H-benzimidazole (compound 1) having the composition listed in table 20 was reconstituted as an oral suspension in 100g of vehicle consisting of PEG 300 (10 g), glyceryl monocaprylate (Capmul MCM,0.40 mg) in sterile rinse water (moderate to 100 g).

Sdd results 5.16.5

A single oral dose of 90mg or 180mg or 270mg of an oral suspension of TQS-168SDD powder is administered to a healthy male subject in a fed or fasted state. Plasma concentrations of TQS-168 and metabolite TQS-621 were measured over time and key pharmacokinetic parameters were determined.

Table 23 and Table 24 present the key pharmacokinetic parameters of TQS-168 and metabolite TQS-621 in subjects following oral administration of the TQS-168SDD formulation.

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* Median (min-max)

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TQS-168 single increment dose (SAD) PK profile: group 3 part 2 received regimen D,180mg of TQS-168SDD powder oral suspension, in a fasted state, and provided 218ng/mL (0.87. Mu.M) of TQS-168C _max . Group 4 received regimen F,90mg of TQS-168SDD powder oral suspension in fed state, and provided 47.1ng/mL (0.19. Mu.M) of TQS-168C _max . Group 5 received regimen G,90mg of TQS-168SDD powder oral suspension in fasted state, and provided 111ng/mL (0.44. Mu.M) of TQS-168C _max . Group 6 received regimen H,270mg TQS-168SDD powderThe suspension was not taken orally, in a fasted state, and provided a maximum TQS-168C of 237ng/mL (0.95. Mu.M) _max . The results are plotted in fig. 44. The recipients of regimen H also exhibited a higher than regimen D (AUC _0-24 ＝608hr*ng/mL,AUC _0-inf =621hr×ng/mL), regimen G (AUC _0-24 ＝192hr*ng/mL,AUC _0-inf =195hr ng/mL) and scheme F (AUC _0-24 ＝168hr*ng/mL,AUC _0-inf Higher exposure of recipients (=171hr ng/mL) (AUC _0-24 ＝736hr*ng/mL,AUC _0-inf ＝836hr*ng/mL)。

Metabolite TQS-621PK profile: scheme D (180 mg TQS 168) provided a higher metabolite TQS-621C than scheme H (621 ng/mL, 2.33. Mu.M), scheme G (214 ng/mL, 0.80. Mu.M) and scheme F (122 ng/mL, 0.46. Mu.M) _max (742 ng/mL, 2.79. Mu.M). The results are plotted in fig. 45. Similarly, the recipients of regimen D also exhibited a higher response than regimen H (AUC _0-24 ＝4250hr*ng/mL,AUC _0-inf =4950hr ng/mL), regimen G (AUC _0-24 ＝1170hr*ng/mL,AUC _0-inf 1200hr ng/mL) and scheme F (AUC _0-24 ＝848hr*ng/mL,AUC _0-inf Higher metabolite exposure (AUC) in recipients =901 hr ng/mL _0-24 ＝5110hr*ng/mL,AUC _0-inf ＝5170hr*ng/mL)。

Comparing 270mg of the TQS-168SDD powder oral suspension, administration in the fasted state (regimen H) with 90mg of the TQS-168SDD powder oral suspension, administration also in the fasted state (regimen G) revealed that 3-fold addition of dose resulted in TQS-168C _max And 2.14-fold and 4.29-fold increases in AUC (0-inf), and TQS-621C _max And AUC (0-inf) increases by 2.90 and 4.13 times. See fig. 38-39.

In a notable comparison, namely 90mg of TQS-168SDD powder oral suspension, administration in the fasted state (regimen G) revealed approximately 136% and 14% TQS-168C compared to administration in the fed state (regimen F) of the same suspension _max And AUC (0-inf), 75% and 33% TQS-621C _max And an increase in AUC (0-inf). See fig. 35-36.

Comparison of 270mg of TQS-168SDD powder oral suspension, administration in the fasted state (regimen H) withAdministration of 180mg TQS-168, also in the fasted state (regimen D), revealed approximately 8.7% and 26% TQS-168C _max And AUC (0-inf), but approximately 16% and 4.3% TQS-621C _max And a decrease in AUC (0-inf). See fig. 38 and 46.

Following oral dosing regimen D (i.e., 180mg TQS168SDD in fasted state), the maximum plasma TQS-168 concentration occurred between 0.5 and 2 hours post-dose, median T _max For 1 hour after administration. The maximum plasma metabolite TQS-621 concentration occurs between 1.5 and 3.0 hours post-administration, median T _max For 2 hours after administration. See fig. 46A-B.

