DISORDERS COMPRISING DEPRESSION

FIELD OF THE INVENTION

The present invention, in at least some aspects, relates to compositions and methods of treatment for neurological disorders, and in particular to compositions containing an inventive molecule as described herein and methods of treatment using same.

BACKGROUND OF THE INVENTION

Depression is a mood disorder that causes a persistent feeling of sadness and loss of interest. Depression has a significant negative impact on the quality of life, including somatic and emotional symptoms. Depression affects how sufferers feel, think and behave and can lead to a variety of emotional and physical problems. Sufferers may have trouble doing normal day-to-day activities, and may also have suicidal ideation.

Depression should be distinguished from sadness or “the blues” as described below, in particular due to the nature of symptoms, their severity or the length of time that they are present.

Various drugs and non-drug therapies are available to treat depression, as described in greater detail below.

BRIEF SUMMARY OF THE INVENTION

The background art provides some therapies that treat depression, but these therapies are not always successful, plus they frequently have deleterious side effects. The present invention, in at least some embodiments, provides compositions comprising inventive molecules as described herein and methods of treatment with same, for treating depression and its subtypes. By“inventive molecule” it is meant a molecule which, as described herein, has been shown to have at least one effect in vitro and/or in vivo, that indicates that it would be useful in the compositions and methods of treatment described herein. Non-limiting examples of such subtypes for which the inventive molecules, compositions, combinations and methods of treatment may be useful include but are not limited to disruptive mood dysregulation disorder (in children up to the age of 18), major depressive disorder (including major depressive episode), persistent depressive disorder (dysthymia), premenstrual dysphoric disorder, substance/medication-induced depressive disorder, depressive disorder due to another medical condition, other specified depressive disorder, unspecified depressive disorder; depression with another feature, including but not limited to anxious distress; mixed features; melancholic features; atypical features (depression that includes the ability to temporarily be cheered by happy events, increased appetite, excessive need for sleep, sensitivity to rejection, and a heavy feeling in the arms or legs); psychotic features; catatonia; seasonal patter depression (also referred to as seasonal affective disorder); and treatment-resistant depression (TRD), which is depression that fails to improve after treatment with a drug or non-drug therapy.

Preferably the treatment comprises an increase of energy metabolism in the nervous system.

Optionally treating comprises one or more of curing, managing, reversing, attenuating, alleviating, minimizing, suppressing, managing, or halting the deleterious effects of the above-described diseases.

Treatment as nrevention of disease and/or symntom onset

According to at least some embodiments, treating also includes at least reducing the rate of onset of symptoms and/or etiology of the disease, for example optionally as determined by measurement of one or more diagnostic markers. Such diagnostic markers would be selected according to the particular neurological disorder.

With regard to the inventive molecules as described herein, without wishing to be limited by a single hypothesis, it is possible that for each disease described herein, prevention or delay of full onset or even symptomatic presentation of these diseases in subjects without symptoms of the disease, or with only minor initial symptoms would be possible by detecting the disease in the subject before full onset or symptomatic presentation, and then administering the inventive molecules as described herein to the subject according to a suitable dosing regimen.

Optionally, managing comprises reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, or reducing the severity of such episodes or a combination thereof.

Individuals at risk of developing a disease can be identified based on various approaches either before disease development or at very early stages in which disease markers can be identified. The identification of individuals at risk as well as diagnosis of early disease can rely on various approaches including genomics, proteomics, metabolomics, lipidomics, glycomics, secretomics, serologic approaches and also optionally tests involving impairment of information processing (see

doi:10.1016/j.psychres.2006.09.014). Family history can also provide information either in combination with one of the previously described approaches or as a standalone approach. Furthermore, over the past decade microbiome composition is becoming recognized as an important factor in health and disease. The advent of new technologies for interrogating complex microbial communities and in the analysis of microbiome and metagenome will provide another approach for identification of individuals at risk of developing a disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the extracellular levels of lactate in astrocytes after treatment with inventive molecules from the Prestwick library;

Figure 2 shows the intracellular levels of glycogen in astrocytes after treatment with lead hits (molecules) from the Prestwick library;

Figure 3 shows the results for the MTT Assay in astrocytes after treatment with lead hits (molecules) from the Prestwick library;

Figure 4 shows mitochondrial activity in astrocytes after treatment with lead hits (molecules) from the Prestwick library;

Figure 5 A shows the extracellular levels of lactate in astrocytes after treatment with 18 hits (molecules) from the CDC54K library; Figure 5B shows levels of intracellular glycogen in astrocytes measured at 3h after stimulation with 18 hits (molecules) from the CDC54K library;

Figure 6 shows the results of weight monitored during a 14-day period after acute administration of the drug (100 mg/kg when not indicated otherwise) in C57B1/6 female mice; n=6;

Figure 7 shows the weight of male and female mice during a 28-day period chronic treatment with GP-01, GP-02, GP-04, GP-05, GP-07 and GP-07 at lOmg/kg, followed by a 14-day recovery period; n>10;

Figure 8 shows the results of anxiety testing: at the end of the chronic treatment, mice were tested for anxiety in an EPM (elevated plus maze). Total distance, frequency of entry and duration in the open arms were measured using Ethovision automatic scoring; n>10;

Figure 9. (A) Localization of the lactate probe implanted in mouse brain. (B) Example of intracerebral lactate probe recording after administration of Vehicle, followed 3h later by GP-07. Area under curve (AUC) were used to calculate treatment effect (Treatment AUC / Veh AUC). (C-D) AUC ratio after administration of Vehicle followed by Vehicle or tested drug at lOmg/kg or lOOmg/kg; n=4-6;

Figure 10 shows glycogen levels in PFC (prefrontal cortex) at 3H after administration of the drug per os at 1, 10 or 100 mg/kg; n>6;

Figures 11A and 11B show the results after GP-04, GP-05, GP-07, GP-P1 and GP-R1 concentrations were measured in tnicrodialysed samples of prefrontal cortex (left panels) and in the plasma (right panels) at 30 min intervals before and after compound’s administration (100 mg/kg), n=5; and

Figure 12 shows the effect of Vehicle (Veh) or GP-07 administration (100 mg/kg, per os) in the grip strength test (A), open field test (B) and forced swim test (C). (D) Forced swim test was also performed after administration of Veh (intraperitoneal; i.p.), or the antidepressants fluoxetine (20 mg/kg, i.p.) or desipramine (20 mg/kg, i.p.); n=11-12.

DETAILED DESCRIPTION OF THE INVENTION The present invention, in at least some embodiments, relates to compositions and methods of treatment comprising same for treatment of a neurological disease, wherein the composition comprises an inventive molecule as described herein. The neurological disease is specifically depression and its subtypes, as described herein.

According to at least some embodiments, there is provided a molecule selected from the group consisting of Families A, C, E, F(7), F(6), G, I, M, PQRV and Y; wherein Family G comprises:

wherein for Family G, R is H, ethyl or methyl; each of R1-R4 is independently H, halogen; alkyl; or alkoxy; wherein Family A comprises:

wherein R1 is H or benzyl unsubstituted or substituted with nitrogen, R2 is H or alkyl, with the proviso that if R2 is H, R1 is not

and with the further proviso that the structure is not that of catalog ID numbers F228-0365, F228-0351, F228-0856 or F228-0541 of Appendix I; wherein Family C comprises:

wherein R1 and R2 are each H or methoxy; each of R3, R4 and R5 are independently alkyl, preferably ethyl, or H; preferably only one of R3-R5 is alkyl, preferably ethyl; more preferably R4 is alkyl, most preferably ethyl; with the proviso that the structure is not that of catalog ID numbers T5464782, F1462-0491, T5463709 or 4052-4279 of Appendix I; wherein Family E comprises: wherein R is pentyl, benzyl, alkyl benzyl or R1; R2 is alkyl, cyclopentyl or cyclobutane; wherein R1 is

or

with the proviso that the structure is not that of catalog ID numbers L287- 1577, or L287-1758 of Appendix I; wherein Family F(7) comprises:

wherein R is alkyl, halogen, or alkoxy; each of R1-R5 is independently H, alkyl, or alkoxy; with the proviso that the structure is not that of catalog ID numbers K404- 0672, K404-0183, K404-0796, F0524-0511, F0524-0507, F0522-0533, F0524-0488, K404-0400, T0507-8442, K404-0906, K404-0842, K404-0852, K404-0914, K404- 0915, K404-0828, K404-0863 or K404-0277 of Appendix I; wherein Family F(6) comprises:

wherein for Family F(6) R is H, halogen; alkyl or alkoxy,;

R1, R2, R3 and R4 are each independently H, alkyl, or alkoxy, with the proviso that if R1 is alkoxy, R is not alkyl and is preferably halogen or alkoxy; with the proviso that the structure is not that of catalog ID numbers K404- 0672, K404-0183, K404-0796, F0524-0511, F0524-0507, F0522-0533, F0524-0488, K404-0400, T0507-8442, K404-0906, K404-0842, K404-0852, K404-0914, K404- 0915, K404-0828, K404-0863 or K404-0277 of Appendix I; wherein Family I comprises:

wherein for Family I, R is

or

wherein for Family I, R1 is cyclopentadiene or benzene, unsubstituted or substituted with S, O or N; R2 is H or a carbonyl; wherein for Family I, R1 is selected from the group consisting of (alternative atoms at each position are indicated in brackets)

wherein each of R3, R4 and R5 is independently H, alkyl (preferably methyl); and

with the proviso that the structure is not that of catalog ID numbers T636- 2007, T636-1250, T636-2391, T636-0054, T636-0027, T636-1243, T636-2360, T636- 0085, T636-0181, D278-0514, T636-1715, T636-2144, T636-1601, or T636-0973 of Appendix I; wherein Family M comprises:

wherein R is H or alkyl; if alkyl, R is methyl or ethyl, unsubstituted or substituted with halogen (preferably F or Cl, more preferably F; preferably up to three halogens), more preferably ethyl; with the proviso that the structure is not that of catalog ID number T5436375 of Appendix I; wherein Family PQRV comprises (brackets indicate that the atom at that position can be C or N):

wherein R1 is benzyl,

or

wherein R2 is alkyl, forms a heterocyclic hexyl moiety with the nitrogen to which it is attached, or is absent; wherein each of R3, R4, R5 and R6 are halogen, H, alkyl, benzyl or alkyl benzyl (unsubstituted or substituted with nitrogen), cyclopentadiene or alky cyclopentadiene (substituted or unsubstituted with S or N) or carbamoyl (optionally alkyated with cyclopropane); R4 and R5 together can be cyclopentadiene, substituted with S and/or N, or unsubstituted, and optionally alkylated; wherein each of R7-R11 is independently halogen, alkyl, or methoxy, and can be the same or different; or is pyrrolidine, optionally formyl pyrrolidine, in which case preferably R7 is pyrrolidine; with the proviso that the structure is not that of catalog ID numbers P025- 0462, P025-0080, P025-0168, T5581430, F0376-0203, or T5246417 of Appendix I; with the proviso that if R1 is:

R2 forms a heterocyclic hexyl moiety with the nitrogen to which it is attached; with the proviso that if R1 is

R7 is pyrrolidine, and [C,N] is C, then R4 is not cyclopentadiene or alky cyclopentadiene substituted with both S and N; with the proviso that if R1 is

[C,N] is N and R3-R6 are H, then none of R7-R11 is methyl, methoxy or halogen; with the proviso that if R1 is

any of R7-R11 is chlorine, and [C,N] is N, then R5 isn’t carbamoyl; with the proviso that if R1 is

[C,N] is C, any of R7-R11 is halogen or methoxy, and R4 and R5 together form cyclopentadiene, substituted with S and/or N, then the cyclopentadiene moiety is not alkylated nor does it feature a benzyl group; wherein Family Y comprises: wherein R is alkyl, S or halogen, preferably S or halogen; if halogen, preferably F; if S, preferably methylthio or ethylthio, most preferably methylthio; with the proviso that the structure is not that of catalog ID numbers L995-0405 or L995-0386 of Appendix I.

