Compositions and methods for inhibiting ACSS2
阅读说明：本技术 用于抑制acss2的组合物和方法 (Compositions and methods for inhibiting ACSS2 ) 是由 菲利普·梅夫斯 谢利·L·贝格尔 杰弗里·D·温克勒 安德鲁·格拉斯 西蒙·戴维·彼得·鲍 于 2018-09-26 设计创作，主要内容包括：本发明提供了用于调节组蛋白乙酰化或者用于治疗或预防神经疾病或病症的用于抑制ACSS2的组合物和方法。(The present invention provides compositions and methods for inhibiting ACSS2 for modulating histone acetylation or for treating or preventing a neurological disease or disorder.)
1. A method for treating or preventing a neurological and cognitive disease or disorder, the method comprising administering to a subject in need thereof a composition comprising an inhibitor of ACSS 2.
2. The method according to claim 1, wherein the neurological and cognitive disease or disorder is selected from the group consisting of post-traumatic stress disorder (PTSD), bipolar disorder, depression, gilles de la tourette syndrome, schizophrenia, obsessive compulsive disorder, anxiety, panic disorder, and phobia.
3. The method of claim 1, wherein the neurological and cognitive disease or disorder is PTSD.
4. The method of claim 1, wherein the inhibitor of ACSS2 is at least one of a compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule compound, a nucleic acid, a vector, an antisense nucleic acid molecule.
5. The method of claim 1, wherein the inhibitor of ACSS2 is a small molecule.
6. The method of claim 5, wherein the small molecule is a compound according to one of formulae (1) to (4):
wherein, X11Is selected from C (R)14)(R15) O, S and NR15；
Each occurrence of X12Is selected from C (R)14)(R15) O, S and NR15；
R11Selected from hydrogen, -OR15Alkyl, cycloalkyl, -C4-C6Heterocyclyl, aryl and-C4-C6Heteroaryl, wherein R11Is optionally substituted;
R12and R13Each independently selected from hydrogen, alkyl, aryl and-C4-C6Heteroaryl, wherein R12And R13Is optionally substituted;
each occurrence of R14And R15Independently selected from hydrogen, halogen, -OH and C1-C6An alkyl group; and
n is an integer of 0 to 8;
wherein the content of the first and second substances,
R21is selected from-C (R)23)mCycloalkyl, heterocyclyl, cycloalkyl-ketone and heterocyclyl-ketone;
R22selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
each occurrence of R23Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
m is an integer of 1 to 3;
wherein R is31Is selected from-C (R)35)pCycloalkyl, heterocyclyl, cycloalkyl-ketone, heterocyclyl-ketone;
R32selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
R33and R34Each independently selected from hydrogen, halogen, alkyl, aryl, heteroaryl;
each occurrence of R35Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
p is an integer from 1 to 3;
wherein the content of the first and second substances,
X41selected from O and S;
R41selected from alkyl, cycloalkyl, heterocyclyl, aryl, heteroarylAnd combinations thereof, wherein R41May be optionally substituted; and
R42and R43Each independently selected from phenyl, thiophenyl and furanyl.
7. The method according to claim 6, wherein the compound represented by formula (1) is selected from the group consisting of
8. The method according to claim 6, wherein the compound represented by formula (2) is selected from the group consisting of
9. The method according to claim 6, wherein the compound represented by formula (3) is selected from the group consisting of
10. The method according to claim 6, wherein the compound represented by formula (4) is selected from the group consisting of
11. A method for treating or preventing an addiction or addiction-related disease or disorder, the method comprising administering to a subject in need thereof a composition comprising an inhibitor of ACSS 2.
12. The method of claim 11, wherein the addiction is alcoholism.
13. The method of claim 11, wherein the addiction-related disease or disorder is acute and/or chronic alcohol-induced memory decline.
14. The method of claim 11, wherein the inhibitor of ACSS2 is at least one of a compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule compound, a nucleic acid, a vector, an antisense nucleic acid molecule.
15. The method of claim 14, wherein the small molecule is a compound according to one of formulae (1) to (4):
wherein, X11Is selected from C (R)14)(R15) O, S and NR15；
Each occurrence of X12Is selected from C (R)14)(R15) O, S and NR15；
R11Selected from hydrogen, -OR15Alkyl, cycloalkyl, -C4-C6Heterocyclyl, aryl and-C4-C6Heteroaryl, wherein R11Is optionally substituted;
R12and R13Each independently selected from hydrogen, alkyl, aryl and-C4-C6Heteroaryl, wherein R12And R13Is optionally substituted;
each occurrence of R14And R15Independently selected from hydrogen, halogen, -OH and C1-C6An alkyl group; and
n is an integer of 0 to 8;
wherein the content of the first and second substances,
R21is selected from-C (R)23)mCycloalkyl, heterocyclyl, cycloalkyl-ketone and heterocyclyl-ketone;
R22selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
each occurrence of R23Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
m is an integer of 1 to 3;
wherein R is31Is selected from-C (R)35)pCycloalkyl, heterocyclyl, cycloalkyl-ketone, heterocyclyl-ketone;
R32selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
R33and R34Each independently selected from hydrogen, halogen, alkyl, aryl, heteroaryl;
each occurrence of R35Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
p is an integer from 1 to 3;
wherein the content of the first and second substances,
X41selected from O and S;
R41selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R is41May be optionally substituted; and
R42and R43Each independently selected from phenyl, thiophenyl and furanyl.
16. The method according to claim 15, wherein the compound represented by formula (1) is selected from the group consisting of
17. The method according to claim 15, wherein the compound represented by formula (2) is selected from the group consisting of
18. The method according to claim 15, wherein the compound represented by formula (3) is selected from the group consisting of
19. The method according to claim 15, wherein the compound represented by formula (4) is selected from the group consisting of
20. A compound according to one of formulae (1) to (4):
wherein, X11Is selected from C (R)14)(R15)、O, S and NR15；
Each occurrence of X12Is selected from C (R)14)(R15) O, S and NR15；
R11Selected from hydrogen, -OR15Alkyl, cycloalkyl, -C4-C6Heterocyclyl, aryl and-C4-C6Heteroaryl, wherein R11Is optionally substituted;
R12and R13Each independently selected from hydrogen, alkyl, aryl and-C4-C6Heteroaryl, wherein R12And R13Is optionally substituted;
each occurrence of R14And R15Independently selected from hydrogen, halogen, -OH and C1-C6An alkyl group; and
n is an integer of 0 to 8;
wherein the content of the first and second substances,
R21is selected from-C (R)23)mCycloalkyl, heterocyclyl, cycloalkyl-ketone and heterocyclyl-ketone;
R22selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
each occurrence of R23Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
m is an integer of 1 to 3;
wherein R is31Is selected from-C (R)35)pCycloalkyl, heterocyclyl, cycloalkyl-ketone, heterocyclyl-ketone;
R32selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
R33and R34Each independently selected from hydrogen, halogen, alkyl, aryl, heteroaryl;
each occurrence of R35Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
p is an integer from 1 to 3;
wherein the content of the first and second substances,
X41selected from O and S;
R41selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R is41May be optionally substituted; and
R42and R43Each independently selected from phenyl, thiophenyl and furanyl.
21. The method according to claim 20, wherein the compound represented by formula (1) is selected from the group consisting of
22. The method according to claim 20, wherein the compound represented by formula (2) is selected from the group consisting of
23. The method according to claim 20, wherein the compound of formula (3)The compound represented by (A) is selected from
24. The method according to claim 20, wherein the compound represented by formula (4) is selected from the group consisting of
Background
Memory formation involves synaptic remodeling (synaptic remodeling) and requires coordinated expression of neuronal genes through a less understood process of modifying chromatin (Kandel, E.R. et al, 2014, Cell,157: 163-. Histone acetylation is a key regulator of memory storage, and remodels chromatin in the hippocampus (hippocampus) most notably in different brain regions already involved in learning and memory (Graff, j. et al, 2013, nat. rev. neurosci.,14: 97-111). Hippocampus memory consolidation requires the transcription factor CREB and coactivator CREB-binding protein (CBP), in particular Histone Acetyltransferase (HAT) activity of CBP (Wood, M.A. et al, 2005, Learn.Mem.,12: 111-972; Korzus, E. et al, 2004, Neuron,42: 961-972). In addition, histone deacetylase inhibitors enhance memory consolidation (Graff, j. et al, 2013, nat. rev. neurosci.,14: 97-111). However, the mechanisms that regulate neuronal protein acetylation in long-term memory are not fully understood.
Chromatin structure and gene expression can be dynamically altered by direct induction of intermediate metabolites of chromatin-modifying enzymes, such as acetyltransferases (Kaelin, W.G.Jr. et al, 2013, Cell,153: 56-69; Katada, S., et al, 2012, Cell,148: 24-28). Alteration of the intracellular acetyl-CoA pool (pools) manipulates histone acetylation (Cai, L. et al, 2011, mol. cell,42: 426-Bush 437; Wellen, K.E. et al, 2009, Science,324: 1076-Bush 1080); thus, the metabolic enzymes that produce nuclear acetyl-CoA can directly control histone acetylation and gene expression (Gut, P. et al, 2013, Nature,502: 489-. In mammalian cells, there are two major enzymes that produce acetyl-CoA for histone acetylation: acetate-dependent acetyl CoA synthase 2(ACSS2) and citrate-dependent ATP-citrate lyase (ACL) (Pietrocola, F. et al 2015, Cell Metab.,21: 805-821). The relative importance of ACSS2 and ACL for nuclear histone acetylation varies depending on tissue type, developmental status and disease (Wellen, K.E. et al, 2009, Science,324: 1076-1080; Pietrocola, F. et al, 2015, Cell Metab.,21: 805-821), but the role of these enzymes in post-mitotic neuronal cells is unknown.
Thus, there remains a need in the art for therapies for treating neurological and cognitive diseases and conditions (disorders). The present invention addresses this unmet need.
Drawings
The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. It is to be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
FIG. 1, comprising FIGS. 1A through 1G, shows the results of an example experiment demonstrating that nuclear ACSS2 supports neuronal gene expression. (FIG. 1A) ACSS2 localizes to the cytoplasm in undifferentiated CAD neurons. ACSS2 was imaged by immunofluorescence microscopy in CAD cells (4', 6-diamidino-2-phenylindole (DAPI) and α -tubulin (α -Tub) immunostaining showed nucleus and cytoplasm, respectively). (FIG. 1B) ACSS2 localizes to the nucleus in differentiated CAD neurons. (FIG. 1C) immunoblot analysis of Cytoplasmic (CE) and Nuclear (NE) extracts from undifferentiated CAD cells (undiff.) and differentiated CAD neurons (diff.) for ACSS2, ACL, and histone H3. Expression of nuclear ACSS2 was higher in differentiated cells (p.d.u., procedure defined unit); t-test P ═ 0.002, n ═ 3, mean ± s.d.). (FIG. 1D) ACSS2 knockdown reduced differentiation-associated upregulation of neuronal gene expression programs. Scatter plots compared fold-change (fold-change) in the number of fragments per million mapping reads (FPKM) per kilobase transcription of the induced genes between wild-type (WT) and ACSS2 Knockdown (KD) cells (fig. 6C). The edge distribution shows the histogram and the kernel density estimate. Normal least squares linear regression is shown with 95% confidence intervals. (FIG. 1E) immunoblots from lysates of differentiated CAD neurons infected with either a lentivirus control (WT) or ACSS2 knock-down vector (shaCSS2) (quantitation shown in FIG. 5G; n-3). (FIG. 1F) ACSS2 knockdown significantly reduced gene upregulation. In wild-type cells (grey), five equal-partitional groups (quintile) (red dots in fig. 6C) of up-regulated genes with the greatest fold change increased. The corresponding gene quintet group showed FPKM fold change in ACSS2 knockdown cells (green) (bars represent mean gene induction values for each quintet group; mean. + -. s.e.m.). (FIG. 1G) ACSS2i treatment of differentiated CAD neurons resulted in decreased expression of differentiation-induced genes. In wild-type CAD differentiation, all genes were mapped in order of fold change and z-scores were calculated for ACSS2i treatment and control, which expressed up-regulation as blue and down-regulation as red (RNA-seq in 24-hour ACSS2 i-treated and DMSO-treated control neurons, excluding genes with z-scores < 0.5). Scale bar, 10 μm (FIG. 1A, FIG. 1B).