Following oral dosing regimen F (i.e., 90mg TQS-168SDD in fed state), the maximum plasma TQS-168 concentration occurs between 0.5 and 3 hours post-dose, median T _max For 1.5 hours after administration. The maximum plasma metabolite TQS-621 concentration occurs between 1.5 and 6.0 hours post-administration, median T _max For 3.5 hours after administration. See FIGS. 34A-B

Following oral dosing regimen G (i.e., 90mg TQS-168SDD in a fasted state), the maximum plasma concentration of TQS-168 occurred between 0.5 and 2.0 hours post-dosing. A median T of 0.5 hours after dosing was observed _max . The maximum plasma metabolite TQS-621 concentration occurs between 1.0 and 3.0 hours post-administration, median T _max For 1.25 hours after administration. See fig. 37A-B.

Following oral dosing regimen H (i.e., 270mg TQS-168SDD in fasted state), the maximum plasma concentration of TQS-168 occurred between 0.5 and 4 hours post-dose, median T _max For 1.50 hours after administration. Metabolite TQS-621 showed maximum plasma concentration between 1.5 and 4 hours post-dose, median T _max For 2 hours after administration. See fig. 38-39.

The terminal half-life of TQS-168 after 90mg fed state, 90mg fasted state, 180mg fasted state and 270mg fasted state TQS-168SDD (T) _1/2 ) Dose-dependent for 3.52, 4.85, 5.64 and 10.4 hours, respectively. The final half-lives of TQS-168 metabolite TQS-621 at doses of 90mg fed state, 90mg fasted state, 180mg fasted state and 270mg fasted state were 5.17, 4.79, 7.12 and respectively 10.4 hours. It should be noted that for all doses of TQS-168, the plasma concentration of TQS-168 was quantifiable starting from 0.5 hours post-dose and remained quantifiable until the final sampling time point of 48 hours post-dose. The concentration of TQS-621 was also quantified starting from 0.5 hours post-dose and was still quantified until the final sampling time point of 48 hours post-dose.

HME formulations and oral suspensions thereof

5.16.6.1 Preparation of Hot Melt Extrudate (HME) formulation of 2- (4-tert-butylphenyl) -1H-benzimidazole (Compound 1) HME

Hot Melt Extrudate (HME) formulations of 2- (4-tert-butylphenyl) -1H-benzimidazole (compound of formula I; TQS-168) having the composition listed in Table 24 were prepared as follows.

The required amounts of compound 1, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus), povidone (Kollidon 17 PF) and copovidone (Kollidon VA 64) were weighed according to table 25 and passed through a 850 μm sieve and transferred into a 2L blender housing. The resulting blender housing was fixed in a blender (Pharmatech blender) and blended for 20 minutes, then added to a double layer Polyethylene (PE) bag ("compound blend of formula I") and transferred to an HME sealing system. The cooler unit was connected to the HME and once the cooler temperature reached 15 ℃, the extrusion process was started using the parameters listed in table 26. The compound blend of formula I was added to the feeder to fill approximately 3/4 of the feeder and the extrudate was collected and discarded during approximately the first 5 minutes of the extrusion process. The feeder is refilled to maintain about 50% of the volume in the feeder throughout the process and extrusion is continued until all of the compound blend of formula I is extruded and collected ("compound HME extrudate of formula I").

The collected extrudate of compound HME of formula I was added to a U5 Quadro mill (provided with a 457 (mm) screen size and an impeller speed of 5000 RPM) until all the extrudate had passed through a 457mm screen to obtain milled particles of compound of formula I. The abrasive particles of compound 1 were then sieved using a 300 micron screen and transferred to a blender housing (Pharmatech 2L blender housing). The resulting blender housing was fixed in a blender (Pharmatech blender), blended for 5 minutes, and collected.

5.16.6.2 Oral suspension of Hot Melt Extrudate (HME) formulation of 2- (4-tert-butylphenyl) -1H-benzimidazole (a compound of formula I)

Hot Melt Extrudates (HMEs) (e.g., 60-1000 mg) of 2- (4-tert-butylphenyl) -1H-benzimidazole (a compound of formula I) having the composition listed in Table 25 were prepared in 100mL of vehicle Ora-Blend(purified water, sucrose, glycerol, sorbitol, flavoring, microcrystalline cellulose, sodium carboxymethyl cellulose, xanthan gum, carrageenan, citric acid, sodium phosphate, simethicone, potassium sorbate and methylparaben) (commercially available oral suspension vehicle prepared by Perrigo Pharmaceuticals) were reconstituted into an oral suspension.

Hme results 5.16.7

In the fasted state, a single oral dose of 180mg of an oral suspension of TQS-168HME powder was administered to healthy male subjects (regimen E). Plasma concentrations of TQS-168 and metabolite TQS-621 were measured over time and key pharmacokinetic parameters were determined.

Table 27 and Table 28 present geometric means of key pharmacokinetic parameters for TQS-168 and metabolite TQS-621 in subjects following oral administration of the TQS-168SDD formulations.

TQS-168 single increment dose (SAD) PK profile: group 3 phase 2 received regimen E,180mg TQS-168HME powder oral suspension in a fasted state. A single dose provided 123ng/mL (0.49. Mu.M) of TQS-168C _max And 356 hr ng/mL AUC0-24. The data are plotted in fig. 47A-B.