Optionally for the above molecule, for Family G, R is methyl or ethyl; for R1- R4, if halogen, one or more of R1-R4 is F or Cl; if alkyl, one or more is ethyl or methyl; if alkoxy, one or more ethoxy or methoxy; wherein for Family A, R1 is nitrogen substituted benzyl or H, and R2 is H; wherein for Family C, R1 and R2 are each methoxy; each of R3-R5, if alkyl, is ethyl; wherein for Family E, R is pentyl or R1; if R2 is alkyl, R2 is methyl or ethyl; wherein for Family F(6) if R is halogen, R is F or Cl; if R is alkyl, R is methyl or ethyl; if R is alkoxy, R is methoxy or ethoxy; if any of R1-R5 is alkyl, then it is methyl; if any of R1-R5 is alkoxy, then it is methoxy or ethoxy; with the proviso that if R1 is alkoxy, R is not alkyl and is preferably halogen or alkoxy; wherein for Family F(7), if R is alkyl, R is ethyl or methyl; if R is halogen, R is Cl or F; if R is alkoxy, R is methoxy or ethoxy; if any of R1-R5 is alkyl, then it is methyl; if any of R1-R5 is alkoxy, then it is methoxy or ethoxy; wherein for Family M, if R is alkyl, R is methyl or ethyl, unsubstituted or substituted with halogen; wherein for Family Y, if R is alkyl, R is ethyl or methyl; if R is S, R is methylthio or ethylthio; if R is halogen, R is F;

Optionally for the above molecule: wherein for Family G, each of R1-R4, if alkyl, is methyl; if alkoxy, is methoxy; wherein for Family C, only one of R3-R5 is ethyl and the remaining are H; wherein for Family M, if R is alkyl, R is ethyl; wherein for Family Y, R is S or halogen;

Optionally for the above molecule: wherein for Family G, at least two of R1-R4 are halogen, at least two are alkyl, one is alkoxy and one is alkyl, one is alkyl and one is H, one is halogen and one is H, or one is alkoxy and one is H; wherein for Family C, R4 is ethyl, and R3 and R5 are H; wherein for Family M, if R is ethyl, R is substituted with F or Cl, more preferably F; preferably up to three halogens; wherein for Family Y, if R is S, R is methylthio.

Optionally for the above molecule: for Family G, the molecule is selected from the group consisting of G1 -G6 of Appendix I (molecules having catalog numbers L924- 1031; L924-1088; L924-0830; L924-0760; L924-0884; or L924-0988); wherein for Family A, the molecule is selected from the group consisting of A1-A3 of Appendix I (molecules having catalog numbers F228-0422, F228- 0350 or F228-0534); wherein for Family C, the molecule is selected from the group consisting of C1-C3 of Appendix I (molecules having catalog numbers T5463586, 4052- 4304 or T5463658); wherein for Family E, the molecule is selected from the group consisting of E1-E4 of Appendix I (molecules having catalog numbers L287-0468, L287- 1641, L287-1221 and L287-0220); wherein for Family F(6), the molecule is selected from the group consisting of F4-F6, F8, F9, F13 of Appendix I (molecules having catalog numbers K404- 0800, K404-0673, F0524-0338, K404-0685, K404-0697, and K404-0394); wherein for Family F(7), the molecule is selected from the group consisting of F1-F3, F7, F10-F12 of Appendix I (molecules having catalog numbers K404- 0834, K404-0838, K404-0885, K404-0910, K404-0855, K404-0860, and F0524-0611); wherein for Family I, the molecule is selected from the group consisting of Ill5 and 17 of Appendix I (molecules having catalog numbers T636-1937, T636- 1114, T636-2387, T636-0134, T636-1210 and T636-2425); wherein for Family M, the molecule is selected from the group consisting of Ml and M2 of Appendix I (molecules having catalog numbers T5599014 and T5653029); wherein for Family PQRV, the molecule is selected from the group consisting of PI, Q1-Q3, R1, VI and V2 of Appendix I (molecules having catalog numbers P025-0159, T5644989, T5599698, T5618591, T5580243, T6937001 and T5511047); and wherein for Family Y, the molecule is selected from the group consisting of Y1 and Y2 of Appendix I (molecules having catalog numbers L995-0125 and L995-0058).

According to at least some embodiments, there is provided a pharmaceutical composition comprising the molecule as described above. The above molecule or pharmaceutical composition may optionally be used as a medicament.

The above molecule or pharmaceutical composition may be used for treatment of a neurological disease, wherein the neurological disease includes depression or a subtype thereof.

Optionally there is provided a method for treating a mammal in need of treatment thereof, comprising administering to the mammal an inventive molecule or a pharmaceutical composition as described above, for treatment of a neurological disease, wherein said neurological disease includes depression or a subtype thereof.

According to at least some embodiments, there is provided an inventive molecule or a pharmaceutical composition comprising same, for treatment of a neurological disease, wherein said neurological disease includes depression or a subtype thereof, wherein said molecule is selected from the group consisting of:

an inventive molecule selected from the group consisting of Families A, C, E, F(7), F(6), G, I, M, PQRV and Y;

wherein a molecule of Family A has the structure:

wherein R1 is H or benzyl unsubstituted or substituted with nitrogen, R2 is H or alkyl, preferably H, with the proviso that if R2 is H, R1 is not

and with the further proviso that the structure is not that of catalog ID numbers F228-0365, F228-0351, F228-0856 or F228-0541 of Appendix I; wherein a molecule of Family C has the structure:

wherein R1 and R2 are each H or methoxy, preferably methoxy; each of R3, R4 and R5 are independently alkyl, preferably ethyl, or H; preferably only one of R3- R5 is alkyl, preferably ethyl, and the remainder are H; more preferably R4 is alkyl, most preferably ethyl, and R3 and R5 are H; with the proviso that the structure is not that of catalog ID numbers T5464782, F1462-0491, T5463709 or 4052-4279 of Appendix I; wherein a molecule of Family E has the structure:

wherein R is pentyl, benzyl, alkyl benzyl or R1, preferably pentyl or R1; R2 is alkyl, cyclopentyl or cyclobutane; if R2 is alkyl, is preferably methyl or ethyl;

wherein R1 is

with the proviso that the structure is not that of catalog ID numbers L287- 1577, or L287-1758 of Appendix I; wherein a Family I has the structure:

wherein for Family I, R is

or

wherein for Family I, R1 is cyclopentadiene or benzene, unsubstituted or substituted with S, O or N; R2 is H or a carbonyl; wherein for Family I, R1 is selected from the group consisting of (alternative atoms at each position are indicated in brackets)

wherein each of R3, R4 and R5 is independently H, alkyl (preferably methyl); and

with the proviso that the structure is not that of catalog ID numbers T636- 2007, T636-1250, T636-2391, T636-0054, T636-0027, T636-1243, T636-2360, T636- 0085, T636-0181, D278-0514, T636-1715, T636-2144, T636-1601, or T636-0973 of Appendix I; wherein a molecule of Family F(6) has the structure:

wherein for Family F(6) R is H, halogen, preferably F or Cl; alkyl, preferably methyl or ethyl; alkoxy, preferably methoxy or ethoxy;

R1 , R2, R3 and R4 are each independently H, alkyl, preferably methyl or ethyl; alkoxy, preferably methoxy or ethoxy; with the proviso that if R1 is alkoxy, R is not alkyl and is preferably halogen or alkoxy; with the proviso that the structure is not that of catalog ID numbers K404- 0672, K404-0183, K404-0796, F0524-0511, F0524-0507, F0522-0533, F0524-0488, K404-0400, T0507-8442, K404-0906, K404-0842, K404-0852, K404-0914, K404- 0915, K404-0828, K404-0863 or K404-0277 of Appendix I; wherein a molecule of Family F(7) has the structure: wherein R is alkyl, preferably ethyl or methyl, halogen, preferably Cl or F, H; alkoxy, preferably methoxy or ethoxy;

Each of R1-R5 is independently H, alkyl, preferably methyl; alkoxy, preferably methoxy or ethoxy; with the proviso that the structure is not that of catalog ID numbers K404- 0672, K404-0183, K404-0796, F0524-0511, F0524-0507, F0522-0533, F0524-0488, K404-0400, T0507-8442, K404-0906, K404-0842, K404-0852, K404-0914, K404- 0915, K404-0828, K404-0863 or K404-0277 of Appendix I; wherein a molecule of Family M has the structure:

wherein R is H or alkyl; if alkyl, R is methyl or ethyl, unsubstituted or substituted with halogen (preferably F or Cl, more preferably F; preferably up to three halogens), more preferably ethyl; with the proviso that the structure is not that of catalog ID number T5436375 of Appendix I; wherein the Family PQRV has the structure (brackets indicate that the atom at that position can be C or N):

wherein R1 is benzyl,

or

wherein R2 is alkyl, forms a heterocyclic hexyl moiety with the nitrogen to which it is attached, or is absent; wherein each ofR3, R4, R5 and R6 are halogen, H, alkyl, benzyl or alkyl benzyl (unsubstituted or substituted with nitrogen), cyclopentadiene or alky cyclopentadiene (substituted or unsubstituted with S or N) or carbamoyl (optionally alkyated with cyclopropane); R4 and R5 together can be cyclopentadiene, substituted with S and/or N, or unsubstituted, and optionally alkylated; wherein each of R7-R11 is independently halogen, alkyl, or methoxy, and can be the same or different; or is pyrrolidine, optionally formyl pyrrolidine, in which case preferably R7 is pyrrolidine; with the proviso that the structure is not that of catalog ID numbers P025- 0462, P025-0080, P025-0168, T5581430, F0376-0203, or T5246417 of Appendix I; wherein a molecule of Family Y has the structure: wherein R is alkyl, S or halogen, preferably S or halogen; if halogen, preferably F; if S, preferably methylthio or ethylthio, most preferably methylthio; with the proviso that the structure is not that of catalog ID number L995-0405 or L995-0386 of Appendix I; an inventive molecule selected from the group consisting of a molecule given in Appendix I, wherein said molecule is selected from the group consisting of catalogID numbers: T0502-5560; T0508-5190, T202-1455, T202-0973, K851- 0113, T5630309, T5672380, T5967389, T5884038, T5231424, T0517-8250, T0511-9200 and T5627721; a molecule as shown in Table 1 herein; and a molecule given in Appendix II, wherein said molecule is selected from the group consisting of catalogID numbers: T6010789, T5993799, T5813085, T6947848, T0517-4117, T5729557, T5705522, Z606-8352, LI 15-0403, T5712071, T5790476, T5788339, G433-0293, T5719257, T5798761, T5821723, T5787526, T5827594, K405-2595, T5274959, M950-1515, T5450239, G508-0015, T5707230, T5710343, 887-0183, T5453923, T0505- 4087, T5673322, T5800607, G869-0071, F2794-0128, T0500-6629,