Fig. 2, comprising fig. 2A through 2J, shows the results of example experiments demonstrating that ACSS2 recruits to transcriptionally active chromatin and promotes neuronal tuple protein acetylation. (FIG. 2A) shows that the Genome browser orbits (Genome browser tracks) of ChIP-seq at the Camk2A locus indicate that upon CAD neuronal differentiation, increased acetylation of H4K5, H4K12 and H3K9 co-occurs with recent ACSS2 enrichment (chromosome 18: 60,920,000-60,990,000). (FIG. 2B) Gene ontology term enrichment analysis of the first 5% of genes that became ACSS 2-bound during CAD neuronal differentiation revealed neuronal pathways. (FIG. 2C) Violin-contour plot (violin-contour plot) shows that the indicated ChIP-seq enrichment of histone acetylation occurs during neuronal differentiation of CAD cells to the top ranked ACSS2 enrichment. (FIG. 2D) ChIP-seq enrichment of 299 genes decreased after ACSS2i treatment (see example 1, methods) showed a strong correlation with histone acetylation in the differentiation status (P for all genes, P)<2.2×10-16) (AUC, area under curve; d, differentiated; u, undifferentiated). (FIG. 2E) previous associations with neuronal differentiationAnalysis of all genes (ND gene, AmiGO annotated set of 1,315 genes) as well as a subset of known ND genes induced during CAD cell differentiation (induced) showed reduced expression in ACSS2 i-treated CAD neurons (inh.) compared to DMSO-treated control neurons (con.). Inhibitor-treatment vs control, P<2.2×10-16. (fig. 2F) nuclear acetyl-CoA levels decreased in response to ACSS2 knockdown (
FIG. 3, comprising FIGS. 3A through 3F, shows the results of an example experiment demonstrating that ACSS2 ChIP-seq localization is associated with in vivo histone acetylation in mouse hippocampus. (FIG. 3A) ChIP-seq of ACSS2 and H3K9ac in mouse hippocampus. Track view for 3 neuronal genes involved in memory: arc, Egr2 and Nr2f2 (chr 15:74,496,025-74,506, 488; chr10:66,991,018-67,006, 804; and chr7:77,488,549-77,516,626, respectively) show ACSS2 and H3K9 ac. (FIG. 3B) in all RefSeq transcripts, the hippocampal ACSS2 and H3K9ac peaks co-localized with the nearest gene TSS (< 1kb from peak) in vivo. (FIG. 3C) RNA-seq expression in the hippocampus dorsalis (dHPC) was associated with hippocampal ACSS2 binding and enrichment for H3K9 acetylation. (FIG. 3D) Gene expression profiles of enriched status classes by their ACSS2 and H3K9 ac. (FIG. 3E) overlap between ACSS 2-targeted genes (hippocampus) and enrichment of CBP and H3K27ac for the entire peak set (ENCODE CBP and H3K27ac ChIP-seq in mouse forebrain and cortex). (FIG. 3F) motif analysis of ACSS2 peak from ChIP-seq in vivo in hippocampus showed the highest enrichment of the neuronal
Fig. 4, comprising fig. 4A through 4F, shows the results of an example experiment demonstrating ACSS2 knockdown in the dorsal hippocampus reduces positional memory of a injurious object and up-regulation of the immediate early gene (immediate early gene) after training. (FIG. 4A) stereotactic surgery was performed to deliver AAV9 knock-down vectors to the dorsal hippocampus (AP, -2.0mm from the forehalogen; DV, -1.4 mm; ML, ± 1.5 mm); after 4 weeks, acclimated mice were trained for subject position memory (OLM; 4 5-min training sessions in the field using 3 different subjects). After 24 hours, the mice were subjected to a retention test in which 1 object was moved to a new position (n-10 per group). (FIG. 4B) immunoblot analysis of hippocampal tissue removed from mice injected with either dorsal (d) or ventral (v) hippocampus with either eGFP control or ACSS2 knock-down vector showed a specific reduction of ACSS2 in the dorsal hippocampus. (FIG. 4C) ACSS 2-knockdown mice had impaired object position memory. During the object position memory training period (TR), eGFP control and
Fig. 5, comprising fig. 5A through 5G, shows the results of an example experiment demonstrating localization of ACSS2 to the nucleus of a neuron. (FIG. 5A) percentage of cells with nuclear staining in the ACSS2 immunofluorescence experiment (undiff., undifferentiated CAD cells; diff., differentiated CAD neurons; hippocampus, primary hippocampal neurons on day 7;
Fig. 6, comprising fig. 6A through 6O, shows the results of an example experiment demonstrating that ACSS2 regulates neuronal gene expression. (FIG. 6A, FIG. 6B) correlation plot of repetitive RNA-seq vs. interference control (scramblecontrol) in undifferentiated CAD cells (FIG. 6A) and differentiated CAD neurons (FIG. 6B). (FIG. 6C) transcriptome analysis by RNA-seq in two highly related biological replicates identified 894 genes that became upregulated in differentiated CAD neurons (red dots show genes with > 1.6-fold elevation). (FIG. 6D) pathway analysis using 894 upregulated genes of StringDB (red dots in FIG. 2A). Protein-protein interaction diagrams show the molecules that play an important role in activity-dependent signal transduction and synaptic plasticity: itpr1, Grin1, Nefh, Dync1h1 and Calm1 centered binding partner networks. (FIG. 2E) Gene ontology enrichment analysis showed upregulation of neuronal pathways. Gene ontology analysis was used for 894 genes that became upregulated in differentiated CAD neurons (FIG. 6C; identified by RNA-seq, Fold Enrichment (FE) >3.5, FDR < 0.005). (FIG. 6F) genome browser views of Nudt from RNA-seq and ChIP-seq (H4K12ac, H4K5ac and H3K9 ac: mm10 chr5:140, 327,500-140,339, 000). (FIG. 6G) relative gene enrichment (> 1.6-fold, grey bars) at genes that were up-regulated during CAD neuronal differentiation for H3K9ac, H4K5ac, and H4K12ac relative to all other genes (black bars). (FIG. 6H, FIG. 6I) correlation of repeated RNA-seq for ACL knockdown (FIG. 6H) and ACSS2 knockdown (FIG. 6I) in undifferentiated CAD cells. (FIG. 6J, FIG. 6K) correlation of repeated RNA-seq for ACL knockdown (FIG. 6J) and ACSS2 knockdown (FIG. 6K) in differentiated CAD neurons. (fig. 6L) ACL knockdown had significantly less effect on differentiation-related upregulation of neuronal gene expression (compared to fig. 1D). Scatter plots compared the fold change in FPKM for the induced genes between wild-type and ACL-knocked-down cells (fig. 6C). The edge distribution shows the histogram and the kernel density estimate. Normal least squares linear regression is shown with 95% confidence intervals. (FIG. 6M) in wild-type cells, the corresponding five-isobaric group of up-regulated genes (red dots in FIG. 6C) with the greatest fold change in FPKM was increased. ACL knockdown showed the same increasing trend as wild-type cells (red bars compared to black bars in fig. 1F), which contrasts with the severe absence in ACSS 2-knockdown cells (green bars; bars represent mean induction values of the gene for each five-aliquot group, error bars represent s.e.m.). (FIG. 6N) boxplots of global mRNA transcript levels in undifferentiated and differentiated CAD neurons from RNA-seq in wild type (interference control knockdown; grey), ACSS 2-knockdown (shaCSS2#25 knockdown; green) and ACL-knockdown (
Fig. 7, comprising fig. 7A through 7P, shows the results of an example experiment demonstrating that ACSS2 is chromatin-binding in differentiated CAD neurons. (FIG. 7A) ChIP-seq was repeated in differentiated CAD neurons using two different anti-ACSS 2 antibodies. The correlation plot shows the relative enrichment with respect to the corresponding MACS peak (default parameters, input as control, 1,598 peaks). (FIG. 7B) correlation plot shows relative genome-wide ChIP-seq enrichment. (FIG. 7C) UCSC genome browser view of ChIP-seq orbits shows that the increase in acetylation of H4K5, H4K12 and H3K9 occurs with ACSS2 enrichment relative to the Nudt1 locus upon CAD neuronal differentiation (chr5:140,326,845-140,339, 655). (FIG. 7D) UCSC genome browser view shows ChIP-seq orbits in undifferentiated CAD cells and differentiated CAD neurons relative to the Tab2 locus (chr10:7,875,000-8,004,000). (FIG. 7E) Gene ontology enrichment analysis of the gene closest to the ACSS2 peak showed enrichment for neuron-specific genes. (FIG. 7F) the frequency of ACSS2 peaks (T-antibodies) located upstream of their target genes correlated with histone acetylation. (FIG. 7G) the frequency of ACSS2 peaks (CS antibodies) located upstream of their target genes correlated with histone acetylation. (FIG. 7H) table shows the percent direct overlap of ACSS2 peak with H3K9ac, H4K5ac and H4K12ac broad MACS peaks. (FIG. 7I, FIG. 7J, FIG. 7K) the deckle diagram shows the enrichment of H3K9ac (FIG. 7I), H4K5ac (FIG. 7J) and H4K12ac (FIG. 7K) relative to the ordered enrichment of ACSS2 peaks (with zeros removed). (FIG. 7L, FIG. 7M, FIG. 7N) differentiation-induced co-enrichment of ACSS2 and acetyl broad peaks (MACS). Peak enrichment correlations are shown for H3K9ac (fig. 7L), H4K5ac (fig. 7M), and H4K12ac (fig. 7N). (FIG. 7O) the discovered de novo scanning (de novo) motif of transcription factor binding sites predicted by HOMER of all ACSS2 ChIP-seq peaks called by MACS in differentiated CAD neurons. (FIG. 7P) ChIP-seq enrichment of differentiation-induced genes as a whole in differentiated CAD neurons showed a correlation with histone acetylation.
Fig. 8, comprising fig. 8A and 8B, shows the results of an example experiment demonstrating that ACSS2 enrichment occurs with histone acetylation at neuronal genes in differentiated CAD neurons. (FIG. 8A) UCSC genome browser view of ChIP-seq orbits showed that upon CAD neuronal differentiation, increased acetylation of H4K5, H4K12, and H3K9 occurred in conjunction with ACSS2 enrichment relative to the Idua (α -l-iduronidase) locus (chr5:108,649,457-108,687, 261). (FIG. 8B) elevated acetylation levels of histones H4K5, H4K12 and H3K9 at the Slc19A1 (solute carrier family 19, member 1) gene in differentiated CAD neurons corresponded to an increase in ACSS2 enrichment (chr10:76,761,141-77,170, 455).