Metabolite TQS-621PK profile: scheme E provides 481ng/mL (1.81. Mu.M) of metabolite TQS-621C _max And AUC of 3090hr ng/mL _0-24 . Illustrated in fig. 47A-B.

Comparison of MC vs. SDD vs. HME PK results

In part 1 of this treatment, subjects received 180mg TQS-168 in the following formulation in a fasted state: methylcellulose (MC), spray Dried Dispersion (SDD) powder oral suspension or Hot Melt Extrudate (HME) powder oral suspension. Plasma concentrations of TQS-168 and metabolite TQS-621 were measured over time and key pharmacokinetic parameters were determined as described previously. For convenience, tables 29-31 compare the results. See fig. 45A-B.

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* Median (min-max)

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^a Dose normalized comparison with regimen C540 mg TQS-168MC powder oral suspension

^b Comparison with regimen D180 mg TQS-168SDD powder oral suspension

Subjects administered 180mg of the TQS-168MC powder oral suspension (regimen B) exhibited quantifiable TQS-168 plasma concentrations from 0.5 hours post-administration that remained quantifiable until between 10 and 48 hours post-administration. The TQS-621 concentration was also quantified starting from 0.5 hours post-dose and remained quantifiable until between 24 and 48 hours post-dose.

Maximum plasma TQS-168 concentration occurs between 1 and 4 hours post-administration, median T _max For 1 hour after administration. T1/2 was obtained as 7.36 hours. Geometric mean (CV%) C _max And AUC (0-inf) values of 53.1ng/mL (55%) and 180ng h/mL (53.1%), respectively.

Maximum plasma TQS-621 concentration occurred between 1 and 4 hours post-dose, median T _max For 1.5 hours after administration. The geometric mean T1/2 obtained was 9.10 hours. Geometric mean (CV%) C _max And AUC (0-inf) values of 199ng hr/mL (33.5%) and 1350ng hr/mL (45.1), respectively.

After 180mg of the TQS-168SDD powder oral suspension (regimen D), the plasma concentration of TQS-168 was quantifiable starting from 0.5 hours post-administration and remained quantifiable until 24 to 36 hours post-administration. It should be noted that one subject exhibited a quantifiable TQS-168 plasma concentration prior to dosing due to some residual of the previous dosing regimen. Due to some residual of the previous dosing regimen, the concentration of TQS-621 can be quantified in all subjects from before dosing. It remained quantifiable until the final sampling time point of 48 hours (day 5) post-dosing. It should be noted that all quantifiable pre-dose concentrations are less than C _max 5% of (C).

Maximum plasma TQS-168 concentration occurs between 0.5 and 2 hours post-administration, median T _max For 1 hour after administration. The geometric mean value T obtained _1/2 For 5.64 hours. The maximum plasma TQS-621 concentration occurred between 1.5 and 3 hours post-dose, medianT _max For 2 hours after administration. The geometric mean value T obtained _1/2 For 7.12 hours.

Geometric mean C of TQS-168 after administration of 180mg of the SDD powder oral suspension compared to its MC counterpart (regimen B) _max AUC (0-last) and AUC (0-inf) resulted in 4.11-fold, 3.64-fold and 3.45-fold increases, respectively. Geometric mean C of TQS-621 after administration of 180mg of the SDD powder oral suspension compared to its MC counterpart (regimen B) _max AUC (0-last) and AUC (0-inf) resulted in 3.73-fold, 3.87-fold and 3.83-fold increases, respectively.

After 180mg of the TQS-168HME powder oral suspension (regimen E), the subject exhibited a quantifiable TQS-168 plasma concentration starting from 0.5 hours post-dose and remained quantifiable until the final sampling time point (day 5) between 16 hours post-dose and 48 hours post-dose. In all subjects, the concentration of TQS-621 was also quantifiable starting from 0.5 hours post-dose and remained quantifiable until the final sampling time point of 48 hours post-dose.

Maximum plasma TQS-168 concentration occurs between 1 and 1.5 hours post-administration, median T _max For 1.5 hours after administration. The geometric mean value T obtained _1/2 7.25 hours. Maximum plasma TQS-621 concentration occurs between 1.5 and 4 hours post-administration, median T _max For 2 hours after administration. The geometric mean value T obtained _1/2 9.17 hours.

Based on C relative to administration of 180mg SDD (regimen D) _max The geometric mean (geometric CV%) of TQS-168 after 180mg HME (regimen E) administration was 56.6% (33.9%), 58.9% (30.6) and 59.6% (30.0%) relative bioavailability, AUC (0-last) and AUC (0-inf). Based on C relative to administration of 180mg SDD _max The geometric mean (geometric CV%) of metabolite TQS-621 after 180mg of TQS-168HME was given, the relative bioavailability was 64.9% (16.4%), 63.5% (13.8) and 63.6% (13.9%), AUC (0-last) and AUC (0-inf). Geometric mean C of TQS-168 after administration of 180mg HME (regimen E) relative to its MC counterpart (regimen B) _max AUC (0-last) and AUC (0-inf) produced 2.32-fold, 2.14-fold and 2.06-fold increases, respectively.