T5832764, M508-0370, T0515-1783, T5393500, T5672380, M381-0730, Z606-8287, G855-0143, Z076-0028, T5311200, E944-0182, L302-0069, T5770640, G869-0064, T5753165, G855-0183, T5329723, T533260, L932- 0267, L302-0181, T5444083, T6125251, T5694329, T0517-2783, T5788545, T5586091, T5967389, T5783794, T5494352, T5477696, P621-1364, Y031- 0361, T5318833, Z606-8351, T5606387, T0516-6894, T5691896, Z606-8298, F5285-0069, T993-1787, Z606-5341, F3394-1364, Y030-2832, T5400234, T5389517, Z603-8037, T0513-0213, and T636-2387; or a molecule that is related to a molecular structure in Appendices I or II, and has a suitable metabolic activity in at least one assay as described herein.

The molecule, or pharmaceutical composition comprising same, as described above, optionally wherein for family PQRV, wherein R2 is alkyl, forms a heterocyclic hexyl moiety with the nitrogen to which it is attached, or is absent; wherein each of R3, R4, R5 and R6 are halogen, H, alkyl, benzyl or alkyl benzyl (unsubstituted or substituted with nitrogen), cyclopentadiene or alky cyclopentadiene (substituted or unsubstituted with S or N) or carbamoyl (optionally alkyated with cyclopropane); R4 and R5 together can be cyclopentadiene, substituted with S and/or N, or unsubstituted, and optionally alkylated; wherein each of R7-R11 is independently halogen, alkyl, or methoxy, and can be the same or different; or is pyrrolidine, optionally formyl pyrrolidine, in which case preferably R7 is pyrrolidine; with the proviso that the structure is not that of catalog ID numbers P025- 0462, P025-0080, P025-0168, T5581430, F0376-0203, or T5246417 of Appendix I; with the proviso that if R1 is:

R2 forms a heterocyclic hexyl moiety with the nitrogen to which it is attached; with the proviso that if R1 is

R7 is pyrrolidine, and [C,N] is C, then R4 is not cyclopentadiene or alky cyclopentadiene substituted with both S and N; with the proviso that if R1 is

[C,N] is N and R3-R6 are H, then none of R7-R11 is methyl, methoxy or halogen; with the proviso that if R1 is

any of R7-R11 is chlorine, and [C,N] is N, then R5 isn’t carbamoyl; with the proviso that if R1 is

[C,N] is C, any of R7-R11 is halogen or methoxy, and R4 and R5 together form cyclopentadiene, substituted with S and/or N, then the cyclopentadiene moiety is not alkylated nor does it feature a benzyl group; wherein for Family I, R6 is absent.

The molecule, or pharmaceutical composition comprising same, as described above, optionally, for Family G, R is methyl or ethyl; for R1-R4, if halogen, one or more of R1-R4 is F or Cl; if alkyl, one or more is ethyl or methyl; if alkoxy, one or more ethoxy or methoxy; wherein for Family A, R1 is nitrogen substituted benzyl or H, and R2 is H; wherein for Family C, R1 and R2 are each methoxy; each of R3-R5, if alkyl, is ethyl; wherein for Family E, R is pentyl or R1; if R2 is alkyl, R2 is methyl or ethyl; wherein for Family F(6) if R is halogen, R is F or Cl; if R is alkyl, R is methyl or ethyl; if R is alkoxy, R is methoxy or ethoxy; if any of R1-R5 is alkyl, then it is methyl; if any of R1-R5 is alkoxy, then it is methoxy or ethoxy; with the proviso that if R1 is alkoxy, R is not alkyl and is preferably halogen or alkoxy; wherein for Family F(7), if R is alkyl, R is ethyl or methyl; if R is halogen, R is Cl or F; if R is alkoxy, R is methoxy or ethoxy; if any of R1-R5 is alkyl, then it is methyl; if any of R1-R5 is alkoxy, then it is methoxy or ethoxy; wherein for Family M, if R is alkyl, R is methyl or ethyl, unsubstituted or substituted with halogen; wherein for Family Y, if R is alkyl, R is ethyl or methyl; if R is S, R is methylthio or ethylthio; if R is halogen, R is F;

The molecule, or pharmaceutical composition comprising same, as described above, optionally, for Family G, each of R1-R4, if alkyl, is methyl; if alkoxy, is methoxy; wherein for Family C, only one of R3-R5 is ethyl and the remaining are H; wherein for Family M, if R is alkyl, R is ethyl; wherein for Family Y, R is S or halogen;

The molecule, or pharmaceutical composition comprising same, as described above, optionally, for Family G, at least two of R1-R4 are halogen, at least two are alkyl, one is alkoxy and one is alkyl, one is alkyl and one is H, one is halogen and one is H, or one is alkoxy and one is H; wherein for Family C, R4 is ethyl, and R3 and R5 are H; wherein for Family M, if R is ethyl, R is substituted with F or Cl, more preferably F; preferably up to three halogens; wherein for Family Y, if R is S, R is methylthio.

The molecule, or pharmaceutical composition comprising same, as described above, optionally, for Family G, the molecule is selected from the group consisting of G1-G6 of Appendix I (molecules having catalog numbers L924- 1031; L924-1088; L924-0830; L924-0760; L924-0884; or L924-0988); wherein for Family A, the molecule is selected from the group consisting of A1-A3 of Appendix I (molecules having catalog numbers F228-0422, F228- 0350 or F228-0534); wherein for Family C, the molecule is selected from the group consisting of C1-C3 of Appendix I (molecules having catalog numbers T5463586, 4052- 4304 or T5463658); wherein for Family E, the molecule is selected from the group consisting of E1-E4 of Appendix I (molecules having catalog numbers L287-0468, L287- 1641, L287-1221 and L287-0220); wherein for Family F(6), the molecule is selected from the group consisting of F4-F6, F8, F9, F13 of Appendix I (molecules having catalog numbers K404- 0800, K404-0673, F0524-0338, K404-0685, K404-0697, and K404-0394); wherein for Family F(7), the molecule is selected from the group consisting of F1-F3, F7, F10-F12 of Appendix I (molecules having catalog numbers K404- 0834, K404-0838, K404-0885, K404-0910, K404-0855, K404-0860, and F0524-0611); wherein for Family I, the molecule is selected from the group consisting of Ill5 and I7 of Appendix I (molecules having catalog numbers T636-1937, T636- 1114, T636-2387, T636-0134, T636-1210 and T636-2425); wherein for Family M, the molecule is selected from the group consisting of Ml and M2 of Appendix I (molecules having catalog numbers T5599014 and T5653029); wherein for Family PQRV, the molecule is selected from the group consisting of P1, Q1-Q3, R1, V1 and V2 of Appendix I (molecules having catalog numbers P025-0159, T5644989, T5599698, T5618591, T5580243, T6937001 and T5511047); and wherein for Family Y, the molecule is selected from the group consisting of Y1 and Y2 of Appendix I (molecules having catalog numbers L995-0125 and L995-0058).

According to at least some embodiments there is provided a method for treating a mammal in need of treatment thereof, comprising administering to the mammal an inventive molecule, or a pharmaceutical composition, as described above, for treatment of a neurological disease, wherein said neurological disease includes depression or a subtype thereof;

The molecule, pharmaceutical composition or method as described above, wherein said subtype includes one or more of disruptive mood dysregulation disorder (in children up to the age of 18), major depressive disorder (including major depressive episode), persistent depressive disorder (dysthymia), premenstrual dysphoric disorder, substance/medication-induced depressive disorder, depressive disorder due to another medical condition, other specified depressive disorder, unspecified depressive disorder; depression with another feature, including but not limited to anxious distress; mixed features; melancholic features; atypical features (depression that includes the ability to temporarily be cheered by happy events, increased appetite, excessive need for sleep, sensitivity to rejection, and a heavy feeling in the arms or legs); psychotic features; catatonia; seasonal pattern depression (also referred to as seasonal affective disorder); or treatment-resistant depression

(TRD).

The molecule, pharmaceutical composition or method as described above, may be used or performed for delaying disease onset in individuals at risk for disease development according to one or more predictive markers.

The molecule, pharmaceutical composition or method as described above, further comprising administering a drug selected from the group consisting of tricydic antidepressant compounds (TCAs), selective serotonin re-uptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs), serotonin-norepinephrine reuptake inhibitors (SNRIs) and atypical antidepressants. The molecule, pharmaceutical composition or method as described above, wherein said TCA is selected from the group consisting of imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine and protriptyline.

The molecule, pharmaceutical composition or method as described above, wherein said SSRI is selected from the group consisting of fluoxetine, paroxetine, sertraline, citalopram, escitalopram, vilazodone and fiuvoxamine.

The molecule, pharmaceutical composition or method as described above, wherein said MAOl is selected from the group consisting of tranylcypromine, phenelzine, selegiline and isocarboxazid.

The molecule, pharmaceutical composition or method as described above, wherein said SNRI is selected from the group consisting of duloxetine, venlafaxine, desvenlafaxine and levomilnacipran.

The molecule, pharmaceutical composition or method as described above, wherein said atypical antidepressant is selected from the group consisting of bupropion, mirtazapine, nefazodone, trazodone and vortioxetine.

The molecule, pharmaceutical composition or method as described above, further comprising administering a non-drug treatment selected from the group consisting of brain stimulation therapy, light therapy and counseling.

The molecule, pharmaceutical composition or method as described above, wherein said brain stimulation therapy is selected from the group consisting of electroconvulsive therapy (ECT), repetitive trans cranial magnetic stimulation (rTMS) and vagus nerve stimulation (VNS).