FIG. 9, comprising FIGS. 9A to 9I, shows the results of an example experiment showing that the enrichment of the gene ACSS2 corresponds to an increase in histone acetylation upon differentiation of CAD neurons. (FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D) pooled gene (gene) enrichment analysis showed ChIP occupancy in differentiated CAD neurons for ACSS 2-enriched first 5% of the genes (first 5% DE; red), ACSS2 (FIG. 9A), H3K9ac (FIG. 9B), H4K5ac (FIG. 9C) and H4K12ac (FIG. 9D). The last 80% of the genes (last 80% DE) are shown in blue, and the average signal of all genes (all gene DE) is shown in green. (FIG. 9E, FIG. 9F, FIG. 9G, FIG. 9H) collective gene enrichment analysis showed ChIP occupancy for ACSS2 (FIG. 9E), H3K9ac (FIG. 9F), H4K5ac (FIG. 9G) and H4K12ac (FIG. 9H) in the first 5% of genes (first 5% DE; red) that became dynamically bound by ACSS2 upon neuronal differentiation. The last 80% of the genes (last 80% DE) are shown in blue, and the average signal of all genes (all gene DE) is shown in green. (FIG. 9I) multiple linear regression analysis was performed to model the interaction between enrichment for gene ACSS2 and changes in wild-type gene expression and to visualize the interaction between differentiation-related gene expression changes and recruitment of ACSS2 to chromatin. The contour plot of the fitted regression model shows high ACSS2 enrichment levels in red and low levels in blue, and is overlaid with scatter plots of the independent gene expression variables. The model shown indicates that high ACSS2 enrichment corresponds to increased gene expression in differentiated CAD neurons.
Fig. 10, comprising fig. 10A through 10C, shows example experimental results demonstrating the function of ACSS2 in the acetylation of neuronal proteins. (fig. 10A) immunoblot analysis of whole cell lysates showed that lentiviral shRNA-mediated knock-down of ACSS2 decreased acetylation of H3K9 and H3K27 (compared to fig. 2G) using ImageJ quantification (n ═ 3, error bars show s.e.m.). (FIG. 10B) immunoblot analysis of the eluates and supernatants of IgG control and ACSS2 co-immunoprecipitation experiments showed that ACSS2 bound to acetylated chromatin. (fig. 10C) immunoblots from lysates of primary hippocampal neurons (day 7) treated with ACSS2i for 24 hours, probed with indicated antibodies (compare fig. 2J) and quantified using ImageJ (n ═ 3, error bars show s.e.m.).
Fig. 11, comprising fig. 11A through 11C, shows example experimental results demonstrating ACSS2 chromatin binding and H3K9ac in the dorsal hippocampus corresponds to H3K27ac and CBP enrichment in neuronal tissue. (FIG. 11A) genome-wide compartmentalization analysis (genome-wide comparative analysis) of hippocampal H3K9ac ChIP-seq and mouse forebrain H3K9ac ChIP-seq in vivo from ENCODE showed similar genome-wide peak distributions: despite their occurrence in different brain regions, the H3K9ac ChIP data are strongly consistent in vivo (Spearman R ═ 0.67). (FIG. 11B) overlap of RefSeq transcripts targeted by the indicated enzymes or modifications (by importing ultrasound efficiency control (GSM1629381), peaks of CBP (GSM1629373) and H3K27ac (GSM1629397) in mouse cortical neurons were recalled using MACS2 (narrow peak, FDR 0.1%); peaks correlated with the most recent TSS in all RefSeq transcripts). (FIG. 11C) Gene ontology enrichment analysis of the conventional CBP-ACSS2 target, indicating that these enzymes co-target genes that regulate synaptic biology and synaptic membrane potential.
FIG. 12, comprising FIGS. 12A through 12H, shows the results of an example experiment demonstrating that ACSS2 expression in the back hippocampus reduces impairment of positional memory of an object. (FIG. 12A) in situ hybridization of ACSS 2RNA to ACSS2 in the sagittal section of the CA1 region of the hippocampus (left panel, reference panel modified according to Allen
FIG. 13, comprising FIGS. 13A through 13F, shows the results of an example experiment demonstrating that ACSS2 regulates extraction-induced, immediate early gene upregulation in vivo. (FIG. 13A) genome-wide RNA-seq was performed on the back hippocampus of eGFP control and shaCSS 2-knockdown mice. This analysis was directed to previously identified and validated gene sets that became upregulated during the sensitive period after memory extraction. Baseline expression of the immediate early gene was unchanged in untrained animals in shacsss 2-AAV9 mice when compared to eGFP-AAV9 control mice (CC, circadian control). (FIG. 13B) early gene upregulation in the dorsal hippocampus of control injected mice during the sensitive phase after contextual memory extraction (RT, 24 hours after conditioned fear, 30min after conditioning chamber exposure). In contrast, there was no dynamic extraction-induced expression of these early response genes in ACSS 2-knockdown mice (P ═ 0.001, paired t test). (FIG. 13C) induction of immediate early gene deficiency (RT/CC) in animals injected with shacsss 2-
FIG. 14 shows the original gel blot of the immunoblots shown in FIGS. 1C, 2G, 1E and 5G. The boxes show the cropped area (cropped area) shown in FIGS. 1C, 2G, 1E, and 5G.
Figure 15 shows the original gel blot of the immunoblots shown in figures 2H and 2J. The boxes show the cropping zones shown in fig. 2H and 2J.
Fig. 16 shows the original gel blot of the immunoblots shown in fig. 4B, 5B and 10B. The boxes show the cropped regions shown in fig. 4B, 5B and 10B.
Figure 17 shows the physiological origin of acetate.
FIG. 18 shows measurement of intraperitoneal injection of EtOH-13C2Thereafter, graphs of histone acetylation in mouse cortex, hippocampus, and liver.
Figure 19 shows a graph measuring histone acetylation in the hippocampus of ACSS 2-knockdown mice.
Figure 20 shows a graph demonstrating the difference in histone acetylation in dorsal, ventral, and muscle of ACSS 2-knockdown mice relative to wild-type mice.
Fig. 21, comprising fig. 21A through 21E, shows that alcohol metabolites provide the starting material for histone acetylation in the brain. FIG. 21A shows in vivo EtOH-d6Experimental summary of mass spectrometry. FIG. 21B shows heavy EtOH-d indicating metabolism6Experimental results for acetylation of histones incorporated in hippocampus. The axis of the spider plot (Arachne plot) represents the% of the third isotope of the acetylated peptide, which corresponds to D3The form of the marker; the natural relative abundance of this isotope was evident in the "none" and "saline 1 h" treatment groups. Fig. 21C shows the results of an experiment showing that marker incorporation in the acetylation of cortical histones shows a pattern similar to that of the hippocampus (patterrn). Fig. 21D shows experimental results, which indicate that marker incorporation in histone acetylation occurs earlier in the liver, which is a major site of alcohol metabolism. FIG. 21E shows experimental results indicating that histone acetylation is relatively independent of liver alcohol metabolism in skeletal muscle (tissue with low ACSS2 expression).
Fig. 22, comprising fig. 22A to 22D, shows histone acetylation in wild-type mice. FIGS. 22A-22C show mass spectra indicating the relative abundance of deuterated histone H4-triacetyl peptide (aa4-17) in the dorsal hippocampus of wild-type mice. Fig. 22A shows the mass spectrum at baseline. FIG. 22B shows d6Mass spectrum at 30min after EtOH injection. FIG. 22C shows d6Mass spectrum 4 hours after EtOH injection. Figure 22D shows experimental results demonstrating that histone acetylation is relatively independent of liver alcohol metabolism in skeletal muscle. Relative abundance of deuterated histone acetylation at 30min and 4h in Wild Type (WT) mice and 30min in hippocampal ACSS2KD mice (compared to fig. 21E).
FIG. 23, comprising FIGS. 23A through 23E, shows EtOH-d Knockdown (KD) in the dorsal hippocampus (dHPC) ACSS26Mass spectrometry analysis of (1). Fig. 23A shows the results of experiments demonstrating that expression of ACSS2 in the dorsal hippocampus was knocked down to prevent incorporation of heavy markers into histone acetylation. Figure 23B shows the results of an experiment showing that incorporation of the heavy marker in the ventral hippocampus (where ACSS2 levels were normal) was unchanged compared to control mice in the same animals. Fig. 23C shows the results of an experiment demonstrating that the acetylation of histones in the dorsal hippocampus was readily marked by the introduction of diacetate intraperitoneally. Figure 23D shows experimental results demonstrating that the introduction of diacetate via intraperitoneal injection readily marks histone acetylation in the cortex. Figure 23E shows the results of experiments demonstrating that neuronal ACSS2 in the brain activates acetate from liver alcohol breakdown and that the acetate readily induces gene-regulated histone acetylation.
Fig. 24, comprising fig. 24A through 24E, shows ACSS 2-mediated acetate-induced transcription in primary hippocampal neurons. FIGS. 24A and 24B show RNA-seq in primary hippocampal neurons isolated from C57/Bl6 mouse embryos and treated with acetate (10mM) in the presence or absence of a small molecule inhibitor of ACSS 2(ACSS2 i). Figure 24A shows a heat map showing 7,600 differentially expressed genes by acetate treatment, and the third column shows the behavior of those genes in the presence of ACSS2 inhibitor. In the presence of ACSS2i, 2107 of 3613 acetate-induced genes were not upregulated (N-4 per group). Figure 24B shows experimental results demonstrating that acetate-induced genes are not regulated by ACSS2i treatment in the absence of acetate. Fig. 24C shows acetate-induced genes in primary hippocampal neurons in blue; the Gene Ontology (GO) terminology analysis of ACSS2 i-sensitive genes (non-overlapping with yellow, acetate + ACSS2i) is shown below. FIG. 24D shows GO terminology analysis of genes sensitive to acetate and directly bound by ACSS2 (according to ACSS2 ChIP-seq). FIG. 24E shows HOMER unsupervised de novo scanning motif analysis of ACSS2 hippocampal binding sites targeting acetate-sensitive genes.
FIG. 25, comprising FIGS. 25A through 25D, shows genes regulated by acetate. Figure 25A shows RNAseq showing differentially regulated genes in primary hippocampal neurons treated with 10mM acetate. Fig. 25B shows Gene Ontology (GO) analysis of significantly up-regulated (red) and significantly down-regulated (blue) genes. Figure 25C shows experimental results demonstrating that ethanol was also upregulated in vitro by acetate in primary hippocampal neurons-81 of 214 genes upregulated in the hippocampus of injected mice (p ═ 6.60 e-17). Fig. 25D shows experimental results demonstrating the cumulative number of ACSS2 peaks near the Transcription Start Site (TSS) of an acetylated ACSS2i sensitive gene, indicating that most ACSS2 binding events occurred at or near the gene promoter.
Fig. 26, comprising fig. 26A and 26B, shows that alcohol metabolites provide the starting material for histone acetylation in fetal brain. FIG. 26A shows the weights d indicating metabolism6Experimental results for histone acetylation with EtOH incorporation into maternal brain. Fig. 26B shows the incorporation of the heavy marker into histone acetylation in fetal brain. Data representation from parent d6Two mixed groups of EtOH injected 4 embryos. The axis of the spider diagram represents the percentage of the third isotope of the acetylated peptide, which corresponds to D3A marker format.
FIG. 27 shows the results of an experiment showing that the peptide H4 aa4-17 has 3 acetyl groups (hippocampus).
FIG. 28 shows experimental results of SILAC-mass spectrometry experiments.
FIG. 29 shows an assay designed to determine the efficacy of decreasing ACSS2 catalytic activity and
Figure 30 shows the chemical structure and activity of ADG-204.
Figure 31 shows the chemical structure and activity of ADG-205.
Figure 32 shows the chemical structure and activity of ADG-206.