Geometric mean C of TQS-621 after 180mg HME administration relative to its MC counterpart _max AUC (0-last) and AUC (0-inf) produced 2.42-fold, 2.46-fold and 2.44-fold increases, respectively.

In summary, the SDD formulations showed a significant improvement in exposure, as well as a significant improvement in exposure of the metabolite TQS-621, compared to the MC and HME formulations at the same TQS-168 dose. See fig. 48A-B.

5.17. Example 17-part 2 multiple dose phase 1 trial-double blind randomization study of subjects receiving multiple doses of TQS-168 or placebo

5.17.1. Test design

Part 2 is a double blind, randomized, placebo controlled clinical study that was conducted to characterize and compare the Pharmacokinetic (PK) profile of TQS-168 and its metabolite TQS-621 following multiple doses of TQS-168 spray-dried dispersion (SDD) powder oral suspension formulations in healthy subjects. The dosing regimen is described in table 32.

IMP, research drug product; N/A, inapplicable.

This randomized, double-blind, placebo-controlled phase 1 multiple dose trial was performed in 18 to 55 year old healthy male subjects having a Body Mass Index (BMI) of 18.0 to 32.0kg/m as measured at screening ² . The subjects all had a weight of at least 55kg at the time of screening. The key criteria for exclusion were the subjects: has the evidence of current SARS-CoV-2 infection, has the clinical manifestations of significant cardiovascular disease, kidney disease, liver disease, skin disease, chronic respiratory disease or gastrointestinal disease, or >Aspartic acid Aminotransferase (AST) or alanine Aminotransferase (ALT) at 1.5 times the Upper Limit of Normal (ULN). Subjects were recruited at one of the study centers in the uk. Each patient provided written informed consent.

Experiments were performed in 3 groups. All subjects were admitted in the morning one day prior to dosing (day-1) and left on site until 48 hours after final dosing (day 9). The screening phase was 4 weeks. After confirmation of eligibility, the subjects were randomized to receive either IMP (TQS-168) or placebo treatment. Subjects received IMP or placebo in the morning (approximately 24 hours apart) on days 1 to 7. Administration was performed in either the fasted state (regimen I) or the fed state (30 minutes after administration of standard pre-doses or high fat meals prior to dosing) after overnight fasting (at least 10 hours). Safety was continuously assessed throughout the trial by monitoring adverse events and concomitant drug use, electrocardiography (ECG), vital signs, laboratory safety assessment and physical examination. Blood samples for pharmacokinetic assessment were collected from each subject on day-1, prior to each dose (.ltoreq.1 hour) and periodically throughout the study up to 48 hours after final dose (where applicable).

Evaluation of pharmacokinetics: blood samples for plasma PK analysis were collected periodically. Venous blood samples were collected from subjects by trained clinical team members. The pre-dosing samples were collected at less than or equal to 1 hour prior to dosing. Samples from 0 to 1 hour post-dose were collected within ±2 minutes of the nominal post-dose sampling time. Samples 1.5 to 12 hours post-dose were collected within + -10 minutes of the nominal post-dose sampling time. Samples from 16 to 48 hours post-dose were collected at a time stamp within ±30 minutes of the nominal post-dose sampling time. Samples were collected in appropriate containers and processed to separate plasma. PK analysis was performed on plasma samples using validated bioanalytical methods.

5.17.2. Spray-dried dispersion (SDD) formulations and oral suspensions thereof

5.17.2.1 SDD preparation of 2- (4-tert-butylphenyl) -1H-benzimidazole (Compound of formula I)

Preparation of the agent

A spray-dried dispersion (SDD) of 2- (4-tert-butylphenyl) -1H-benzimidazole (compound of formula I) having the composition listed in table 20 was prepared by spray-drying the starting formulation shown in table 21.

Preparation procedure of 5.17.2.2 spray-dried raw material preparation

The compound of formula I (45.0 g) was slowly added to 2-propanol (1791.1 g) with stirring and placed under a homogenizer (Silverson SL2 homogenizer) with stirring for 5 minutes or more until the compound of formula I was completely dissolved. The reaction mixture was then removed from the homogenizer and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) (60.0 g) was slowly added with stirring, placed back under the homogenizer and stirred for 10 minutes or more until the Soluplus was completely dissolved. The reaction mixture was then removed from the homogenizer and slowly added with stirring to amorphous silica @244 FP) was returned to the homogenizer and stirred for an additional 15 minutes or more until the amorphous silica was completely dispersed. The resulting suspension is referred to herein as a "stock formulation"

Procedure for the preparation of spray-dried dispersion (SDD) formulations of compounds of formula I5.17.2.3

The raw material formulation is stirred under a homogenizer at a suitable speed to maintain a uniform dispersion without generating bubbles. The raw formulation was then sprayed as a fine spray through a nozzle into the collection chamber of a spray dryer unit (prosept 4M8 spray dryer, using the parameters set forth in table 3 and set forth with blank solution) where the solvent was rapidly evaporated to produce a solution containing the compound of formula I, polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus) and silica @244 FP) (SDD formulation of the compound of formula I). Once all of the stock formulation has been sprayed and collected, the stock formulation is replaced with 2-propanol (blank solution) and sprayed through the nozzle of the spray dryer for 5 minutes or more to allow any remaining "stock formulation" to collect within the gas stream.