The molecule, pharmaceutical composition or method as described above, wherein the molecule is in the Family PQRV, with the proviso that the molecule does not include one or more of: Thieno[3,2-c]pyridine-2-sulfonamide, 5-acety 1-4, 5,6,7- tetrahydro-N-(phenylmethyl)-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl- 4,5,6,7-tetrahydro-N-|(3-methoxyphenyl)methyl|-; Thieno[3,2-c]pyridine-2- sulfonamide, 5-(cyclopropylcarbonyl)-4,5,6,7-tetTahydro-N-[3-(methylthio) phenyl]-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4, 5,6,7- tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)- 4,5,6,7-tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-(cyclopropylcarbonyl)- N-(3-fluoro-4-methylphenyl)-4,5,6,7-tetrahydro-; Thieno [ 3 ,2-c|pyridine-2- sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-; Thieno[3,2- c]pyridine-2-sulfanamide, 5-acetyl-N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-;

Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4, 5,6,7- tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-(cyclopropylcarbonyl)-4, 5,6,7- tetrahy dro-N - [3 -(methy lthio)phenyl] Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl- N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5- (cyclopropylcarbonyl)-N-(3-fluoro-4-methylphenyl)-4,5,6,7-te trahydro-.

Optionally the molecule, pharmaceutical composition or method provides a treatment that comprises an increase of energy metabolism in the nervous system.

It is understood that molecules shown in Appendix I that are toxic or inactive in one or more assays, for example as shown by the test results given herein, are not inventive molecules as described herein. However it is possible that even such molecules could be active if given at lower amounts (for toxic molecules) or at higher amounts or a different form (for molecules that are inactive in one or more assays).

The present invention also provides different forms, including variations and derivatives, of the above compounds, including tautomers, resolved enantiomers, diastereomers, solvates, metabolites, salts and pharmaceutically acceptable prodrugs thereof.

The molecule, pharmaceutical composition or method as described above, wherein the molecule is in the Family PQRV, with the proviso that the molecule does not include one or more of: Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-4, 5,6,7- tetrahydro-N-(phenylmethyl)-; Thieno[3 ,2-c] pyridine-2-sulfonamide, 5-acetyl- 4,5,6,7-tetrahydro-N-[(3-methoxyphenyl)methyl]-; Thieno[3,2-c]pyridine-2- sulfonamide, 5-(cyclopropylcarbonyl)-4,5,6,7-tetrahydro-N-[3-(methylthio) phenyl]-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4, 5,6,7- tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)- 4,5,6,7-tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-(cyclopropylcarbonyl)- N-(3-fluoro-4-methylphenyl)-4,5,6,7-tetrahydro-; Thieno [3 ,2-c]pyridine-2- sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-; Thieno[3,2- c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-;

Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl-N-(2,5-dimethylphenyl)-4, 5,6,7- tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-(cyclopropylcarbonyl)-4, 5,6,7- tetrahydro-N-[3-(methyl1hio)phenyl]-; Thieno[3,2-c]pyridine-2-sulfonamide, 5-acetyl- N-(2,5-dimethylphenyl)-4,5,6,7-tetrahydro-; Thieno[3,2-c]pyridine-2-sulfonamide, 5- (cyclopropylcarbonyl)-N-(3-fluoro-4-methylphenyl)-4,5,6,7-te trahydro-.

Optionally the molecule, pharmaceutical composition or method provides a treatment that comprises an increase of energy metabolism in the nervous system.

It is understood that molecules shown in Appendix I that are toxic or inactive in one or more assays, for example as shown by the test results given herein, are not inventive molecules as described herein. However it is possible that even such molecules could be active if given at lower amounts (for toxic molecules) or at higher amounts or a different form (for molecules that are inactive in one or more assays).

The present invention also provides different forms, including variations and derivatives, of the above compounds, including tautomers, resolved enantiomers, diastereomers, solvates, metabolites, salts and pharmaceutically acceptable prodrugs thereof.

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

As used herein, if a plurality of serial integral values is given, then the series is assumed to include all integral values in between each integral value.

The terms "individual", "host", "subject", and "patient" are used interchangeably herein, and refer any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in the following subsections.

METHODS OF TREATMENT

As mentioned hereinabove the inventive molecules described herein can be used to treat a neurological disorder as described herein.

Thus, according to an additional aspect of the present invention there is provided a method of treating a neurological disorder. Specifically the neurological disorder is depression, including its subtypes as described herein.

As used herein the term“treating” refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the above-described diseases, disorders or conditions. It also includes managing the disease as described above. By "manage" it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes and the like.

Treating, according to the present invention, can be effected by specifically administering at least one of the inventive molecules of the present invention in the subject.

The inventive molecule may optionally be administered in as part of a pharmaceutical composition, described in more detail below.

Methods of Therapeutic Use

According to at least some embodiments, there is provided new uses and methods of treatment for neurological diseases by administering the inventive molecule to a subject in need of treatment thereof, in a therapeutically effective amount.

The amount to be administered depends upon the therapeutic need and could easily be determined by one of ordinary skill in the art according to the efficacy of the molecule as described herein. Neurological diseases and disorders to Be Treated

Neurological diseases and disorders that may be treated using the inventive molecules are described herein.

Depression - symptoms and prognosis

Non-limiting examples of a depressive symptom which can be treated or prevented with the inventive molecules as described herein include depressed mood; sadness; anxiety; "empty" feelings; loss of interest or pleasure; irritability;

restlessness; changes in appetite or weight; sleep disturbances; lack of or decreased energy; feelings of worthlessness; guilt; helplessness; anger and hostility; difficulty in thinking, concentrating, or making decisions; hopelessness; tiredness; fatigue;

memory difficulties; tearfulness; brooding; phobias; excessive worry over physical health; sexual dysfunction; persistent physical symptoms that do not respond to treatment; and combinations thereof. These depressive symptoms can be associated with a depressive state or depressive disorder or can be found sub-clinically or not associated with these depressive states/disorders.

According to the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV), periods of sadness are inherent aspects of the human experience. These periods should not be diagnosed as a Major Depressive Episode unless criteria are met for severity (i.e., five out of nine symptoms), duration (i.e., most of the day, nearly every day for at least 2 weeks), and clinically significant distress or impairment. The diagnosis "Depressive Disorder Not Otherwise Specified" may be appropriate for presentations of depressed mood with clinically significant impairment that do not meet criteria for duration or severity. "Depressive Disorder Not Otherwise Specified" includes disorders with depressive features that do not meet the criteria for Major Depressive Disorder. Examples of "Depressive Disorder Not Otherwise Specified" include minor depressive disorder, recurrent brief depressive disorder, and situations in which the clinician has concluded that a depressive disorder is present but is unable to determine whether the depressive disorder is primary, due to a general medical condition, or substance-induced. Optionally, subjects with such depressive symptoms that do not fulfill a diagnosis of depression may also be treated with the inventive molecules as described herein, alone or in combination with an additional therapy as described in greater detail below.

Without wishing to be limited by a single hypothesis, the inventive molecules are believed to potentially provide a therapeutic effect more quickly than known treatments, whether alone or in synergistic combination with one or more known treatments.

Depression mechanism of action

Depression is related to impaired neurotransmission, e.g. low neurotransmitter levels. As a result, compounds that increase neurotransmitter levels in the brain and thus enhance their transmission exhibit anti-depressant properties as well as beneficial effects for a variety of other mental disorders. The main neurotransmitters are serotonin, dopamine, norepinephrine, noradrenaline, acetylcholine, glutamate, gamma-amino-butyric acid, as well as neuropeptides. Increased neurotransmission is achieved by increasing the concentration of the neurotransmitter in the synaptic cleft, thus making the neurotransmitter available for increased or prolonged

neurotransmission through inhibition of re-uptake into the pie-synaptic nerve end, or by preventing neurotransmitter catabolism by inhibition of degrading enzymes such as monoaminooxidase A and B (MAO A and MAO B, respectively).

Recent evidence has indicated that neurotransmitter transmission is not the sole cause for depression, but that other mechanims, including brain metabolism, are also impaired in the brain of depressed patients. This evidence has led to the search for novel classes of antidepressants that act on alterative mechanisms including brain energy metabolism. In particular, impaired lactate metabolism has been implicated in depression. Carrard et al (“Peripheral administration of lactate produces

antidepressant-like effects”, Molecular Psychiatry advance online publication, 18 October 2016) demonstrated that administration of lactate seemed to reverse symptoms of depression in different animal models of depression that respond to acute and chronic antidepressant treatment.

COMPOUNDS OF THE PRESENT INVENTION

The compounds of the present invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)— or (S)- stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and diastereomers, and mixtures, racemic or otherwise, thereof. Accordingly, this invention also includes all such isomers, including diastereomeric mixtures, pure diastereomers and pure enantiomers of the compounds of this invention. The term“enantiomer” refers to two stereoisomers of a compound which are non-superimposable mirror images of one another. The term

“diastereomer” refers to a pair of optical isomers which are not mirror images of one another. Diastereomers have different physical properties, e.g., melting points, boiling points, spectral properties, and reactivities.

The compounds of the present invention may also exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. The term “tautomer” or“tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, then all stereoisomers are contemplated and included as the compounds of the invention. Where stereochemistry is specified by a solid wedge or dashed line representing a particular configuration, then that stereoisomer is so specified and defined.

The compounds of the present invention include solvates, pharmaceutically acceptable prodrugs and salts (including pharmaceutically acceptable salts) of such compounds. The phrase“pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

A“solvate” refers to an association or complex of one or more solvent molecules and a compound of the invention. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term“hydrate” can also be used to refer to a complex wherein the solvent molecule is water.

A“prodrug” is a compound that may be converted under physiological conditions or by solvolysis to the specified compound or to a salt of such compound. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of a compound of the present invention. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes phosphoserine, phosphothreonine, phosphotyrosine, 4- hydroxyproline, hydroxylysine, demosine, isodemosine, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statine, 1, 2,3,4- tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobulyric acid, cirtulline, homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine, phenylglycine,

propargylglycine, sarcosine, methionine sulfone and tert-bulylglycine.