Detailed Description
The present invention relates to compositions and methods for treating neurological and cognitive diseases and disorders. In some embodiments, the present invention provides compositions and methods for treating memory-related diseases and disorders. In various embodiments, the compositions and methods described herein are useful in the treatment of anxiety disorders and conditions, such as phobias, panic disorders, psychosocial stress (e.g., as seen in disaster, catastrophe, or violent victims), obsessive-compulsive disorders, generalized anxiety disorder, and post-traumatic stress disorder (PTSD). In some embodiments, the compositions and methods described herein are useful for modulating long term memory storage or consolidation.
The invention also relates to compositions and methods for treating addiction and/or addiction-related diseases or disorders. In various embodiments, the compositions and methods described herein are useful for preventing or treating acute alcohol-induced memory impairment (alcohol-induced memory impairment) and chronic alcohol-induced memory impairment.
In some embodiments, the methods of the invention comprise modulating chromatin acetylation. In one embodiment, the methods of the invention reduce chromatin acetylation. In one embodiment, the chromatin is neuronal chromatin. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor.
In certain instances, the compositions and methods described herein relate to inhibiting acetate-dependent acetyl CoA synthase 2(ACSS 2). In one embodiment, the compositions of the present invention comprise an inhibitor of ACSS2. In one embodiment, the inhibitor of ACSS22 inhibits expression, activity, or both of ACSS2.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.
Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to methods conventional in the art and various general references (e.g., Sambrook and Russell,2012, Molecular Cloning, laboratory apparatus Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY and Ausubel et al, 2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout the document.
As used herein, each of the following terms has the meaning associated therewith in this section.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.
As used herein, "about" when referring to a measurable value, such as an amount, time interval, etc., is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, or ± 0.1% of the stated value, as such variations are suitable for practicing the disclosed methods.
The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof having at least one observable or detectable characteristic (e.g., age, treatment, time, etc.) that is different from those organisms, tissues, cells or components thereof that exhibit the "normal" (expected) individual characteristic. Characteristics that are normal or expected for one cell or tissue type may be abnormal for a different cell or tissue type.
"antisense" specifically refers to a nucleic acid sequence of the non-coding strand of a double-stranded DNA molecule encoding a protein, or to a sequence that is substantially homologous to said non-coding strand. As defined herein, an antisense sequence is complementary to a sequence of a double-stranded DNA molecule encoding a protein. The antisense sequence need not be complementary to only the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to a regulatory sequence, indicated on the coding strand of the protein-encoding DNA molecule, which controls expression of the coding sequence.
A "disease" is a health state of an animal, wherein the animal may not maintain homeostasis, and wherein the health of the animal continues to deteriorate if the disease is not improved.
In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is not as good as it would be in the absence of the disorder. The condition does not necessarily lead to a further reduction in the health status of the animal if left untreated.
A disease or disorder is "alleviated (relieved)" if the severity of signs or symptoms of the disease or disorder, the frequency with which a patient experiences such signs or symptoms, or both, is reduced.
An "effective amount" or "therapeutically effective amount" of a compound is an amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered.
"encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (i.e., rRNA, tRNA and mRNA) or having defined amino acid sequences and biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be referred to as the protein or other product encoding the gene or cDNA.
The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cell thereof, whether in vitro or in vivo, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.
"therapeutic" treatment is treatment administered to a subject who exhibits signs or symptoms of a disease or disorder for the purpose of reducing or eliminating those signs or symptoms.
As used herein, "treating a disease or disorder" means reducing the severity and/or frequency of signs or symptoms of a disease or disorder experienced by a patient.
For antibodies, the term "specifically binds" as used herein refers to antibodies that recognize a specific antigen, but do not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, this cross-species reactivity does not itself alter the classification of antibodies as specific. In another example, an antibody that specifically binds to an antigen may also bind to a different allelic form of the antigen. However, this cross-reactivity does not itself alter the classification of antibodies as specific.
In some cases, the term "specific binding" may be associated with the interaction of an antibody, protein or peptide with a second chemical, used to indicate that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical; for example, in general, antibodies recognize and bind to specific protein structures, not proteins. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody in the reaction between labeled "A" and the antibody.
A "coding region" of a gene includes the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene, which are homologous or complementary, respectively, to the coding region of an mRNA molecule produced by transcription of the gene.
The "coding region" of an mRNA molecule also includes the nucleotide residues of the mRNA molecule that match the anticodon region of the transfer RNA molecule during translation of the mRNA molecule or that encode a stop codon. Thus, the coding region may include nucleotide residues of codons that include amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
"complementary" as used herein to denote nucleic acids refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. Adenine residues of a first nucleic acid region are known to be capable of forming specific hydrogen bonds ("base pairing") with residues of a second nucleic acid region that are antiparallel to the first region (if the residues are thymine or uracil). Similarly, a cytosine residue of a first nucleic acid strand is known to be capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if the two regions are arranged in an antiparallel manner and at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In one embodiment, the first region comprises a first portion and the second region comprises a second portion, wherein at least about 50% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion when the first and second portions are arranged in an anti-parallel manner. In one embodiment, at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In one embodiment, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term "DNA" as used herein is defined as deoxyribonucleic acid.
The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
The term "expression vector" as used herein refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product capable of being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, sirnas, ribozymes, and the like. Expression vectors can contain a variety of control sequences which represent nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.
The term "fusion polypeptide" refers to a chimeric protein comprising a protein of interest (e.g., luciferase) linked to a heterologous sequence (e.g., a non-luciferase amino acid or protein).
The term "homology" refers to a degree of complementarity. Partial homology or complete homology (i.e., identity) may exist. Homology is typically measured using Sequence Analysis Software (e.g., Sequence Analysis Software Package of the genetics Computer group. university of Wisconsin Biotechnology center.1710university Avenue. Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications. Conservative substitutions typically include substitutions within the following ranges: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
"isolated" means altered from or removed from the native state. For example, a nucleic acid or peptide that is naturally present in a living animal in its normal background is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the material with which it coexists in its natural background is "isolated. An isolated nucleic acid or protein may be in a substantially purified form, or may be present in a non-natural environment, such as, for example, a host cell.
When used in conjunction with a nucleic acid, as in "an isolated oligonucleotide" or "isolated polynucleotide," the term "isolated" refers to a nucleic acid sequence that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated nucleic acid exists in a form or environment that is different from the form or environment in which it exists in nature. In contrast, non-isolated nucleic acids (e.g., DNA and RNA) exist in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is present on the host cell chromosome adjacent to an adjacent gene; RNA sequences (e.g., a particular mRNA sequence encoding a particular protein) are present in a cell as a mixture with a variety of other mrnas that encode a number of proteins. However, for example, an isolated nucleic acid includes such nucleic acid in a cell that normally expresses the nucleic acid, where the nucleic acid is in a chromosomal location different from that of the native cell, or is otherwise flanked by nucleic acid sequences different from those found in nature. An isolated nucleic acid or oligonucleotide may exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide contains at least the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both the sense and antisense strands (i.e., the oligonucleotide may be double-stranded).
When used in conjunction with a polypeptide, as in "isolated protein" or "isolated polypeptide," the term "isolated" refers to a polypeptide that is identified and separated from at least one contaminant with which it is ordinarily associated in its source. Thus, an isolated polypeptide exists in a form or environment that is different from the form or environment in which it exists in nature. In contrast, non-isolated polypeptides (e.g., proteins and enzymes) exist in the state they exist in nature.
"nucleic acid" means any nucleic acid, whether it consists of deoxyribonucleosides or ribonucleosides, and whether it consists of phosphodiester linkages or modified linkages, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of these linkages. The term nucleic acid also specifically includes nucleic acids consisting of bases other than the 5 biologically occurring bases adenine, guanine, thymine, cytosine and uracil. The term "nucleic acid" generally refers to large polynucleotides.
Polynucleotide sequences are described herein using conventional notation: the left end of the single-stranded polynucleotide sequence is the 5' -end; the left-hand orientation of a double-stranded polynucleotide sequence is referred to as the 5' -orientation.
The direction of 5 'to 3' addition of nucleotides to nascent RNA transcripts is referred to as the direction of transcription. A DNA strand having the same sequence as mRNA is called "coding strand"; sequences on the DNA strand located 5' to a reference point on the DNA are referred to as "upstream sequences"; sequences on the DNA strand 3' of the reference point on the DNA are referred to as "downstream sequences".
By "expression cassette" is meant a nucleic acid molecule comprising a coding sequence operably linked to promoter/regulatory sequences necessary for transcription and optionally translation of the coding sequence.
The term "operably linked" as used herein denotes the linkage of nucleic acid sequences in a manner that results in a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. The term also refers to the linkage of sequences encoding amino acids in a manner that produces a functional (e.g., enzymatic activity, ability to bind to a binding partner, ability to inhibit, etc.) protein or polypeptide.
As used herein, the term "promoter/regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other examples, the sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in an inducible manner.
An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, results in the significant production of the gene product only in the presence of an inducing agent corresponding to the promoter.
A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, results in the production of the gene product in a cell under most or all of the physiological conditions of the cell.
The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. The person skilled in the art has the following general knowledge: nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides may be hydrolyzed to nucleosides. Polynucleotides as used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including without limitation, by recombinant means, i.e., cloning of the nucleic acid sequence from a recombinant library or cell genome using conventional cloning techniques and PCR or the like, and by synthetic means.
In the context of the present invention, the following abbreviations for common nucleobases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.
As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and does not limit the maximum number of amino acids that a protein or peptide sequence can contain. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of which there are a variety of types. "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide includes a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.
As used herein, a "peptidomimetic" is a compound containing a non-peptide structural component capable of mimicking the biological effect of a parent peptide. The peptidomimetic may or may not comprise a peptide bond.
The term "RNA" as used herein is defined as ribonucleic acid.
"recombinant polynucleotide" refers to a polynucleotide having sequences that are not naturally associated together. The amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector may be used to transform a suitable host cell.
Recombinant polynucleotides may also serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.).
The term "recombinant polypeptide" as used herein is defined as a polypeptide produced by using recombinant DNA methods.
As used herein, "associated" refers to the covalent attachment of one molecule to a second molecule.
As used herein, the term "transdominant negative mutant gene" refers to a gene that encodes a polypeptide or protein product that prevents other copies of the same gene or gene product that are not mutated (i.e., have the wild-type sequence) from functioning properly (e.g., by inhibiting the function of the wild-type protein). The product of a transdominant negative mutant gene is referred to herein as "dominant negative" or "DN" (e.g., a dominant negative protein or DN protein).
The phrase "inhibit," as used herein, means to reduce expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and/or protein by a measurable amount or to prevent altogether. Inhibitors are compounds, e.g., antagonists, that bind to, partially or completely block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate protein, gene, and mRNA stability, expression, function, and activity.
As the term is used herein, a "variant" is a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but which retains the essential biological properties of the reference molecule. Sequence changes of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Sequence variations of peptide variants are often limited or conserved, such that the sequences of the reference peptide and the variant are very similar overall and identical in multiple regions. The variant and reference peptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. Variants of a nucleic acid or peptide may be naturally occurring, such as allelic variants, or may be variants that are known not to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be prepared by mutagenesis techniques or by direct synthesis.
A "vector" is a composition of matter that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. A variety of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be taken to include non-plasmid and non-viral compounds that facilitate delivery of nucleic acids to cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful within the scope of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. There are a variety of techniques in the art for administering compounds including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.
As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the composition components in which it is contained.