5.17.2.1 Oral suspension of spray-dried dispersion (SDD) formulation of 2- (4-tert-butylphenyl) -1H-benzimidazole (a compound of formula I)

A spray-dried dispersion (SDD) (e.g., 60-1000 mg) of 2- (4-tert-butylphenyl) -1H-benzimidazole (a compound of formula I) having the composition listed in table 20 was reconstituted as an oral suspension in 100g of vehicle consisting of PEG 300 (10 g), glyceryl monocaprylate (Capmul MCM, 0.40 mg) in sterile rinse water (moderate to 100 g).

Scheme I Subjects received an oral dose of 120mg of TQS-168 spray-dried dispersion (SDD) powder oral suspension or placebo in a fasted state, 1 time per day for 7 consecutive days.

Scheme J Subjects received an oral dose of 90mg of an oral suspension of TQS-168 spray-dried dispersion (SDD) powder or placebo in the fed state, 1 time per day for 7 consecutive days. Subjects were provided with high fat breakfast on days 1 and 7 and standard breakfast on days 2-6.

Scheme K Subjects received an oral dose of 300mg of an oral suspension of TQS-168 Spray Dried Dispersion (SDD) powder or placebo in the fed state, 1 time per day for 7 consecutive days. Subjects were provided with high fat breakfast on days 1 and 7 and standard breakfast on days 2-6.

1.1.1.5.17.3. Results

Healthy male subjects were given multiple oral doses of 120mg of TQS-168 spray-dried dispersion (SDD) powder, either in the fasted state (regimen I) or 90mg of TQS-168SDD, in the fed state (regimen J). Plasma concentrations of TQS-168 and metabolite TQS-621 were measured over time and key pharmacokinetic parameters were determined.

Table 33 presents the geometric mean of the key pharmacokinetic parameters of TQS-168 and metabolite TQS-621 in the subject following oral administration of TQS-168.

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TQS-168 protocol IPK curve: subjects in cohort 1 received a single 120mg TQS-168SDD orally 1 day for 7 consecutive days in a fasted state.

On day 1, after a single administration of regimen I, the plasma concentrations of TQS-168 for all subjects were quantifiable starting from 0.5 hours post-administration and remained quantifiable until 16 to 24 hours post-administration. In all subjects, the concentration of TQS-621 on day 1 was also quantifiable starting from 0.5h post-dose and remained quantifiable until 24 h post-dose.

Maximum plasma TQS-168 concentration on day 1 occurred between 0.5 and 2.0 hours post-dose, median T _max For 1.0 hour after administration. Geometric mean (CV%) C _max And AUC (0-tau) values were 172ng/mL (49.3%) and 438ng h/mL (55.1%), respectively. Maximum plasma TQS-621 concentration on day 1 The degree of onset is between 1.0 and 4.0 hours after administration, median T _max For 1.5 hours after administration. Geometric mean (CV%) C _max And AUC (0-tau) values were 331ng/mL (40.3%) and 2270ng h/mL (35.7%), respectively. See fig. 49-50.

After multiple dosing, the day 7 plasma concentrations of TQS-168 and metabolite TQS-621 in all subjects were quantifiable prior to dosing, except that one subject became quantifiable at 0.5 hours post-dosing. The subjects remained quantifiable until between the final sampling time points of 16 hours post-dose to 48 hours post-dose.

Maximum plasma TQS-168 concentration on day 7 occurred between 0.5 and 1.5 hours post-dose, median T _max For 0.75 hours after administration. The concentration then drops, resulting in an average elimination half-life of 7.7 hours. Geometric mean (CV%) C _max And AUC (0-tau) values were 273ng/mL (105.8%) and 692ng h/mL (89.8%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.59 (88.1%) and 1.58 (51.6%), respectively. See fig. 49-50.

Maximum plasma TQS-621 concentration on day 7 occurred between 1.0 and 4.0 hours post-dose, median T _max For 1.75 hours after administration. The concentration then drops, resulting in an average elimination half-life of 10.2 hours. Geometric mean (CV%) C _max And AUC (0-tau) values were 340ng/mL (39.5%) and 3320ng h/mL (42.1%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.30 (38.7%) and 1.46 (30.8%), respectively. See fig. 49-50.