Additional types of prodrugs are also encompassed. For instance, a free carboxyl group of an inventive compound can be derivatized as an amide or alkyl ester. As another example, compounds of this invention comprising free hydroxy groups may be derivatized as prodrugs by converting the hydroxy group into a group such as, but not limited to, a phosphate ester, hemisuccinate, dimethylaminoacetate, or phosphoiyloxymethyl-oxycarbonyl group, as outlined in D. Fleisher, Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group may be an alkyl ester optionally substituted with groups including, but not limited to, ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem, 1996, 39, 10. More specific examples include replacement of the hydrogen atom of the alcohol group with a group such as (C1-C6)alkanoyloxymethyl, 1-((C1- C6)alkanoyloxy)ethyl, 1 -methyl-1 -((C1-C6)alkanoyloxy)ethyl, (C1- C6)alkoxycarbonyloxymethyl, N— (C1-C6)alkoxycarbonylamino-methyl, succinoyl, (C1-C6)alkanoyl, a-amino(Cl-C4)alkanoyl, arylacyl and a-aminoacyl, or (a- aminoacyl-a-aminoacyl, where each a-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(0)(0H)2,— P(0)(0(C 1 -C6)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

Free amines of such compounds can also be derivatized as amides, sulfonamides or phosphonamides. All of these moieties may incorporate groups including, but not limited to, ether, amine and carboxylic acid functionalities. For example, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as R-carbonyl, RO-carbonyl, NRR'-carbonyl, wherein R and R' are each independently (C1-C10)alkyl, (C3-C7)cycloalkyl, or benzyl, or R- carbonyl is a natural a-aminoacyl or natural a-aminoacyl-natural a-aminoacyl,— C(0H)C(0)0Y wherein Y is H, (C1-C6)alkyl or benzyl,— C(OYO)Y1 wherein YO is (C1-C4) alkyl and Y1 is (C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C 1 -C4)alky 1 or mono-N— or di-N,N— (C1-C6)alkylaminoalkyl, or— C(Y2)Y3 wherein Y2 is H or methyl and Y3 is mono-N— or di-N,N-(C 1 -C6)alkylamino, morpholino, piperidin-1- yl or pyrrolidin-l-yl.

For additional examples of prodrug derivatives, see, for example, a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) and Methods in Enzymology,

Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985); b) A

Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and H. Bundgaard, Chapter 5“Design and Application of Prodrugs,” by H. Bundgaard p. 113-191 (1991); c) H. Bundgaard, Advanced Drug Delivery Reviews, 8:1-38 (1992); d) H. Bundgaard, et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and e) N. Kakeya, et al., Chem. Pharm. Bull., 32:692 (1984), each of which is specifically incorporated herein by reference.

Alternatively or additionally, compound of the invention may possess a sufficiently acidic group, a sufficiently basic group, or both functional groups, and accordingly react with any of a number of inorganic or organic bases or acids to form a salt. Examples of salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base, such salts including, but not limited to, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4- dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, g-hydroxybulyrates, glycollates, tartrates, methanesulfonates,

propanesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates. Since a single compound of the present invention may include more than one acidic or basic moiety, the compounds of the present invention may include mono, di or tri-salts in a single compound.

If the inventive compound is a base, the desired salt may be prepared by any suitable method available in the art, for example, by treatment of the free base with an acidic compound, for example an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid such as glucuronic acid or galacturonic acid, an alpha hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. If the inventive compound is an acid, the desired salt may be prepared by any suitable method, for example, by treatment of the free acid with an inorganic or organic base. Examples of suitable inorganic salts include those formed with alkali and alkaline earth metals such as lithium, sodium, potassium, barium and calcium. Examples of suitable organic base salts include, for example, ammonium,

dibenzylamrnonium, benzylammonium, 2-hydroxyethylammonium, bis(2- hydroxyethyl)ammonium, phenylethylbenzylamine, dibenzylethylenediamine, and the like salts. Other salts of acidic moieties may include, for example, those salts formed with procaine, quinine and N-methylglucosamine, plus salts formed with basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine.

In certain embodiments, the salt is a“pharmaceutically acceptable salt” which, unless otherwise indicated, includes salts that retain the biological effectiveness of the corresponding free acid or base of the specified compound and are not biologically or otherwise undesirable.

The compounds of the present invention as described herein also include other salts of such compounds which are not necessarily pharmaceutically acceptable salts, and which may be useful as intermediates for preparing and/or purifying such compounds and/or for separating enantiomers of such compounds.

PHARMACEUTICAL COMPOSITIONS

The present invention, in some embodiments, features a pharmaceutical composition comprising a therapeutically effective amount of a therapeutic agent according to the present invention. According to the present invention the therapeutic agent is an inventive molecule as described herein. The therapeutic agents of the present invention can be provided to the subject alone, or as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, mucosal (including intra-nasal) or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl -substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N' -di benzy lethy lenediamine, N- methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition according to at least some embodiments of the present invention also may include a pharmaceutically acceptable anti-oxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. A pharmaceutical composition according to at least some embodiments of the present invention also may include additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)) and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol).

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions according to at least some embodiments of the present invention include water, buffered saline of various buffer content (e.g., Tris- HC1, acetate, phosphate), pH and ionic strength, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.

Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions according to at least some embodiments of the present invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Optionally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety- nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms according to at least some embodiments of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

A composition of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. Preferred routes of administration for therapeutic agents according to at least some embodiments of the present invention include intravascular delivery (e.g. injection or infusion), intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, oral, enteral, rectal, pulmonary (e.g. inhalation), nasal, topical (including transdermal, buccal and sublingual), intravesical, intravitreal, intraperitoneal, vaginal, brain delivery (e.g. intra-cerebroventricular, intra-cerebral, and convection enhanced diffusion), CNS delivery (e.g. intrathecal, perispinal, and intra-spinal) or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous (IV) and intradermal), transdermal (either passively or using iontophoresis or electroporation), transmucosal (e.g., sublingual administration, nasal, vaginal, rectal, or sublingual), administration or administration via an implant, or other parenteral routes of administration, for example by injection or infusion, or other delivery routes and/or forms of administration known in the art.

The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion or using bioerodible inserts, and can be formulated in dosage forms appropriate for each route of administration. In a specific embodiment, an inventive molecule or a pharmaceutical composition comprising same according to at least some embodiments of the present invention can be administered intraperitoneally or intravenously.

Compositions of the present invention can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acom P nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

In some in vivo approaches, the compositions disclosed herein are administered to a subject in a therapeutically effective amount. As used herein the term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected. For the inventive molecules and compositions comprising same as described herein, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing.

The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on tire duration of the treatment desired. For example, dosage levels of 0.0001 to 100 mg/kg of body weight daily may be administered to mammals and more specifically 0.001 to20 mg/kg. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Generally, for intravenous injection or infusion, dosage may be lower. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms according to at least some embodiments of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Optionally the pharmaceutical formulation may be administered in an amount between 0.0001 to 100 mg/kg weight of the patienVday, preferably between 0.001 to 20.0 mg/kg/day, according to any suitable timing regimen. A therapeutic composition according to at least some embodiments according to at least some embodiments of the present invention can be administered, for example, three times a day, twice a day, once a day, three times weekly, twice weekly or once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8 weeks. Moreover, the composition can be administered over a short or long period of time (e.g., 1 week, 1 month, 1 year, 5 years).

Alternatively, therapeutic agent can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the therapeutic agent in the patient. The half-life for molecules may vary widely. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A "therapeutically effective dosage" of an inventive molecule preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifepan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction.

One of ordinary skill in the art would be able to determine a therapeutically effective amount based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

In certain embodiments, the pharmaceutical compositions are administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the pharmaceutical compositions which is greater than that which can be achieved by systemic administration. For example, in the case of a neurological disorder, the inventive molecule may be administered locally to a site near the CNS.

Pharmaceutical compositions of the present invention may be administered with medical devices known in the art. For example, in an optional embodiment, a pharmaceutical composition according to at least some embodiments of the present invention can be administered with a needle or other hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices known in the art. For example, in an optional embodiment, a therapeutic composition according to at least some embodiments of the present invention can be administered with a needle or hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multichamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, therapeutic agents according to at least some embodiments of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds according to at least some embodiments of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J Physiol. 1233:134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

Formulations for parenteral administration

In a further embodiment, pharmaceutical compositions disclosed herein are administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of an inventive molecule as described herein, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-oxidants (e.g., water soluble antioxidants such as ascorbic acid, sodium metabisulfite, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDT A), sorbitol, tartaric acid, phosphoric acid), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are ethanol, propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be freeze dried (lyophilized) or vacuum dried and redissolved/resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating thecompositions.

Formulations for topical administration Inventive molecules disclosed herein can be applied topically, preferably to one or more of the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa

Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acom II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc.,

Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator. Oral formulations may be in the form of chewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations will require the inclusion of penetration enhancers.

Controlled delivery polymeric matrices

Inventive molecules disclosed herein may also be administered in controlled release formulations. Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where the inventive molecules are dispersed within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the inventive molecule is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably.

Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used for delivery of inventive molecules, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275- 283 (1987); and Mathiowitz, et al., J. Appl Polymer ScL, 35:755-774 (1988).

The devices can be formulated for local release to treat the area of

implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.

COMBINATION THERAPY It will be appreciated that treatment of the above-described diseases according to the present invention may be combined with other treatment methods known in the art (i.e., combination therapy). Thus the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, according to at least some embodiments of the present invention can also be used in combination with one or more of the following agents.

Optionally any of the therapies described below may be used with any inventive molecule as described herein.

For drug therapies, optionally the drug is included in a single dosage or composition with any inventive molecule as described herein. Alteratively, the drug is administered separately from the inventive molecule as described herein. Preferably, a reduced amount of the drug is administered, as compared to the normal dose that would be given to the subject, given his/her health parameters (e.g., weight, age, resistance of symptoms to treatment).

One class of antidepressant drugs are tricyclic antidepressant compounds (TCAs), for example imipramine, amitriptyline, and clomipramine, inhibit the reuptake of serotonin and noradrenaline. Non-limiting examples of TCAs include imipramine, nortriptyline, amitriptyline, doxepin, trimipramine, desipramine and protriptyline. These compounds are widely regarded as among the most effective antidepressants available, but they have a number of disadvantages because they interact with brain receptors such as cholinergic receptors. Most importantly, risks associated with overdoses of TCAs include acute cardiotoxicity.

Another class of antidepressant drugs is the selective serotonin re-uptake inhibitors (SSRls), non-limiting examples of which include fluoxetine, paroxetine, sertraline, citalopram, escitalopram, vilazodone and fiuvoxamine. SSRls block the serotonin transporter (SERT), a high affinity sodium chloride-dependent neurotransmitter transporter that terminates serotonergic neurotransmission by uptake of serotonin, thus inhibiting re-uptake of serotonin alone. These compounds have been demonstrated to be effective in the treatment of depression and anxiety and are usually better tolerated than TCAs. These medications are typically started at low dosages and dosage level may be increased until they reach a therapeutic level. A common side effect is nausea Other possible side effects include decreased appetite, dry mouth, sweating, infection, constipation, yawning, tremors, sleepiness and sexual dysfunction.