As used herein, the language "pharmaceutically acceptable salt" refers to a salt of an administered compound prepared from a pharmaceutically acceptable non-toxic acid, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, hexafluorophosphoric acid, citric acid, gluconic acid, benzoic acid, propionic acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid, fumaric acid, succinic acid, tartaric acid, glycyrrhizic acid hydrate (amsonic acid), pamoic acid (pamoic acid), p-toluenesulfonic acid and methanesulfonic acid. Suitable organic acids may be selected from, for example, fatty acids, aromatic acids, carboxylic acids and sulfonic acids of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, camphorsulfonic acid, citric acid, fumaric acid, gluconic acid, isethionic acid, lactic acid, malic acid, mucic acid (mucic acid), tartaric acid, p-toluenesulfonic acid, glycolic acid, glucuronic acid, maleic acid, furoic acid, glutamic acid, benzoic acid, anthranilic acid, salicylic acid, phenylacetic acid, mandelic acid, pamoic acid, methanesulfonic acid, ethanesulfonic acid, pantothenic acid, benzenesulfonic acid (benzenesulfonate), stearic acid, sulfanilic acid (sulfanilic acid), alginic acid, galacturonic acid and the like. In addition, by way of non-limiting example, pharmaceutically acceptable salts include alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-related or potassium), and ammonium salts.
As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent or encapsulating material, that is involved in carrying or delivering a compound useful within the scope of the invention to a patient, so that it may exert its intended effect. Typically, these constructs are carried or delivered from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compounds useful within the invention, and not deleterious to the patient in question. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl dodecanoate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; a surfactant; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials used in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, that are compatible with the activity of the compounds useful within the scope of the present invention and that are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may also include pharmaceutically acceptable salts of the compounds useful within the scope of the present invention. Other additional ingredients that may be included in Pharmaceutical compositions used in the practice of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (master catalog of Genaro, mack publishing co.,1985, Easton, PA), which is incorporated herein by reference.
As used herein, the term "efficacy" refers to the dose required to produce a half maximal response (ED)50)。
As used herein, the term "pharmacodynamic" refers to the maximal effect (Emax) achieved within the assay.
The term "alkyl" as used herein, alone or as part of another substituent means having the indicated number of carbon atoms (i.e., C), unless otherwise specified1-6Represents 1 to 6 carbon atoms) and includes straight-chain, branched-chain or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl and cyclopropylmethyl.
As used herein, the term "substituted alkyl" denotes an alkyl group as defined above substituted with 1,2 or 3 substituents selected from halogen, -OH, alkoxy, -NH2Amino group, azido group, -N (CH)3)2-C (═ O) OH, trifluoromethyl, -C.ident.N, -C (═ O) O (C)1-C4) Alkyl, -C (═ O) NH2、-SO2NH2、-C(＝NH)NH2and-NO2. Examples of substituted alkyls include, but are not limited to, 2-difluoroPropyl, 2-carboxycyclopentyl and 3-chloropropyl.
As used herein, unless otherwise specified, the term "heteroalkyl," alone or in combination with another term, means a stable straight or branched chain alkyl group consisting of the indicated number of carbon atoms and one or two heteroatoms selected from O, N and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom may be located anywhere in the heteroalkyl group, including between the remaining heteroalkyl group and the segment to which it is attached, and may be attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH2-CH2-CH3、-CH2-CH2-CH2-OH、-CH2-CH2-NH-CH3、-CH2-S-CH2-CH3and-CH2CH2-S(＝O)-CH3. Up to two heteroatoms may be consecutive, such as (e.g.) -CH2-NH-OCH3or-CH2-CH2-S-S-CH3。
The term "alkoxy" as used herein, alone or in combination with other terms, is used, unless otherwise specified, to denote an alkyl group as defined above having the indicated number of carbon atoms, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy), and higher carbon (higher) homologs and isomers, which are linked to the rest of the molecule through an oxygen atom.
As used herein, unless otherwise specified, the term "halo" or "halogen", alone or as part of another substituent, denotes a fluorine, chlorine, bromine or iodine atom.
As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group in which each atom (i.e., backbone atom) forming the ring is a carbon atom. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl is fused to an aromatic ring. Cycloalkyl groups include groups having 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include (but are not limited to) the following moieties:
monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl groups include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyl groups include adamantane and norbornane. The term cycloalkyl includes "unsaturated non-aromatic carbocyclic" or "non-aromatic unsaturated carbocyclic" groups, both of which represent non-aromatic carbocyclic rings as defined herein, which contain at least one carbon double bond or one carbon triple bond.
As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a heteroalicyclic group containing 1 to 4 ring heteroatoms independently selected from O, S and N. In one embodiment, each heterocycloalkyl group has from 4 to 10 atoms in its ring system, provided that the ring of the group does not contain two adjacent O or S atoms. In another embodiment, the heterocycloalkyl group is fused to an aromatic ring. In one embodiment, the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. Unless otherwise specified, the heterocyclic ring system may be attached at any heteroatom or carbon atom that provides a stable structure. Heterocycles can be aromatic or non-aromatic in nature. In one embodiment, the heterocycle is heteroaryl.
Examples of 3-membered heterocycloalkyl include, and are not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and β lactam. Examples of 5-membered heterocycloalkyl groups include, but are not limited to, pyrrolidine, oxazolidine, and thiazolidinedione. Examples of 6-membered heterocycloalkyl include, but are not limited to, piperidine, morpholine, and piperazine. Other non-limiting examples of heterocycloalkyl groups are:
examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, propylene oxide, thiazabutane, pyrrolidine, pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane, 2, 3-dihydrofuran, 2, 5-dihydrofuran, tetrahydrofuran, thiophenane, piperidine, 1,2,3, 6-tetrahydropyridine, 1, 4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2, 3-dihydropyran, tetrahydropyran, 1, 4-dioxane, 1, 3-dioxane, homopiperazine, homopiperidine, 1, 3-dioxane (1,3-dioxepane), 4,7-dihydro-1, 3-dioxane (4,7-dihydro-1, 3-dioxapin) and cyclohexene oxide.
As used herein, the term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings and having aromaticity, i.e., having (4n +2) delocalized pi (pi) electrons, where n is an integer.
The term "aryl" as used herein, alone or in combination with other terms, means, unless otherwise specified, a carbocyclic aromatic system containing one or more rings (typically 1,2 or 3 rings) wherein the rings may be linked together in a pendant manner, such as biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracyl, and naphthyl.
As used herein, the term "aryl- (C)1-C3) Alkyl "denotes a functional group in which a 1-to 3-carbaalkylene chain is attached to an aryl group, e.g., -CH2CH2-phenyl. In one embodiment, aryl- (C)1-C3) Alkyl is aryl-CH2-or aryl-CH (CH)3) -. The term "substituted aryl- (C)1-C3) Alkyl represents aryl- (C) wherein aryl is substituted1-C3) An alkyl functional group. Similarly, the term "heteroaryl- (C)1-C3) Alkyl "represents a functional group in which a1 to 3 carbon alkylene chain is attached to a heteroaryl group, e.g., -CH2CH2-a pyridyl group. The term "substituted heteroaryl- (C)1-C3) Alkyl represents heteroaryl- (C) wherein heteroaryl is substituted1-C3) An alkyl functional group.
As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heterocyclic ring having aromatic character. The polycyclic heteroaryl group may include one or more rings that are partially saturated. Examples include the following:
examples of heteroaryl groups also include pyridyl, pyrazinyl, pyrimidinyl (specifically 2-and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (specifically 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (specifically 3-and 5-pyrazolyl), isothiazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, 1,3, 4-triazolyl, tetrazolyl, 1,2, 3-thiadiazolyl, 1,2, 3-oxadiazolyl, 1,3, 4-thiadiazolyl, and 1,3, 4-oxadiazolyl.
Examples of the polycyclic heterocycles and heteroaryls include indolyl (specifically, 3-, 4-, 5-, 6-and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (specifically, 1-and 5-isoquinolyl), 1,2,3, 4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (specifically, 2-and 5-quinoxalinyl), quinazolinyl, 2, 3-diazanaphthyl (phthalazinyl), 1, 8-naphthyridinyl, 1, 4-benzodioxane, coumarin, dihydrocoumarin, 1, 5-naphthyridinyl, benzofuranyl (specifically, 3-, 4-, 5-, 6-and 7-benzofuranyl), 2, 3-dihydrobenzofuranyl, 1, 2-benzisoxazolyl, dihydrocoumarinyl, cinnolinyl, quinoxalinyl, 2-and 5-quinoxalinyl, Benzothienyl (specifically, 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (specifically, 2-benzothiazolyl and 5-benzothiazolyl), purinyl (purinyl), benzimidazolyl (specifically, 2-benzimidazolyl), benzotriazolyl, thioxanthyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidyl, and quinolizidinyl.
As used herein, the term "substituted" refers to an atom or group of atoms replacing a hydrogen as a substituent attached to another group. The term "substituted" also means any level of substitution, i.e., mono-, di-, tri-, tetra-, or penta-substitution, wherein such substitution is permitted. Substituents are independently selected, and substitutions may be in any chemically accessible position. In one embodiment, the number of substituents varies between 1 and 4. In another embodiment, the number of substituents varies between 1 and 3. In another embodiment, the number of substituents varies between 1 and 2.
As used herein, the term "optionally substituted" means that the group referred to may be substituted or unsubstituted. In one embodiment, a group referred to is optionally substituted with zero substituents, i.e., the group referred to is unsubstituted. In another embodiment, the mentioned groups are optionally substituted with one or more other groups individually and independently selected from the groups described herein.
In one embodiment, the substituents are independently selected from oxygen, halogen, -CN, -NH2、-OH、-NH(CH3)、-N(CH3)2Alkyl (including linear, branched and/or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoroalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S (═ O)2Alkyl, -C (═ O) NH [ substituted or unsubstituted alkyl or substituted or unsubstituted phenyl]-C (═ O) N [ H or alkyl]2-OC (═ O) N [ substituted or unsubstituted alkyl group]2-NHC (═ O) NH [ substituted or unsubstituted alkyl or substituted or unsubstituted phenyl-]-NHC (═ O) alkyl, -N [ substituted or unsubstituted alkyl]C (═ O) [ substituted or unsubstituted alkyl group]-NHC (═ O) [ substituted or unsubstituted alkyl group]-C (OH) [ substituted or unsubstituted alkyl group]2and-C (NH)2) [ substituted or unsubstituted alkyl group]2. In another embodiment, for example, the optional substituents are selected from oxygen, fluorine, chlorine, bromine, iodine, -CN, -NH2、-OH、-NH(CH3)、-N(CH3)2、-CH3、-CH2CH3、-CH(CH3)2、-CF3、-CH2CF3、-OCH3、-OCH2CH3、-OCH(CH3)2、-OCF3、-OCH2CF3、-S(＝O)2-CH3、-C(＝O)NH2、-C(＝O)-NHCH3、-NHC(＝O)NHCH3、-C(＝O)CH3、-ON(O)2and-C (═ O) OH. In another embodiment, the substituents are independently selected from C1-6Alkyl, -OH, C1-6Alkoxy, halo, amino, acetamido, oxygen, and nitro. In another embodiment, the substituents are independently selected from C1-6Alkyl radical, C1-6Alkoxy, halo, acetamido, and nitro. As used herein, when the substituent is alkyl or alkoxy, the carbon chain may be branched, straight, or cyclic.
The range is as follows: throughout this disclosure, various aspects of the invention may exist in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a range such as1 to 6 is to be considered to have specifically disclosed sub-ranges such as1 to 3,1 to 4,1 to 5, 2 to 4, 2 to 6,3 to 6, etc., as well as individual numerical values within that range, e.g., 1,2, 2.7, 3,4, 5, 5.3, and 6. This is applicable regardless of the width of the range.