The protocol IPK summaries, subjects in cohort 1 were given protocol I in a fasted state and received 120mg TQS-168SDD 1 time daily for 7 consecutive days. Day 1 provided 172ng/mL (0.69. Mu.M) TQS-168C _max . Day 7 of continuous dosing provided a much larger TQS-168C of 273ng/mL (1.09. Mu.M) _max . The results are plotted in figures 40-41. A similar increase was observed in exposure measured by AUC (day 1 AUC (0-tau) =438 hr ng/mL, day 7 AUC (0-tau) =692hr ng/mL). Metabolite TQS-621PK profile: group 1 showed day 1 metabolite TQS-621C at 331ng/mL (1.24. Mu.M) _max And 340ng/mL (1.27. Mu.M) day 7C _max . The results are plotted in fig. 42-43. Metabolite exposure on day 1 was recorded as AUC (0-tau) =2270 hr ng/mL. Metabolite exposure of AUC (0-tau) =3220 hr ng/m is provided on day 7. See fig. 49-50.

TQS-168 protocol J PK profile: subjects in cohort 2 received a single 90mg TQS-168SDD in the fed state, orally 1 time a day for 7 consecutive days.

On day 1, after a single administration of 90mg of an oral suspension of TQS-168 spray-dried dispersion (SDD) powder, the plasma concentration of TQS-168 was quantifiable for all subjects starting from 0.5 hours post-administration and remained quantifiable until 16 to 24 hours post-administration. In all subjects, the concentration of TQS-621 on day 1 was also quantifiable starting from 0.5h post-dose and remained quantifiable until 24h post-dose.

Maximum plasma TQS-168 concentration on day 1 occurred between 1.5 and 4.0 hours post-dose, median T _max For 3.0 hours after administration. Geometric mean (CV%) C _max And AUC (0-tau) values of 47.4ng/mL (27.4%) and 223ng.h/mL (30.9%), respectively. A comparison with scheme I is illustrated in fig. 40A-B.

Maximum plasma TQS-621 concentration on day 1 occurred between 4.0 and 6.0 hours post-dose, median T _max For 4.0 hours after administration. Geometric mean (CV%) C _max And AUC (0-tau) values of 189ng/mL (40.1%) and 1400ng h/mL (42.6%), respectively. A comparison with scheme I is illustrated in fig. 42A-B.

On day 1, no single subject exceeded maximum allowable C _max Or a maximum allowable AUC (0-24) (based on AUC (0-tau), where tau=24 h) value. Maximum individual TQS-168C on day 1 _max 65.8ng/mL, which is C _max 21.6% of the exposure limit. The maximum TQS-168AUC (0-tau) alone was 299ng h/mL, which represents 10.9% of the exposure limit of AUC (0-24).

After multiple administrations of 90mg of the TQS-168 spray-dried dispersion (SDD) powder oral suspension to healthy subjects in the fed state for 7 days, the plasma concentration of TQS-168 in all subjects was quantifiable prior to administration, except that three subjects became quantifiable at 0.5 hours post-administration, and remained quantifiable until 16 to 36 hours post-administration. In all subjects, the concentration of TQS-621 was quantifiable at the pre-dosing time point and was still quantifiable until the final sampling time point of 48 hours post-dosing.

Maximum plasma TQS-168 concentration on day 7 occurred between 1.0 and 4.0 hours post-dose, median T _max For 3.0 hours after administration. The geometric mean elimination half-life was 4.46h. Geometric mean (CV%) C _max And AUC (0-tau) values were 48.8ng/mL (48.3%) and 227ng.h/mL (46.7%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.03 (37.5%) and 1.24 (22.8%), respectively. A comparison with scheme I is illustrated in fig. 41A-B.

Maximum plasma TQS-621 concentration on day 7 occurred between 4.0 and 6.0 hours post-dose, median T _max For 5.0 hours after administration. The concentration then decreases in a generally two-phase fashion, resulting in a geometric mean elimination half-life of 8.65 h. Geometric mean (CV%) C _max And AUC (0-tau) values of 218ng/mL (37.6%) and 2060ng.h/mL (54.8%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.15 (12.4%) and 1.47 (18.0%), respectively. A comparison with scheme I is illustrated in fig. 43A-B.

On day 7, no single subject exceeded the maximum allowable C of TQS-168 _max Value or maximum allowable AUC (0-24) (based on AUC (0-tau), where tau=24h value. Maximum individual TQS-168C on day 7 _max 94.1ng/mL of C _max 30.9% of the exposure limit. The maximum TQS-168AUC (0-tau) alone was 535ng.h/mL, which represents 19.5% of the exposure limit of AUC (0-24).

Scheme J PK summary: subjects in cohort 2 were administered regimen J in the fed state and received 1 dose per day of 90mg TQS-168SDD and provided a 1 st day TQS-168C of 47.4ng/mL (0.19. Mu.M) _max . On day 7 of continuous dosing of 90mg TQS-168SDD in the fed state, 48.8ng/mL (0.19. Mu.M) of TQS-168C was observed _max . The results are plotted in figures 40-41.