In addition, compounds that prevent the catabolism of neurotransmitters more broadly by inhibiting MAO A and MAO B exhibit antidepressant effects. The MAOs catalyse the oxidation of amino group containing neurotransmitters such as serotonin, noradrenaline, and dopamine. Compounds that prevent such catabolism are referred to as monoamine oxidase inhibitors (MAOIs). MAOIs include but are not limited to tranylcypromine, phenelzine, selegiline and isocarboxazid. These drugs are typically prescribed when other drugs haven't worked, because they can have serious side effects. Using MAOIs requires a strict diet because of dangerous (or even deadly) interactions with foods— such as certain cheeses, pickles and wines— and some medications and herbal supplements.

Another class of drugs include serotonin-norepinephrine reuptake inhibitors (SNRIs). Examples of SNRIs include duloxetine, venlafaxine, desvenlafaxine and levomilnadpran. SNRIs block the reabsorption (reuptake) of the neurotransmitters serotonin and norepinephrine in the brain.

Atypical antidepressants represent a class of drugs that do not belong to any of the other antidepressant categories. They have a variety of mechanisms of action. Non-limiting examples include bupropion, mirtazapine, nefazodone, trazodone and vortioxetine.

As noted above, these drugs can lead to several side-effects such as dry mouth, blurry vision, lowered gastrointestinal motility or constipation, urinary retention, cognitive and/or memory impairment, drowsiness, confusion, restlessness, dizziness, hypersensitivity, changes in appetite and weight, sexual dysfunction, nausea and vomiting, hypotension, tachycardia, and occasionally irregular heart rhythms. Hallucinations, delirium and coma are some of the toxic effects caused by overdose. Therefore being able to administer a reduced amount of the drug, when administered along with an inventive molecule as described herein, is clearly beneficial.

In addition to side effects, available antidepressants have major drawbacks that include in particular their slow mode of action. Indeed, they take on average 3 to 5 weeks before showing effects in patients, which results in considerable morbidity at the beginning of patients’ treatment due to elevated risk of suicide. In addition, 30 to 50% of patients are resistant to any available antidepressant. However, as noted above, optionally combining a known therapy with an inventive molecule may reduce the length of time before therapeutic onset and/or may also overcome therapeutic resistance.

Other combinations will be readily appreciated and understood by persons skilled in the art. In some embodiments, the therapeutic agents can be used to attenuate or reverse the activity of a drug suitable for treatment of a neurological disease as described herein, and/or limit the adverse effects of such drugs.

As persons skilled in the art will readily understand, the combination can include the therapeutic agents and/or a pharmaceutical composition comprising same, according to at least some embodiments of the invention and one other drug; the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, with two other drugs, the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, with three other drugs, etc. The determination of the optimal combination and dosages can be determined and optimized using methods well known in the art.

The therapeutic agent according to the present invention and one or more other therapeutic agents can be administered in either order or simultaneously.

Where the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, according to at least some embodiments of the invention are administered in conjunction with another therapy, e.g. as herein above specified, dosages of the co-administered drug will of course vary depending on the type of codrug employed, on the specific drug employed, on the condition being treated and so forth.

Treatment of neurological diseases using the agents of the present invention may be combined with other treatment methods known in the art that are non-drug treatments. Non-drug therapies include but are not limited to brain stimulation therapy, light therapy and counseling or“talk therapy” Non-limiting examples of brain stimulation therapy include electroconvulsive therapy (ECT), repetitive trans cranial magnetic stimulation (rTMS) and vagus nerve stimulation (VNS). Optionally such non-drug therapies are administered in a coordinated manner with the inventive molecule as described herein. Diagnosis of Depression

Diagnosis involves a physical exam, to rule out physical causes; and a psychiatric exam, to determine that depression is the correct diagnosis.

Depression biomarkers

A review by Strawbridge et al (‘Biomarkers for depression: recent insights, current challenges and future prospects”, Neuropsychiatr Dis Treat. 2017; 13: 1245- 1262) indicated that a number of biomarkers have been considered in depression. The most reliable biomarkers that are associated with depression are IL-6 and CRP (C- reactive Protein), which have been repeatedly shown to be reliably elevated in depression. Other biomarkers that may be useful include but are not limited to tumor necrosis factor alpha (TNFa), IL-1b, selected interleukins IL-2, IL-4, IL-8, IL-10, interferon gamma, macrophage inhibitory protein la, IL-la, IL-7, IL-12p70, IL-13, 1L-15, eotaxin, granulocyte macrophage colony -stimulating factor, IL-5, 1L-16, IL-17, monocyte chemoattractant protein-4, thymus and activation-regulated chemokine, eotaxin-3, TNFb, interferon gamma-induced protein 10, serum amyloid A, soluble intracellular adhesion molecule and soluble vascular cell adhesion molecule. For the selected interleukin biomarkers, the initial level of the interleukin may not be significantly higher in depressed patients, but the level decreases with symptom remission.

Therefore according to at least some embodiments of the present invention, there are provided biomarkers for diagnosis of depression and/or for determining treatment efficacy, selected from the group consisting of IL-6, CRP, tumor necrosis factor alpha (TNFa), IL-1b, selected interleukins IL-2, IL-4, IL-8, IL-10, interferon gamma, macrophage inhibitory protein la, IL-la, IL-7, IL-12p70, IL-13, IL-15, eotaxin, granulocyte macrophage colony-stimulating factor, IL-5, IL-16, IL-17, monocyte chemoattractant protein-4, thymus and activation-regulated chemokine, eotaxin-3, TNFb, interferon gamma-induced protein 10, serum amyloid A, soluble intracellular adhesion molecule and soluble vascular cell adhesion molecule, or a combination thereof. Preferably the biomarkers comprise one or both of IL-6 and CRP. Example 1 - Testing of Inventive Molecules for Denression

Material and Methods

1. Mouse animal experimentation

All experiments were carried out in accordance with the Swiss Federal Guidelines for Animal Experimentation and were approved by the Cantonal Veterinary Office for Animal Experimentation in Switzerland.

2. Cell cultures

Primary cultures of cerebrocortical astrocytes were obtained from 1 -2-day-old OF1 mouse pups (Charles River Laboratories). Briefly, cortices were isolated and minced in small pieces under a dissecting microscope. The cells were incubated for 30min at 37°C in a solution containing 20U/ml of papain enzyme (Worthington Biochemical), L-cysteine ImM (Sigma), and lOkU/ml DNase I (Worthington Biochemical) for an enzymatic dissociation. Papain activity was stopped by the addition of fetal calf serum (FCS) to the solution, and a single-cell suspension was then obtained by mechanical dissociation, which consisted in cells trituration in a DMEM (D7777, Sigma-Aldrich) medium (supplemented with 44 mm NaHCO ₃ , and 10 ml/L antibiotic/antimycotic solution) containing 10% FCS. The cells were seeded at an average density of 6 x 10 ⁴ cells/cm ² in poly-D-lysine-coated 96, 12 or 6-well culture plates, depending on their use, and incubated at 37°C in a humidified atmosphere containing 5% CO ₂ / 95% air. Culture medium was renewed twice per week. Cells were stimulated and harvested between DIV14 and DIV17, when confluence and cell growth were optimal.

2.1 High throughput screening (HTS) for lactate secretion

Secretion of lactate in a high-throughput screening (HTS) fashion was measured indirectly through the acidification of extracellular medium. To this end, primary astrocytes grown in 96- well plates for 17 days were stimulated with the compounds as listed herein.

After washing the cells twice with stimulation medium (DMEM (D5030, Sigma), 3mM NaHCO ₃ and 5mM Glucose) at 37°C, cells were stimulated with the compounds at a final concentration of lOuM (1% DMSO final) in 50ul per well of stimulation medium supplemented with lOuM of the extracellular pH sensor SNARF- 5F %-(AND-6)-CAR (Life Technologies Corporation). Each compound was tested in two different plates for duplicates.

After 90 min stimulation, fluorescence was read at exc. (excitation) 480 nm / emm. (emission) 580 nm and at exc 480 nm / emm 630 nm The ratio of

fluorescence between 630nm and 580 emission values, which represents extracellular pH, was calculated.

In each plate, 8 wells were used for negative controls (DMSO) and 8 wells were used for positive controls (CCCP 2uM in DMSO). Z prime values were calculated for each of the plates tested and values < 0 were discarded.

The average and SD of compounds’ values tested in duplicates were calculated, and compound was noted as HIT when the difference between

compound’s average and negative control’s average was greater than three times the sum of compound’s SD and negative controls’ SD. Only molecules with scores greater than 40% were considered as lead hits for the CDC54K library; for the remaining libraries, all hits were considered. Scores are calculated as the % of activity compared to the positive control in each plate (which is 100%).

Primary screening hits were cherry picked on new plates and confirmed for SNARF5 effect, after having discarded those compounds that are fluorescent (exc.

480 nm / emm. 580 nm or 630 run) before stimulation. Extracellular medium was next analyzed for extracellular lactate quantification for secondary screening.

2.2 Extracellular lactate quantification

Secretion of L-lactate was determined in the extracellular medium of 96-well plated astrocytes after 90 min stimulation (at 37°C, in 5% CO ₂ / 95% air conditions) with the drug of interest. The stimulation medium was composed of D5030 medium (completed with D-glucose 5mM and 44mM sodium bicarbonate) for 90min in concentrations ranging from 0 to 100mM.

Briefly, 200ml of a Glycine (Sigma)-Semicarbazide (Acres) 0.2M pH 10 buffer containing 3mM NAD (Roche) and LDH 14U/ml (Roche) was added to each well of a 96-well plate containing 30ml aliquots complemented with 20ml fresh complete D5030 medium Samples were incubated at 37°C for lh. After samples cooled down at room temperature, the fluorescence intensity (340nm excitation / 450nm emission) that represents the amount of NADH produced was measured, and lactate concentration values were determined from a standard curve of L-lactate.

2.3 Intracellular glycogen quantification

For glycogen dosage, a protein dosage was first performed in order to assess whether harvested astrocytes from primary cell cultures yielded enough and equivalent amounts of proteins comparing each replicate, and to ensure that the obtained differences in glycogen quantities were due to drug action and not to inner protein quantities.

Astrocytes used for these dosages were previously grown in 6-well plates for 17 days and stimulated with Vehicle (DMSO) or drug of interest (ImM to 100mM) for 180niin, at 37°C 5% CO ₂ / 95% air in D5030 complete medium. Medium was removed and replaced with 600ml of 30mM Tris HC1, and stored at -20°C.

Proteins were dosed using the micro BCA Protein Assay kit (Thermo Scientific), as described in the manufacturer’s instructions. Briefly, thawed cells were sonicated and 5ml aliquots were placed in a transparent 96- well plate, to which we added 25ml 30mM Tris HC1, 70ml H ₂ O and 100mI of a BCA mix (made as described in manufacturer’s guidelines). After a 120min-incubation at 37°C, absorbance was measured with Satire 2 spectrophotometer at a wavelength of 562nm, and protein quantities were determined from a standard curve of Bovine Serum Albumin (BSA).