Description of the invention
The present invention relates to compositions and methods for treating or preventing memory-related diseases or disorders, such as (but not limited to) PTSD, addiction and addiction-related diseases or disorders. The present invention is based in part on the following findings: ACSS2 regulates histone acetylation and neuronal gene transcription. Inhibition of ACSS2 expression (e.g., by RNA interference) or ACSS2 activity (e.g., by small molecules) reduces histone acetylation and impairs long-term (long-term) spatial memory. Accordingly, the present invention relates to compositions and methods for inhibiting ACSS2 to inhibit histone acetylation and treat memory-related diseases or disorders.
In some embodiments, the compositions of the present invention comprise an inhibitor of ACSS2 activity. In some embodiments, the composition comprises an inhibitor of ACSS2 expression. Thus, in various embodiments, the compositions comprise an isolated nucleic acid (e.g., siRNA, miRNA, ribozyme, antisense RNA, etc.) that reduces the expression level of ACSS2 in a cell.
In some embodiments, the composition comprises an inhibitor of ACSS2 activity. Thus, in various embodiments, the composition comprises a small molecule, nucleic acid, peptide, antibody, antagonist, aptamer, or peptidomimetic that reduces the activity of ACSS2.
In some embodiments, the present invention provides methods for treating a neurological or cognitive disease or disorder in a subject. In one embodiment, the neurological or cognitive disease or disorder is a memory-related disease or disorder. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor. In one embodiment, the method is useful in treating PTSD.
In another embodiment, the invention provides a method for treating an addiction or addiction related disease or disorder in a subject. In some embodiments, the methods of the invention are useful in treating acute alcohol-induced memory impairment. In other embodiments, the methods of the invention are useful in treating memory decline caused by chronic alcohol. In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor.
Inhibitors
In some embodiments, the present invention provides compositions for treating a neurological or cognitive disease or disorder in a subject. In one embodiment, the neurological or cognitive disease or disorder is a memory-related disease or disorder. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor. In another embodiment, the invention provides a composition for treating an addiction or addiction related disease or disorder in a subject. In some embodiments, the methods of the invention are useful in treating acute alcohol-induced memory impairment. In other embodiments, the methods of the invention are useful in treating memory decline caused by chronic alcohol. In certain embodiments, the composition inhibits expression, activity, or both of ACSS2 in the subject.
In one embodiment, the compositions of the present invention comprise an inhibitor of ACSS2. In various embodiments, the inhibitor of ACSS2 is any compound, molecule, or agent that reduces, inhibits, or prevents the expression, activity, or function of ACSS2. Thus, an inhibitor of ACSS2 is any compound, molecule, or agent that reduces expression, activity, or both of ACSS2. In various embodiments, the inhibitor of ACSS2 is a nucleic acid, a peptide, a small molecule, an siRNA, a ribozyme, an antisense nucleic acid, an antagonist, an aptamer, a peptidomimetic, or any combination thereof.
Small molecule inhibitors
In some embodiments, the inhibitor is a small molecule. When the inhibitor is a small molecule, the small molecule can be obtained using standard methods known to the skilled person. These methods include chemical organic synthesis or biological methods. Biological methods include purification from biological sources, recombinant synthesis, and in vitro translation systems using methods well known in the art. In one embodiment, the small molecule inhibitors of the present invention comprise organic molecules, inorganic molecules, biomolecules, synthetic molecules, and the like.
Combinatorial libraries of molecularly diverse compounds and methods of making libraries that are potentially useful in the treatment of a variety of diseases and conditions are well known in the art. The methods can use a variety of techniques well known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of individual compounds, synthesis of chemical mixtures, rigid core structures (rigid corestractures), flexible linear sequences, deconvolution strategies (deconvolution strategies), tagging techniques, and generation of unbiased molecular maps (unbiased structures) vs. for lead compound discovery.
In a general approach to small library synthesis, activated core molecules are condensed with a plurality of building blocks (building blocks), resulting in the generation of a combinatorial library of covalently linked core-building block assemblies. The shape and rigidity of the core determines the orientation of the building blocks in shape space. Libraries can be biased to target characterized biological structures by changing cores, bonds, or building blocks ("targeted libraries") or synthetic libraries can be biased with lower structure using flexible cores.
Even though salts are not shown, the small molecules and small molecule compounds described herein may exist as salts, and it is to be understood that the invention encompasses all salts and solvates of the inhibitors shown herein as well as non-salts and non-solvated forms of the inhibitors, as is well known to the skilled artisan. In some embodiments, the salts of the inhibitors described herein are pharmaceutically acceptable salts.
When tautomeric forms may exist for any of the inhibitors described herein, each tautomeric form is intended to be encompassed by the invention, although only one or some tautomeric forms may be explicitly shown. For example, when a 2-hydroxypyridinyl moiety is shown, the corresponding 2-pyridone tautomer is also expected to be shown.
The invention also includes any or all stereochemical forms, including any enantiomeric or diastereomeric form of the inhibitor. The recitation of structures or names herein is intended to encompass all possible stereoisomers of the indicated inhibitors. The invention also encompasses all forms of the inhibitor, such as crystalline or amorphous forms of the inhibitor. Also contemplated are compositions comprising an inhibitor of the invention, such as compositions comprising a substantially pure inhibitor in its particular stereochemical form, or compositions comprising a mixture (including two or more stereochemical forms) of inhibitors of the invention in any ratio, such as a racemic or non-racemic mixture.
In one embodiment, the small molecule inhibitors of the invention include analogs or derivatives of the inhibitors described herein.
In one embodiment, the small molecules described herein are candidate molecules for derivatization. As such, in certain instances, analogs of the small molecules described herein having modulated potency, selectivity, and solubility are included herein and provide useful lead compounds for drug discovery and drug development. Therefore, in some cases, new analogs were designed during optimization, taking into account drug delivery, metabolism, novelty, and safety issues.
In some cases, the small molecule inhibitors described herein are derivatized/similarly materialized as is well known in the art of combinatorial and medicinal chemistry. Analogs or derivatives can be prepared by adding and/or substituting functional groups at different positions. As such, the small molecules described herein can be converted into derivatives/analogs using well-known chemical synthesis procedures. For example, all hydrogen atoms or substituents may be selectively modified to produce new analogs. In addition, the linking atom or group may be modified to have a longer or shorter linker with a carbon backbone or heteroatom. In addition, the ring groups may be altered to have different numbers of atoms in the ring and/or to include heteroatoms. In addition, aromatics may be converted to rings, and vice versa. For example, the ring may be 5 to 7 atoms, and may be a homocyclic or heterocyclic ring.
As used herein, the term "analog" or "derivative" is intended to mean a compound or molecule prepared from a parent compound or molecule by one or more chemical reactions. As such, an analog may be a structure having a structure similar to the small molecule inhibitors described herein or may be based on the backbone of the small molecule inhibitors described herein, but differs therefrom with respect to certain components or structural compositions, which may have a similar or opposite effect metabolically. Any analog or derivative of a small molecule inhibitor according to the invention may be used to treat an autoimmune disease or disorder.
In one embodiment, the small molecule inhibitors described herein may be independently derivatized/similarly materialized by modifying hydrogen groups independently of each other to other substituents. That is, each atom on each molecule can be independently modified relative to other atoms on the same molecule. Any conventional modification for producing derivatives/analogs may be used. For example, the atoms and substituents can independently be comprised of hydrogen, alkyl, aliphatic, linear aliphatic, aliphatic with chain heteroatoms, branched aliphatic, substituted aliphatic, cycloaliphatic, heterocycloaliphatic with one or more heteroatoms, aromatic, heteroaromatic, polyaromatic, polyamino acid, peptide, polypeptide, combinations thereof, halogen-substituted aliphatic, and the like. In addition, any ring group on the compound can be derivatized to increase and/or decrease ring size and to change the backbone atoms to carbon or heteroatoms.
In one embodiment, the small molecule inhibitor is a compound represented by formula (1)
Wherein, X11Is selected from C (R)14)(R15) O, S and NR15；
Each occurrence of X12Is selected from C (R)14)(R15) O, S and NR15；
R11Selected from hydrogen, -OR15Alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein R11Is optionally substituted;
R12and R13Each independently selected from hydrogen, alkyl, aryl and heteroaryl, wherein R is12And R13Is optionally substituted;
each occurrence of R14And R15Independently selected from hydrogen, halogen, -OH and alkyl; and
n is an integer of 0 to 8.
In one embodiment, n is 0. In one embodiment, n is 1. In one embodiment, n is 2. In one embodiment, n is 3.
In one embodiment, R11Is OR15. In one embodiment, R15Is an alkyl group. In one embodiment, R15Is methyl.
In one embodiment, R11Is piperidinyl.
In one embodiment, R11Is morpholinyl.
In one embodiment, R11Is pyrrolidinyl.
In one embodiment, R11Is furyl.
In one embodiment, R11Substituted by hydroxyl.
In one embodiment, R12Is an alkyl group. In one embodiment, R12Is methyl.
In one embodiment, R12Is an aryl group. In one embodiment, R12Is phenyl.
In one embodiment, R12Is C5-C6A heteroaryl group. In one embodiment, R12Is furan. In one embodiment, R12Is phenylthio. In one embodiment, R12Is a pyridyl group.
In one embodiment, R13Is an alkyl group. In one embodiment, R13Is methyl.
In one embodiment, R13Is an aryl group. In one embodiment, R13Is phenyl.
In one embodiment, R13Is C5-C6A heteroaryl group. In one embodiment, R13Is furan. In one embodiment, R13Is phenylthio. In one embodiment, R13Is a pyridyl group.
In one embodiment, R12And R13Are the same.
In one embodiment, the compounds represented by formula (1) include (but are not limited to):
in one embodiment, the small molecule inhibitor is a compound represented by formula (2):
wherein the content of the first and second substances,
R21is selected from-C (R)23)mCycloalkyl, heterocyclyl, cycloalkyl-ketone (cycloalkylketone) and heterocyclyl-ketone (heterocyclylcyclo-ketone);
R22selected from the group consisting of alkyl, aryl, heteroaryl, alkyl-aryl, and alkyl-heteroaryl;
each occurrence of R23Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
m is an integer of 1 to 3.
In one embodiment, each occurrence of R23Independently selected from phenyl, -OH, methyl, ethyl and-CN.
In one embodiment, when m is 3, two occurrences of R23Is identical and one occurrence of R23Is different. In one embodiment, when m is 3, each occurrence of R23Are the same.
In one embodiment, R1Is a group represented by the formula (2a)
Wherein X21Selected from O, N or S; and
R24selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl.
In one embodiment, the compounds represented by formula (2) include (but are not limited to):
in one embodiment, the small molecule inhibitor is a compound represented by formula (3)
Wherein R is31Is selected from-C (R)35)pCycloalkyl, heterocyclyl, cycloalkyl-ketone (cycloalkylalkinyl-one), heterocyclyl-ketone (heterocyclylcarbonyl-one);
R32selected from alkyl, aryl, heteroaryl, -C1-C3Alkyl radical- (C)3-C6Aryl) and-C1-C3Alkyl radical- (C)3-C6Heteroaryl);
R33and R34Each independently selected from hydrogen, halogen, alkyl, aryl, heteroaryl;
each occurrence of R35Independently selected from hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, -OH and-CN; and
p is an integer of 1 to 3.
In one embodiment, R32Is ethyl.
In one embodiment, R33And R34Each independently selected from hydrogen and-Cl.
In one embodiment, R33And R34Are the same.
In one embodiment, each occurrence of R35Independently selected from phenyl, -OH, methyl, ethyl and-CN.
In one embodiment, when p is 3, two occurrences of R35Is identical and one occurrence of R35Is different. In one embodiment, when p is 3, each occurrence of R35Are the same.
In one embodiment, the compounds represented by formula (3) include (but are not limited to):
in one embodiment, the small molecule inhibitor is a compound represented by formula (4):
wherein the content of the first and second substances,
X41selected from O and S;
R41selected from the group consisting of alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and combinations thereof, wherein R is41May be optionally substituted; and
R42and R43Each independently selected from phenyl, thiophenyl and furanyl.