A similar profile was observed compared to the exposure profile(1 st day AUC (0-tau) =223 hr ng/mL, 7 th day AUC (0-tau) =227 hr ng/mL. With respect to TQS-621PK profile, group showed 189ng/mL (0.71 μm) of the 1 st day metabolite TQS-621C) _max And 218ng/mL (0.82. Mu.M) of day 7C _max . The results are plotted in fig. 42-43. Metabolite exposure was recorded for AUC (0-tau) =1400 hr ng/mL on day 1. Metabolite exposure of AUC (0-tau) =2060 hr ng/m was provided on day 7.

TQS-168 protocol K PK profile: subjects in cohort 3 received a single 300mg TQS-168SDD 1 time daily for 7 consecutive days in the fed state.

On day 1, after a single administration of 300mg of an oral suspension of TQS-168 spray-dried dispersion (SDD) powder, the plasma concentrations of TQS-168 for all subjects were quantifiable starting from 0.5 hours post-administration and remained quantifiable in all subjects until the final sampling point 24 hours post-administration.

Maximum plasma TQS-168 concentration on day 1 occurred between 1.5 and 4.0 hours post-dose, median T _max For 2.0 hours after administration. Geometric mean (CV%) C _max And AUC (0-tau) values were 229ng/mL (38.3%) and 630 ng h/mL (55.1), respectively. See fig. 51A-B for a comparison with the illustrations of schemes I and J.

Maximum plasma TQS-621 concentration on day 1 occurred between 4.0 and 10.0 hours post-dose, median T _max For 4.0 hours after administration. Geometric mean (CV%) C _max And AUC (0-tau) values of 1000ng/mL (24.6%) and 9730ng h/mL (39.9%), respectively. See fig. 53A-B for a comparison with the illustrations of schemes I and J.

After multiple dosing, the day 7 plasma concentrations of TQS-168 and metabolite TQS-621 were quantifiable in all subjects prior to dosing and remained quantifiable until 24 to 48 hours post-dosing. The concentration of TQS was also quantifiable in all subjects at the pre-dosing time point and remained quantifiable until the final sampling time point of 48 hours post-dosing.

Maximum plasma TQS-168 concentration on day 7 occurred between 0.50 and 4.0 hours post-dose, median T _max For 4.0 hours after administration. The concentration then decreased, resulting in an average value of 5.67 hoursElimination half-life. Geometric mean (CV%) C _max And AUC (0-tau) values of 400ng/mL (79.3%) and 2010ng h/mL (88.3%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.74 (54.6%) and 1.66 (29.5%), respectively. See fig. 52A-B for a comparison with the illustrations of schemes I and J.

Maximum plasma TQS-621 concentration on day 7 occurred between 4.0 and 6.0 hours post-dose, median T _max For 5.0 hours after administration. The concentration then drops, resulting in an average elimination half-life of 7.29 hours. Geometric mean (CV%) C _max And AUC (0-tau) values of 1300ng/mL (36.7%) and 1490 ng h/mL (61.2%), respectively. Based on C _max And AUC (0-tau), geometric mean (CV%) cumulative ratio was 1.30 (32.9%) and 1.53 (26.1%), respectively. See fig. 54A-B for a comparison with the illustrations of schemes I and J.

Scheme K PK summary: subjects in cohort 1 were dosed with regimen K and received 300mg TQS-168SDD 1 time daily for 7 consecutive days in a fed state. Day 1 provided 229ng/mL (0.91. Mu.M) of TQS-168C _max . Day 7 of continuous dosing provided a much larger TQS-168C of 400ng/mL (1.60. Mu.M) _max . The results are plotted in figures 40-41. Similar increases were observed in AUC-measured exposure (day 1 AUC (0-tau) =630 hr ng/mL, day 7 AUC (0-tau) =2010 hr ng/mL). Group 1 showed 1000ng/mL (3.76. Mu.M) of day 1 metabolite TQS-621C _max And 1300ng/mL (4.88. Mu.M) day 7C _max . The results are plotted in fig. 42-43. Metabolite exposure was recorded for AUC (0-tau) =9730 hr ng/mL on day 1. Metabolite exposure of AUC (0-tau) =149900 hr ng/mL is provided on day 7.

In summary, part 2 of the trial provided 7 consecutive 1-daily doses of TQS-168 in different doses of TQS-168SDD powder oral suspension to healthy male subjects in a fed or fasted state. The data show that the increase in dose corresponds to an increase in plasma concentrations of TQS-168 and TQS-168 metabolite TQS-621.