Glycogen was quantified using a 250ml-aliquot of the same stimulated, thawed, and sonicated cells. After an incubation period of 30min at 90°C and 400rpm, 28ml of an acetic acid/sodium acetate (both from Sigma) 0.1M pH 4.6 buffer was added to each aliquot, which was then separated in two. Each split aliquot received whether 5ml of amyloglucosidase (Roche) or 5ml H ₂ O, and all cell solutions were incubated for 120min in a shaking 37°C-waterbath. After a centrifugation at 16’ 000G for 5min, 20ml of superatant were placed 96-well plate, to which 150ml of a mix containing 0.67mM ATP (Roche), 0.67mM NADP (Roche), 1.8% hexokinase/glucose-6-phosphate dehydrogenase (Roche) and 0.1M Tris Buffer-HCl / 3.3mM magnesium (Fluka) / pH 8.1 buffer was added. Fluorescence was measured with Safire 2 spectrophotometer (340nm excitation / 440nm emission). Glycogen concentration was obtained by substracting glucose value of samples with amyloglucosidase to samples without amyloglucosidase, and were expressed relative to the amount of proteins previously determined.

2.4 MTT viability assay

For cell toxicity determination, astrocytes in 96-well plates were stimulated 24h (37°C 5% CO ₂ / 95% air) with a gradient ranging from 0.1 to 200mM of tested compounds. After stimulation, 5 mg/ml thiazol blue tetrazolium bromide (MTT, Sigma-Aldrich) in warm D5030 complete medium was added to each well, and cells were incubated for 4h at 37°C (5% CO ₂ ). The medium was then removed by aspiration, and the reaction was stopped by the addition of 50ml DMSO per well.

The amount of reduced MTT (formazan) solubilized in DMSO was then determined spectrophotometrically using absorbance at 570 nm (Safire 2; Tecan).

2.5 Production of reactive oxygen species (ROSY

Hydrogene peroxide (H202) released in the supernatant is detected enzymatically with Amplex red (Zhou, Diwu et al. 1997). Oxidation of Amplex red is catalysed by the horseradish peroxidase in presence of H202 into highly fluorescent resorufin. Fluorescence measure is read at 545 nm extinction, 590nm emission. The amount of H202 was expressed relatively to the proteins content extracted from the cells in culture. 3. In vivo testing

3.1 Mice

For in vivo acute toxicity, in vivo chronic toxicity, pharmacodynamics experiments, and pharmacokinetics experiments, adult male or female C57B1/6J mice weighting 18-28g (8 weeks of age) were used (Charles River or Harlan).

All experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council 2011) and were approved by relevant animal care authorities.

Animals were housed in groups of 3-5 in polypropylene cages (30 X 40 X 15 cm) with wire mesh top in a temperature (22 ± 2 °C) and humidity (55 ± 15%) controlled environment on a 12 hour light cycle (07.00 - 19.00h lights on), except after surgeries when animal were housed individually.

3.2 In vivo drug administration

Drugs were administered per os (gavage) in a solution made of water supplemented with 0.4% hydroxypropyl methylcellulose (HPMC) Methocel 4KM (w/v) and 0.25% Tween-20 (v/v), as previously described. The compound was administered at lOmg/kg. Concentrations of drugs tested ranged from 10 to 100 mg/kg.

3.3 In vivo acute toxicity

In vivo acute toxicity was assessed with a starting maximal concentration of 100 mg/kg. If at any point toxic effects were observed, a second 10-times lower concentration was tested, and so forth until non-toxic concentration was reached, hence providing optimal dose of our compound for in vivo testing. Groups of 6-8 female mice were monitored for 14 days after single oral administration of the drug, weighted every day, and a macroscopic histological examination was performed at the end of the experiment. Clinical evaluation included the observation of mice’ ability to feed, hydrate, notification of any visible pain, unusual grooming or respiration, blood loss, evidence of microbial infection, and/or significant loss of weight.

3.4 In vivo chronic toxicity

Chronic toxicity was assessed in groups of 10 male and 10 female C57BL/6J mice over a period of 28 days. Drugs or Vehicle were administered per os, once a day, as previously described. During this period, clinical symptoms and weight were recorded. At the end of the 28-day period, 3 mice per group were sacrificed for histopalhological analyses. The other mice were kept for another 14 days without treatment to assess for late-coming toxic effects, followed by the same analyses. Histopathology was performed by specialized platform of mouse pathology facility at the CHUV hospital (Lausanne, Switzerland).

3.5 In vivo pharmacodynamics - Lactate biosensors

Extracellular levels of lactate were monitored in vivo using lactate biosensors (Pinnacle Technology), according to the manufacturer’s instructions. Cannulae were surgically implanted in mice cerebral cerebral motor cortex areas M1/M2 (coordinates: +1.94 mm (to bregma), lateral -1.4mm (to mideline), ventral -1.0mm (to dura)) 5-7 days before administration of the compounds. Drugs were administered per os as previously described, and cerebral levels of extracellular lactate were dynamically recorded for 6 hours. Mice were administered vehicle alone first, followed 3 hours later by vehicle or drug (10 or 100 mg/kg). Area Under the Curve (AUC) quantifying the fluctuations of extracellular lactate concentrations for each of the compound tested was calculated using Graphad Prism and the ratio of AUC after drug over Vehicle administration was calculated. Groups of 8 male mice were used for each condition.

3.6 In vivo pharmacodynamics - Glycogen quantification

To measure intracerebral levels of glycogen, mice were euthanized at different time points after drug administration, using a microwave beam (lsec, 6kW) focused directly on mice brains. This method of fixation results in the rapid inhibition of enzymatic reactions, thereby preserving intact metabolic state in the brain of euthanized animals. Glycogen concentration was quantified using standard biochemical procedure. Groups of 8 male mice were used for each condition.

3.7 In vivo pharmacokinetics

3.7.1 Surgery

Mice were anesthetized using isoflurane (2% and 800 mL/min O ₂ ). Before surgery, Finadyne (1 mg/kg, s.c.) was administered for analgesia during surgery and the post-surgical recovery period. A mixture of bupivacaine and epinephrine was used for local anesthesia of the incision site of the periost of the skull.

3.7.2 Microdialysis probe implantation into the prefrontal cortex (PFC)

The animals were placed in a stereotaxic frame (Kopf instruments, USA). MetaQuant microdialysis probes with a 3 mm exposed polyacrylonitrile membrane (MQ-PAN 3/3, Brainlink, the Netherlands) were implanted into the prefrontal cortex (coordinates for the tip of the probe: AP = +2.0 mm (to bregma), lateral = -0.7 mm (to midline), ventral = -3.3 mm (to dura), with the incisor bar set at 0.0 mm and an angle of 8°). All coordinates were based on“The mouse brain in stereotaxic coordinates” by Paxinos and Franklin (2004). The probes were attached to the skull with a stainless steel screw and dental cement (Fuji Plus Capsules, Henry Schein, the Netherlands).

3.7.3 Jugular vein cannulation

In the same surgical procedure, a catheter was placed into the jugular vein to accommodate blood sampling. An indwelling cannula was inserted into the right jugular vein, and exteriorized through an incision on top of the skull. The end of the jugular vein catheter was fixed in position with dental acrylic cement and attached to the skull with two stainless steel screws.

3.7.4 Experimental design

Experiments were initiated one day after surgery. The MetaQuant microdialysis probes were connected with flexible PEEK tubing (Western Analytical Products Inc. USA; PK005-020) to a microperfusion pump (CMA Microdialysis) and perfused with aCSF + 0.2% BSA at a flow rate of 0.12 mL/min. Ultrapurified water + 0.2% BSA was used as the carrier flow at a flow rate of 0.80 mL/min. After a minimum of 1.5 hours of prestabilization, microdialysis samples were collected in 30 minute intervals. Samples were collected into polystyrene microvials (Microbiotech/se AB, Sweden; 4001029) using an automated fraction collector (UV 8301501, TSE, Univentor, Malta). After collection of three basal samples, at t = 0 minutes, drug of interest was administered per os. Eight additional samples were collected after compound administration. All samples were stored at -80 °C until offline analysis.

In parallel, blood samples (50 mL) were taken from the jugular vein through the cannula. These samples were collected at specified intervals into vials containing 5 mL heparin (500 IE /mL in saline). The samples were mixed by inverting the tubes and, subsequently, centrifuged at 4000 rpm (1500xg) for 10 min at 4 °C. The superatant was stored as plasma in 1.5 mL Eppendorf vials (Sarstedt, Germany) at - 80 °C until off-line analysis.

At the end of the experiment, the animals were euthanized and terminal brain tissue was collected for visual histological verification of the probe positions.

3.8 Therapeutic effect - depression mouse model

The depression mouse model features wild-type C57B1/6 laboratory mice without any transgenic changes. To ensure that the actions of the tested molecules in the animal models of depression are not due to effects on locomotor activity or muscle strength in animals, molecules are administered to mice that are then tested in the grip strength test (GST) and in the open-field test (OFT) following the protocols described in Carrard et al. (Garrard, Elsayed, Margineanu, Bouiy-Jamot, Frangière, Meylan, Petit, Fiuemlli, Magistretti and Martin. Peripheral administration of lactate produces antidepressant-like effects. Molecular Psychiatry, 2016 Oct 18, e-pub). If alterations in muscle strength and/or locomotor activity are measured following administration drug, no further analyses are performed with this molecule.

3.8.1 Grip Strength Test (GST) GST is a test used to identify molecules that could modulate mouse grip strength, which might affect results in the FST. 1.5 hr after a single oral molecules administration, mice are placed on a grid dynamometer and the muscle strength (N) or their two front paws or all their four paws is measured in 3 consecutive sessions, and the best score is recorded.

3.8.2 Open Field Test (OFT)

OFT is a test used to identify molecules that could modulate mouse activity, which might affect results in the FST. 1.5 hr after a single oral molecules

administration, mice are placed in a 39 cm (long) x 33 cm (large) x 33 cm (high) open box and their activity is videotaped for 10 minutes. Mouse activity is then

automaticcaly scored using Ethovision pro video tracking system (Noldus).

3.8.3 Forced swim test (FST)

FST is a test used to identify molecules that produce an acute antidepressantlike behavioral response.

Drugs that do not affect locomotor activity and/or neuromuscular strength are assessed for their acute antidepressant effect using the FST. 1.5 hr after a single oral (vehicle or lactate-enhancing molecules) or intraperitoneal (vehicle, fluoxetine or despratmine) administration, mice are placed in a cylindrical container filled to a depth of 15 cm with water (23-25°C). 6-min swim session are videotaped, and time spent immobile, which reflects the animal depressive-like behavior, is scored subsequently by an experimenter blind to the conditions used.