In one embodiment, R42And R43Are the same.
In one embodiment, R41Is an adamantyl group.
In one embodiment, R41Is piperidinyl.
In one embodiment, R41Is morpholinyl.
In one embodiment, R41Is pyrrolidinyl.
In one embodiment, R41Is furyl.
In one embodiment, R41Is an alkyl group. In one embodiment, R41Is C1-C25An alkyl group. In one embodiment, the alkyl group is a branched alkyl group. In one embodiment, the alkyl group is a straight chain alkyl group.
In one embodiment, R21is-C3-C10Cycloalkyl, which may be optionally substituted. In one embodiment, the cycloalkyl group is substituted. In one embodiment, the cycloalkyl group is unsubstituted. In one embodiment, the cycloalkyl group is monocyclic. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. In another embodiment, the cycloalkyl is polycyclic. For example, two or more-C can be connected3-C10Cycloalkyl groups to form polycyclic cycloalkyl groups. Non-limiting examples of polycyclic cycloalkyl groupsExamples include adamantane and norbornane. In one embodiment, the cycloalkyl group is adamantane, which may be optionally substituted. Cycloalkyl groups may also be bicyclic, including (but not limited to) tetrahydronaphthyl, indanyl, and tetrahydropentalene. In one embodiment, the cycloalkyl group is saturated or partially unsaturated. Non-limiting examples of saturated or partially unsaturated cycloalkyl groups include cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, cyclooctatetraenyl, cyclononenyl, cyclodecenyl, cyclooctynyl, cyclononynyl, cyclodecynyl and the like. In one embodiment, the cycloalkyl is fused to an aromatic ring.
In one embodiment, the compound represented by formula (4) is selected from
Preparation of the small molecule inhibitor
The compounds represented by formulas (1) - (4) can be prepared by the general schemes described herein using synthetic methods known to those skilled in the art. The following examples illustrate non-limiting embodiments of the invention.
In a non-limiting embodiment, the synthesis of the compounds represented by formulas (1) and (4) is accomplished by treating 4-nitro-o-phenylenediamine (a) with a diketone (b) to form 6-nitroquinoxaline (C), which is subsequently reduced by Pd/C-catalyzed hydrogenation to produce 6-aminoquinoxaline (d). Diketones (a) can be produced using methods known in the art (Tet. Lett.,1995,36:7305-7308, which is incorporated herein by reference in its entirety).
Then, the quinoxaline d is treated with an isocyanate to form a compound represented by the formula (1) or (4).
In another non-limiting embodiment, quinoxaline d is first treated with triphosgene and then an amine is added to form the compound represented by formula (1) or (4).
The compounds of the present invention may have one or more stereocenters, and each stereocenter may independently exist in the R or S configuration. In one embodiment, the compounds described herein are present in an optically active form or in a racemic form. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisomeric and stereoisomeric forms, or combinations thereof, having therapeutically useful properties as described herein. The preparation of the optically active form is effected in any suitable manner, including by way of non-limiting example, resolution of the racemic form by recrystallization techniques, synthesis from optically-active starting materials, chiral synthesis, or chromatographic separation using a chiral stationary phase. In one embodiment, a mixture of one or more isomers is used as a therapeutic compound described herein. In another embodiment, the compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including stereoselective synthesis, enantioselective synthesis and/or separation of mixtures of enantiomers and/or diastereomers. Separation of the compounds and isomers thereof is achieved by any means, including by non-limiting examples chemical, enzymatic, fractional crystallization, distillation and chromatography.
The methods and formulations described herein include N-oxides (if appropriate), crystalline forms (also known as polymorphs), solvates, amorphous phases and/or pharmaceutically acceptable salts of the compounds having the structure of any of the compounds described herein, as well as metabolites and active metabolites of these compounds having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates, and the like. In one embodiment, the compounds described herein are present in the form of solvates with pharmaceutically acceptable solvents, such as water and alcohols. In another embodiment, the compounds described herein are present in a non-solvated form.
In one embodiment, the compounds of the present invention may exist as tautomers. All tautomers are included within the scope of the compounds provided herein.
Compounds described herein also include isotopically-labeled compounds in which one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include, and are not limited to2H、3H、11C、13C、14C、36Cl、18F、123I、125I、13N、15N、15O、17O、18O、32P and35and S. Isotopically-labeled compounds are prepared by any suitable method or by using an appropriate isotopically-labeled reagent in place of a non-labeled reagent which is otherwise used.
In one embodiment, the compounds described herein are labeled by other means, including (but not limited to) the use of chromophores or fluorescent moieties, bioluminescent tags or chemiluminescent tags.
Using the techniques and materials described herein, and as (for example) Fieser & Fieser's Reagents for organic Synthesis, volumes 1-17 (John Wiley and Sons, 1991); rodd's Chemistry of CarbonCompounds, volumes 1-5 and supples (Elsevier Science Publishers, 1989); organic Reactions, Vol.1-40 (John Wiley and Sons,1991), Larock's Comprehensive Organic Transformations (VCH Publishers Inc.,1989), March,
The compounds described herein are synthesized using any suitable procedure starting from compounds obtained from commercial sources, or prepared using the procedures described herein.
In one embodiment, reactive functional groups, such as hydroxyl, amino, imino, thio, or carboxyl groups, are protected to avoid them from undesirably participating in the reaction. Protecting groups are used to block some or all of the reactive moieties and to prevent these groups from participating in chemical reactions until the protecting group is removed. In another embodiment, each protecting group is removable by a different means. Protecting groups cleaved under completely unrelated reaction conditions meet the requirement for differential removal.
In one embodiment, the protecting group is removed by acid, base, reducing conditions (such as, for example, hydrogenolysis), and/or oxidizing conditions. Groups such as trityl, dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile and are used to protect carboxyl and hydroxyl reactive moieties in the presence of amino groups protected with a Cbz group removable by hydrogenolysis and an Fmoc group labile with a base. Blocking of carboxylic acid and hydroxyl reactive moieties with base labile groups such as (but not limited to) methyl, ethyl and acetyl groups occurs in the presence of amines blocked with acid labile groups such as t-butyl carbamate, or with acid and base stable but hydrolysis removable carbamate.
In one embodiment, the carboxylic acid and hydroxyl reactive moieties are blocked with hydrolysis of a removable protecting group, such as benzyl, while the amine group capable of forming a hydrogen bond with an acid is blocked with a base labile group, such as Fmoc. The carboxylic acid reactive moiety is protected by conversion to a simple ester compound as exemplified herein, including conversion to an alkyl ester, or blocked with an oxidation-removable protecting group, such as 2, 4-dimethoxybenzyl, while the coexisting amino groups are blocked with a fluoride-labile silyl carbamate.
Allyl end capping groups are useful in the presence of acid-and base-protecting groups, since the former are stable and are subsequently removed by metal or pi-acid catalysts. For example, in the presence of acid-labile t-butyl carbamate or base-labile acetate amine protecting groups, the allyl-blocked carboxylic acid is deprotected by a palladium-catalyzed reaction. Another form of protecting group is a resin to which a compound or intermediate is attached. As long as the residue is attached to the resin, the functional group is blocked and does not react. Once released from the resin, the functional groups are available for reaction.
Typically, the blocking/protecting group may be selected from:
further protecting Groups and detailed descriptions of techniques suitable for the generation of protecting Groups and their removal are described in Greene & Wuts, Protective Groups in Organic Synthesis, 3 rd edition, John Wiley & Sons, New York, NY,1999 and Kocienski, Protective Groups, Thieme Verlag, New York, NY,1994, which disclosures are incorporated herein by reference.
Nucleic acid inhibitors
In some embodiments, the inhibitor is a nucleic acid. In various embodiments, the inhibitor is an siRNA, miRNA, shRNA, or antisense molecule that inhibits ACSS2. In one embodiment, the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is capable of directing expression of the inhibitor nucleic acid. Thus, the present invention encompasses expression vectors and methods for introducing exogenous DNA into a cell, accompanied by expression of the exogenous DNA in the cell, such as, for example, those described in Sambrook et al (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in Ausubel et al (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), as well as elsewhere herein.
In another aspect of the invention, ACSS2 may be inhibited by inactivating ACSS2 and/or sequestering ACSS2. As such, inhibition of ACSS2 activity can be accomplished by using a transdominant negative mutant.
In one embodiment, the siRNA is used to reduce the level of ACSS2 protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsrna) into a wide range of organisms and cell types results in the degradation of complementary mRNA. In cells, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs or siRNAs by ribonucleases called Dicer. Subsequently, the siRNA and protein components are assembled into an RNA-induced silencing complex (RISC), which unwinds during this process. The activated RISC is then bound to the complementary transcript by base pairing interaction between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence-specific degradation of the mRNA results in gene silencing. See, for example, U.S. Pat. Nos. 6,506,559; fire et al, 1998, Nature 391(19): 306-; timmons et al, 1998, Nature395: 854; montgomery et al, 1998, TIG 14(7): 255-258; david R.Engelke, Main edition, RNAInterference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); hannon Master, RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al (2004, Nature432:173-178) describe chemical modification of siRNA for assisted intravenous systemic delivery. Optimization of siRNA includes consideration of overall G/C content, terminal C/T content, Tm, and nucleotide content of the 3' overhang. See, for example, Schwartz et al, 2003, Cell,115: 199-. Thus, the invention also includes methods of reducing levels of ACSS2 using RNAi technology.
In another aspect, the invention includes a vector comprising an siRNA or an antisense nucleic acid. In one embodiment, the siRNA or antisense polynucleotide is capable of inhibiting the expression of a target polypeptide, wherein the target polypeptide is ACSS2. Incorporation of the desired polynucleotide into a vector and selection of vectors are well known in the art, as described, for example, in Sambrook et al (2012) and Ausubel et al (1997) and elsewhere herein.
In certain embodiments, the expression vectors described herein encode short hairpin rna (shrna) inhibitors. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby reducing the expression of the target. In certain embodiments, the encoded shRNA is expressed by the cell and then processed into siRNA. For example, in some cases, the cell has a native enzyme (e.g., dicer) that cleaves the shRNA to form the siRNA.
The siRNA, shRNA or antisense nucleic acid can be cloned into some type of vector as described elsewhere herein. For expression of siRNA or antisense polynucleotides, at least one module (module) in each promoter functions to locate the start site for RNA synthesis.
To assess the expression of the siRNA, shRNA or antisense nucleic acid, the expression vector to be introduced into the cells may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a population of cells that are attempted to be transfected or infected with the viral vector. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance, and the like.
Thus, in another aspect, the invention relates to a vector comprising a nucleotide sequence of the invention or a construct of the invention. The choice of vector will depend on the host cell into which it is subsequently introduced. In a specific embodiment, the vector of the present invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In particular embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. Prokaryotic-and/or eukaryotic-vector based systems can be used with the present invention to produce polynucleotides or their homologous polypeptides. Many such systems are commercially available and widely available.
Furthermore, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2012) and in Ausubel et al (1997) and in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers that are functional in at least one organism. (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No.6,326,193).
By way of illustration, the vector into which the nucleic acid sequence is introduced may be a plasmid, which, when introduced into a cell, is integrated or not integrated into the genome of the host cell. Illustrative, non-limiting examples of vectors into which the nucleotide sequences of the invention or the genetic constructs of the invention may be inserted include tet-on inducible vectors for expression in eukaryotic cells.
The vector can be obtained by conventional methods known to the person skilled in the art (Sambrook et al, 2012). In a specific embodiment, the vector is a vector for transforming animal cells.