6. Equivalent and incorporated by reference

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

All references, issued patents and patent applications cited in the text of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method of reducing neuroinflammation and/or treating a neurodegenerative disease in a human subject, the method comprising:

the amount and/or dose of which provides an average peak concentration of TQS-168 in plasma of at least 750ng/mL (C _max )。

2. The method of claim 1, wherein TQS-168 or a salt thereof is administered in an amount that provides a mean plasma C of TQS-168 of at least 1000ng/mL after administration _max 。

3. The method of claim 2, wherein TQS-168 or a salt thereof is administered in an amount that provides a mean plasma C of TQS-168 of at least 1250ng/mL after administration _max 。

4. The method of claim 3, wherein TQS-168 or a salt thereof is administered in an amount that provides a mean plasma C of TQS-168 of at least 1500ng/mL after administration _max 。

5. The method of claim 4, wherein TQS-168 or a salt thereof is administered in an amount that provides a mean plasma of TQS-168 of at least 1750ng/mL after administrationC _max 。

6. The method of any one of claims 1-5, wherein TQS-168 or a salt thereof is administered in an amount that provides a TQS-168AUC of at least 3000ng hr/ml after administration _0-t 。

7. The method of claim 6, wherein the TQS-168 or salt thereof is administered in an amount that provides a TQS-168AUC of at least 4000 ng-hr/ml after administration _0-t 。

8. The method of claim 7, wherein the TQS-168 or salt thereof is administered in an amount that provides a TQS-168AUC of at least 5000 ng-hr/ml after administration _0-t 。

9. The method of claim 8, wherein the TQS-168 or salt thereof is administered in an amount that provides a TQS-168AUC of at least 5500ng hr/ml after administration _0-t 。

10. The method of claim 9, wherein the TQS-168 or salt thereof is administered in an amount that provides a TQS-168AUC of at least 6000ng hr/ml after administration _0-t 。

11. The method of claim 10, wherein the TQS-168 or salt thereof is administered in an amount that provides a TQS-168AUC of about 7000 ng-hr/ml after administration _0-t 。

12. The method of any one of claims 1-11, wherein plasma C to TQS-168 _max Time (T) _max ) No more than 2 hours.

13. The method of claim 12, wherein the T _max No more than 90 minutes.

14. The method of claim 13, wherein the T _max No more than 75 minutes.

15. Claim(s)The method of claim 14, wherein said T _max Is about 60 minutes.

16. A method of reducing neuroinflammation and/or treating a neurodegenerative disease in a human subject, the method comprising:

17. The method as claimed in claim 16, wherein TQS-168 or a salt thereof is administered in an amount that provides 200-2750ng/mL of plasma C of TQS-621 after administration _max 。

18. The method as claimed in claim 17, wherein TQS-168 or a salt thereof is administered in an amount that provides plasma C of TQS-621 of 300-2200ng/mL after administration _max 。

19. The method as claimed in claim 18, wherein TQS-168 or a salt thereof is administered in an amount that provides plasma C of TQS-621 of 400-1800ng/mL after administration _max 。

20. A method of reducing neuroinflammation and/or treating a neurodegenerative disease in a human subject, the method comprising:

the amounts thereof are provided after administration:

And

21. The method of any one of claims 1-20, wherein TQS-168 or a salt thereof is administered at a daily oral dosage of 200-800 mg.

22. The method as claimed in claim 21, wherein TQS-168 or a salt thereof is administered in a daily oral dosage of 300-700 mg.

23. The method as claimed in claim 22, wherein TQS-168 or a salt thereof is administered in a daily oral dosage of 400-600 mg.

24. The method as claimed in claim 23, wherein TQS-168 or a salt thereof is administered in a daily oral dosage of 400-500 mg.

25. The method of claim 24, wherein TQS-168 or a salt thereof is administered at a daily oral dosage of 400mg or 450 mg.

26. The method of any one of claims 1-25, wherein TQS-168 or a salt thereof is administered in a liquid suspension.

27. The method of any one of claims 1-25, wherein TQS-168 or a salt thereof is administered in a liquid solution.

28. The method of any one of claims 1-25, wherein TQS-168 or a salt thereof is administered in a solid dosage form.

29. The method of claim 28, wherein TQS-168 or a salt thereof is crystalline.

30. The method of claim 28, wherein TQS-168 or a salt thereof is amorphous.

31. The method as claimed in claim 30, wherein TQS-168 or a salt thereof is in the form of a spray dried dispersion.

32. The method as claimed in claim 30, wherein TQS-168 or a salt thereof is in the form of a hot melt extrudate.

33. The method of any one of claims 28-32, wherein the solid dosage form is a sachet.

34. The method of any one of claims 28-32, wherein the solid dosage form is a capsule.

35. The method of any one of claims 28-32, wherein the solid dosage form is a tablet.

36. The method of any one of claims 1-35, wherein the subject has a neurodegenerative disease selected from the group consisting of motor neuron disease, amyotrophic Lateral Sclerosis (ALS), alzheimer's disease, vascular dementia, frontotemporal lobar degeneration (frontotemporal lobar dementia), dementia with lewy bodies, parkinson's disease, huntington's disease, demyelinating disease, and Multiple Sclerosis (MS).

37. The method of claim 36, wherein the subject has a motor neuron disease.

38. The method of claim 37, wherein the subject has ALS.

39. The method of claim 36, wherein the subject has alzheimer's disease.

40. The method of any one of claims 1-35, wherein the subject is at least 40 years old and is free of a past diagnosed neurodegenerative disease.

41. The method of claim 40, wherein the subject is at least 60 years old.

42. The method of claim 41, wherein the subject is at least 65 years old.