4. Statistical analyses

Statistical analyses were done using Graphpad prism v.6 using unpaired or paired 2-way Student’s t-test for pairwise comparisons, or one-way or two- ways ANOVA followed by Dunnett, Bonferroni or Tukey HSD post-hoc tests when appropriate for multiple pair-wise comparisons.

Results Summary 1. High Throughput Cellular Screening

Identification of lactate-enhancing drugs with high throughput screening (HTS) experiments on astrocytes primary cultures using extracellular pH dye (SNARF5F 5-(and-6)-carboxylic acid) for 90 min. The procedure is described in Material and Methods (2. Cell culture, 2.1. HTS for lactate secretion and 2.2. Extracellular lactate quantification).

The procedure was performed as follows:

Primary screening: acidification of the extracellular medium

Primary screening confirmation: acidification of the extracellular medium, removal of compounds with fluorescent activity at exc. 480nm / emm. 580 nm or 630 nm.

Secondary screening: dosage of extracellular lactate

The first library screened was the Prestwick library, composed of 1240 FDA- approved drugs (available from Prestwick Holding and Chemical Inc., USA). The best stimulators of release of lactate were found to be the following 19 hits in Table 1.

Table 1 - Prestwick hits

The next library tested was the CDC54K library composed of 54,000 compounds (from the Bioscreening facility at EPFL, Lausanne, Switzerland), grouped into chemical families. Appendix I features a list of chemical motifs, based upon structural analysis of the full list of hits. Appendix II features a list of molecules that were shown to be active but that may be additional to the molecules of Appendix I. The molecules listed in Table 1 above, as well as in Appendix II, are termed herein ‘Inventive molecules”.

Any molecule featuring a motif or that is related to a molecular structure given in Appendix I, and has suitable metabolic activity in at least one assay as described herein, may also be termed herein an“inventive molecule”.

2. In vitro characterization

Hits were characterized in vitro on primary astrocytes cultures for their effect on lactate secretion (EC50), glycogen degradation, H ₂ O ₂ production (to avoid molecules that stimulate glycolysis through blocking of mitochondrial respiration) and cellular toxicity (LD50). The molecules were also characterized for their ‘druggabilily’ through Pfizer rule of 5 and theoretical crossing of the blood brain barrier (polar surface area < 90 A).

Technical procedures are described in Material and Methods (2. Cell culture).

a. Hits from Prestwick Library

i. Secretion of lactate

Levels of lactate secreted by astrocytes were measured in the extracellular medium at 90 min after stimulation with 20 hits (molecules) from the Prestwick library (10 mM each), as shown in Figure 1. n=6-10; Ctrl pos. is CCCP (2uM).

Statistical analysis consisted in ANOVA followed by Fisher LSD post-hoc test for pair-wise comparisons. In addition, a range of concentrations of the Prestwick compounds (0 - 100 mM) was used to calculate EC50, as shown in Table 1 below.

ii. Degradation of Glycoeen

Levels of intracellular glycogen in astrocytes were measured at 3h after stimulation with 20 hits from the Prestwick libraiy (10 mM each), as shown in Figure 2. n=6-10; Ctrl pos. is glutamate (0.5mM). Statistical analysis consisted in ANOVA followed by Fisher LSD post-hoc test for pair-wise comparisons.

iii. Cellular Toxicity by MTT

MTT cellular viability assay was performed on astrocytes exposed to molecules from the Prestwick libraiy (concentrations ranging from OuM to 200uM). Examples for lead molecules are shown in Figure 3. The cellular toxicity results are summarized in Table 2 below.

iv. Mitochondrial activity

Mitochondrial respiration in astrocytes was measured through production of H ₂ O ₂ at 90 min after stimulation with Prestwick hits (10 uM each). Figure 4 shows mean absorbance + SEM; n=4. CCCP (2uM) was used as positive control.

v. List Summary

Table 2 shows a summary of Prestwick hits activity, including HTS score, lactate effect (EC50), statistical significance of glycogen degradation (* p<0.05, ** p0.Ol, *** p0.001, **** p0.0001), cellular toxicity measured by MTT (IC50), Pfizer Rule of 5 and total polar surface area (PSA).

b. Hits from the CDC54K library

i. Lactate secretion

Levels of lactate secreted by astrocytes were measured in the extracellular medium at 90 min after stimulation with hits (molecules) from the CDC54K library. Concentrations ranging from 0 to lOOuM were used to calculate EC50, as shown in Table 3 below. Lead hits from the CDC54K library that have been tested consisted in one member of each of the 18 CDC54K families. The results are shown in Figure 5 A.

ii. Glycogen degradation

Figure 5B shows levels of intracellular glycogen in astrocytes that were measured at 3h after stimulation with 18 hits from the CDC54K library (10 mM each). n=6-10; Ctrl pos. is Glutamate. Statistical analysis consisted in ANOVA followed by Fisher LSD post-hoc test for pair-wise comparisons.

iii. Cellular Toxicity by MTT

MTT cellular viability assay was performed on astrocytes exposed to

CDC54K hits (concentrations ranging from OuM to 200uM). IC50 data are summarized in Table 3.

iv. Mitochondrial activity

Mitochondrial respiration in astrocytes was measured through production of H ₂ O ₂ at 90 min after stimulation with CDC54K hits (ranging from 0 to 200uM). IC50 data are summarized in Table 3.

v. List Summary Table 3 shows a summary of CDC54K hits activity, including HTS score, lactate effect (EC50), statistical significance of glycogen degradation, cellular toxicity measured by MTT (IC50), effect on H202, Pfizer Rule of 5 and total polar surface area (PSA).

3. In vivo characterization

a. Acute toxicity

Lead molecules from in vitro were tested in vivo, starting with acute toxicity/dose optimization on wild-type C57B1/6 female mice for a period of 14 days following administration. For this period, mice were weighted and clinically monitored (feeding, hydration, pain, grooming, respiration, blood loss, microbial infection). At the end of the 14-day evaluation, mice were sacrificed and high level organ analysis was performed. Drugs were always administered per os (gavage) in solution composed of Methocel 4KM 0.4%, Tween 0.25%. The results are shown in Figure 6.

Summary

GP-03 was toxic at 100mg/kg but not at 10mg/kg (dose optimization)

None of the other tested molecules (GP-01 - GP-07; GP-A, I, P, Q, R, V) were toxic at lOOmg/kg

b. Chronic toxicity

Chronic toxicity was assessed. C57B1/6 male and female mice were used with GP- 01, 02, 04, 05, 06 and 07 at lOmg/kg. GP-03 was not tested as was already toxic after acute administration at 100mg/kg, and did not show good PD effect at lOmg/kg (see below for more information).

Mice were treated for 28-day and monitored for their weight and dinical symptoms, and were next tested for anxiety in an elevated plus maze (EPM). Half of mice were then sacrificed and pathological analysis was performed on a number of organs (brain, tongue, esophagus, diaphragm, stomach, small intestine, pancreas, large intestine, kidneys, adrenal, liver, spleen, pancreas, mesentheric lymph nodes, spinal cord, bone marrow, muscle), while half of mice were sacrificed 14-day later to assess for recovery effects and/or remote toxicity and same pathological analysis ways performed. Results are shown in Figures 7 and

8.

Summary GP-06 chronic administration at lOmg/kg was toxic and interrupted when weight loss was > 20%. Therefore chronic administration of GP-06 at 10mg/kg will not be used.

GP-01, GP-02, GP-04, GP-05 and GP-07 are safe when administered chronically at

10mg/kg.

• EPM (elevated plus maze) analysis revealed increased anxiety of GP-06 treated mice at the end of the treatment, which correlates with toxicity of the chronic treatment. None of the other chronic treatments resulted in significantly elevated anxiety.

• Pathological analysis performed by mouse pathology facility at the CHUV revealed minor treatment-related effects in GP-07-treated mice, including leukocyte cell infiltrates, single cell necrosis in the liver and bulbe duct proliferation. The same was true for a focal amorphous, intratubular vacuole in the kidney of one male mouse treated with GP-07.

c. Pharmacodynamics - lactate biosensors

To measure biological effect of lead molecules in vivo in the brain, lactate levels were quantified after administration of the drug by using lactate biosensors implanted in the cortex of freely moving mice. The results are shown in Figure 9.

Summary

Significant increase of cerebral lactate with GP-04. GP-05. GP-06 and GP-07 at 10 mg/kg (Prestwick library), and family GP-I3, GP-P1 and GP-R1 at 100 mg/kg (10mg/kg not yet tested; CDC54K library).

d. Pharmacodynamics - glycogen levels

Glycogen levels were measured in microwave-fixed PFC (prefrontal cortex, 6kW, lsec), which ensures enzymatic inhibition and stops glycogen degradation. Samples were then flash frozen before dosage.

First, glycogen levels were analyzed at lh, 3h and 6h after drug administration. The highest decreases in PFC glycogen were observed at 3H. This time point was subsequently used for dose-response experiments. Glycogen levels were quantified at 3H after administration with GP-01 to GP-07 at 1, 10 or 100 mg/kg. The results are shown in Figure

10 Summary

All tested molecules showed significant decrease of cerebral levels of glycogen at lOmg/kg and/or lOOmg/kg, except for GP-03.

e. Pharmacokinetics (PK)

PK was measured for GP-04, GP-05, GP-07, GP-R1 and GP-P1 in the PFC

(prefrontal cortex) and plasma of wild type C56B1/6 mice by CRO Brainsonline. The results are shown in Figures 11 A and 11B.

Summary

• Levels of GP-04, GP-05, GP-07 and GP-R1 are at therapeutic range (100nM to luM) and sustained over several hours in the PFC after gavage with 100mg/kg.

GP-01, GP-02 and GP-P1 need chemical improvement to reach their target at therapeutic dose in the brain

Test of GP-07 on antidepressant effects in the Forced Swim Test procedure

To assess for antidepressant effect of lactate-stimulating drugs, depressive behavior was assesses in the classicaly used forced swim test (FST) paradigm. Mouse muscle strength and activity, which disturbance may affect FST, were also analysed after administration of lactate-stimulating drugs in the grip strength test (GST) and open field test (OFT), respectively. To compare antidepressant effect of lactate-stimulating drugs with classical antidepressants, FST was performed on mice after administration of classical

antidepressants Fluoxetine or Desipramine. The results are shown in Figure 12.

Summary

• GP-07 does not affect grip strength or activity, as measured with the grip strength test (GST) and open field test (OFT), respectively

• GP-07 leads to significant reduction in the time spent immobile in the forced swim test (FST), indicating a potential antidepressant effect, similar to the effect of the classical antidepressants Fluoxetine and Desipramine. It will be appreciated that various features of the invention which are, for clarity, described in the contexts of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. It will also be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the invention is defined only by the claims which follow.