In one embodiment, the recombinant expression vector may further contain a nucleic acid molecule encoding a peptide or peptidomimetic inhibitor of the invention as described elsewhere herein.
The promoter may be one which is naturally associated with the gene or polynucleotide sequence, e.g.as may be obtained by isolating the 5' non-coding sequence upstream of the coding segment and/or exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a polynucleotide sequence, either downstream or upstream of the sequence. Alternatively, certain advantages are obtained by placing the encoding polynucleotide segment under the control of a recombinant or heterologous promoter, that is, a promoter that is not normally associated with the polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with the polynucleotide sequence in its natural environment. These promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are not "naturally occurring," i.e., promoters or enhancers containing different elements of different transcriptional regulatory regions and/or mutations that alter expression. In addition to nucleic acid sequences that synthetically produce promoters and enhancers, sequences may be generated using recombinant cloning and/or nucleic acid amplification techniques, including PCR, in conjunction with the compositions disclosed herein (U.S. patent 4,683,202, U.S. patent 5,928,906). In addition, it is contemplated that control sequences which direct the transcription and/or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, and the like, can also be used.
Naturally, it will be important to use promoters and/or enhancers which effectively direct the expression of a DNA segment in the cell type, organelle, and organism selected for expression. It is generally known to those skilled in the art of molecular biology how to use a combination of promoters, enhancers and cell types for protein expression, see, for example, Sambrook et al (2012). The promoters used may be constitutive, tissue-specific, inducible and/or useful under appropriate conditions to direct high level expression of the introduced DNA segment, as is advantageous in large scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.
The recombinant expression vector may also contain a selectable marker gene that facilitates selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, β -galactosidase, chloramphenicol acetyltransferase, luciferin luciferase or immunoglobulins or parts thereof, such as the Fc part of an immunoglobulin, e.g. IgG, which confer resistance to certain drugs. The selectable marker may be introduced on a separate vector from the nucleic acid of interest.
After production of the siRNA polynucleotide, the skilled artisan will appreciate that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Thus, siRNA polynucleotides may also be designed to resist degradation by modification to include phosphorothioate or other linkages, methyl phosphonate, sulfone, sulfate, carbonyl, phosphorodithioate, phosphoramidate, phosphate, and the like (see, e.g., Agrwal et al, 1987, Tetrahedron Lett.28: 3539-3542; Stec et al, 1985Tetrahedron Lett.26: 2191-2194; Moody et al, 1989Nucleic acids sRs.12: 4769-4782; Eckstein,1989Trends biol. Sci.14: 97-100; Stein, In: Oligoxynyuchemotides. Antisenhibitors of Gene Expression, Cohen's major edition, Macmil Press, London, pp.97-117 (1989)).
Any polynucleotide may also be modified to improve its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'and/or 3' ends; the use of phosphorothioate or 2' O-methyl in the backbone rather than phosphodiester linkages; and/or contain unconventional bases such as inosine, Q nucleoside (queosine), and wyutin, among others, as well as acetyl-, methyl-, thio-, and other modified forms of adenine, cytosine, guanine, thymine, and uracil.
In one embodiment of the invention, antisense nucleic acid sequences expressed by plasmid vectors are used to inhibit expression of the ACSS2 protein. Antisense expression vectors are used to transfect mammalian cells or the mammal itself, thereby resulting in reduced endogenous expression of ACSS2.
Antisense molecules and their use for inhibiting gene expression are well known In the art (see, e.g., Cohen,1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene expression, CRC Press). As the term is defined elsewhere herein, an antisense nucleic acid is a DNA or RNA molecule that is complementary to at least a portion of a particular mRNA molecule (Weintraub,1990, Scientific American 262: 40). In a cell, an antisense nucleic acid hybridizes to a corresponding mRNA, thereby forming a double-stranded molecule, thereby inhibiting translation of the gene.
The use of antisense methods to inhibit gene translation is known in the art and described, for example, in Marcus-Sakura (1988, anal. biochem.172: 289). DNA encoding antisense molecules can be used to provide these antisense molecules to cells by gene expression, as taught by Inoue,1993, U.S. Pat. No.5,190,931.
Alternatively, antisense molecules of the invention can be prepared synthetically and then provided to cells. In one embodiment, the antisense oligomer is between about 10 to about 30 nucleotides. In one embodiment, the antisense oligomer is about 15 nucleotides. In one embodiment, antisense oligomers of about 10 to about 30 nucleotides are readily synthesized and introduced into target cells. Synthetic antisense molecules contemplated by the present invention include oligonucleotide derivatives known in the art having improved biological activity as compared to unmodified oligonucleotides (see U.S. Pat. No.5,023,243).
In one embodiment of the invention, ribozymes are used to inhibit the expression of ACSS2 protein. Ribozymes for inhibiting the expression of a target molecule can be designed by introducing a target sequence, which is complementary to, for example, the mRNA sequence encoding ACSS2, into the basic ribonuclease structure. Ribozymes targeting ACSS2 can be synthesized using commercially available reagents (Applied Biosystems, inc., Foster City, CA), or they can be expressed genetically from the DNA encoding them.
In one embodiment, the inhibitor of ACSS2 can comprise one or more components of a CRISPR-Cas system. The CRISPR method uses a CRISPR-associated (Cas) nuclease, which is complexed with a small rna (grna) as a guide, to cleave the DNA upstream of the Promiscuous Adjacent Motif (PAM) in any genomic position in a sequence-specific manner. CRISPR can use separate guide RNAs called crRNA and tracrRNA. These two different RNAs have been combined into a single RNA, enabling site-specific mammalian genomes to be cut through the design of short guide RNAs. Cas and guide rna (grna) can be synthesized by known methods. Cas/guide-RNA (gRNA) uses non-specific DNA to cleave protein Cas and oligo RNA to hybridize to target and recruit Cas/gRNA complexes. In one embodiment, a guide rna (grna) targeting the gene encoding ACSS2 and a CRISPR-associated (Cas) peptide form a complex to cause a mutation within the target gene. In one embodiment, the inhibitor includes a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the inhibitor comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.
Polypeptide inhibitors
In some embodiments, the inhibitor is a peptide or polypeptide inhibitor that inhibits ACSS2. For example, in one embodiment, the peptide inhibitors of the present invention inhibit ACSS2 by binding directly to ACSS2, thereby preventing the normal functional activity of ACSS2. In another embodiment, the peptide inhibitors of the invention inhibit ACSS2 by competing with endogenous ACSS2. In another embodiment, the peptide inhibitors of the present invention inhibit the activity of ACSS2 by acting as a transdominant negative mutant.
Variants of the peptides and polypeptides according to the invention may be (i) variants in which one or more amino acid residues are replaced by a conserved or non-conserved amino acid residue, and the replaced amino acid residue may or may not be an amino acid residue encoded by the genetic code, (ii) variants in which one or more modified amino acid residues are present, e.g. a residue modified by attachment of a substituent group, (iii) variants in which the polypeptide is an alternatively spliced variant of a polypeptide according to the invention, (iv) fragments of the polypeptide and/or (v) variants in which the polypeptide is fused to another polypeptide, such as a leader or secretory sequence or a sequence for purification (e.g. His-tag) or for detection (e.g. Sv5 epitope tag). Such fragments include polypeptides produced by proteolytic cleavage (including multi-site proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variations are considered to be within the purview of those skilled in the art in light of the teachings herein.
Antibody inhibitors
In some embodiments, the inhibitor is an antibody or antibody fragment. In some embodiments, the inhibitor is an antibody or antibody fragment that specifically binds to ACSS2. That is, the antibody may inhibit ACSS2 to provide a beneficial effect.
The antibody may beIntact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., Fab or (Fab)2Fragments), antibody heavy chains, antibody light chains, humanized antibodies, genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778) or chimeric antibodies, e.g., antibodies that contain the binding specificity of a murine antibody, but in which the remainder are of human origin. Antibodies, including monoclonal and polyclonal antibodies, humanized antibodies, fragments, and chimeras, can be made using methods known to those skilled in the art.
The antibody may comprise sets of heavy and light chain complementarity determining regions ("CDRs") inserted between sets of heavy and light chain frameworks ("FRs"), respectively, that provide support for the CDRs and define the spatial relationship of the CDRs relative to each other. A CDR set may contain three hypervariable regions of either the heavy or light chain V regions. Starting from the N-terminus of the heavy or light chain, these regions are denoted "
The antibody may be an immunoglobulin (Ig). Ig may be, for example, IgA, IgM, IgD, IgE, and IgG. Immunoglobulins may include heavy chain polypeptides and light chain polypeptides. The heavy chain polypeptide of an immunoglobulin may include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The light chain polypeptide of an immunoglobulin can include a VL region and a CL region.
The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, or a fully human antibody. The humanized antibody may be an antibody from a non-human species that binds a desired antigen having one or more Complementarity Determining Regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
The antibody may be a dual specificity antibody. The dual specific antibody can bind to or react with two antigens, e.g., two of the antigens as described in more detail below. The dual specific antibody may be comprised of fragments of two of the antibodies described herein, thereby enabling the dual specific antibody to bind to or react with two desired target molecules, which may include an antigen, a ligand (including a ligand for a receptor), a receptor (including a ligand-binding site on a receptor), a ligand-receptor complex, and a marker as described in more detail below. Dual specific antibodies may comprise a first antigen binding site that specifically binds to a first target and a second antigen binding site that specifically binds to a second target, with particularly advantageous properties such as producibility, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, and low toxicity. In some cases, there is a dual specific antibody, wherein the dual specific antibody binds to a first target with high affinity and binds to a second target with low affinity. In other examples, there are dual specific antibodies, wherein the dual specific antibody binds to a first target with low affinity and binds to a second target with high affinity. In other examples, there is a dual specific antibody, wherein the dual specific antibody binds to a first target with a desired avidity and binds to a second target with a desired avidity.
Antibodies can be made using intact polypeptides or fragments containing the immunological antigen of interest. The polypeptide or oligopeptide used to immunize the animal may be derived from translation or chemical synthesis of RNA and, if desired, may be bound to a carrier protein. Suitable carriers that can be chemically coupled to the peptide include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The conjugated polypeptide can then be used to immunize an animal (e.g., a mouse, rat, or rabbit).
Combination of
In some embodiments, the compositions of the present invention comprise a combination of ACSS2 inhibitors described herein. In certain embodiments, a composition comprising a combination of inhibitors described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual inhibitor. In other embodiments, the compositions comprising a combination of inhibitors described herein have a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual inhibitor.
In some embodiments, the compositions of the present invention comprise a combination of an ACSS2 inhibitor and a second therapeutic agent. For example, in one embodiment, the second therapeutic agent includes (but is not limited to) PTSD therapy, anxiety therapy, and substance abuse therapy.
In some embodiments, the second therapeutic agent is a PTSD therapy. Exemplary therapeutic agents include, but are not limited to, anti-anxiety treatments, antidepressants, and adrenergic agents. In one embodiment, the PTSD treatment is a therapeutic treatment. For example, in one embodiment, the PTSD treatment includes psychotherapy, behavioral or cognitive behavioral therapy, ocular desensitization reprocessing (EMDR) collective therapy, transcranial magnetic stimulation, deep brain stimulation, and neurofeedback techniques, as well as drug therapy, including ketamine and d-cycloserine.
In one embodiment, administration of the ACSS2 inhibitor in an emergency or intensive care unit can be used for PTSD prevention. During peritraumatic periods, reactivated memory tracks are vulnerable to rupture, and therefore ACSS2 inhibition offers the potential to affect restorability of the traumatic memory.
In some embodiments, the second therapeutic agent is a substance abuse therapy. For example, in one embodiment, substance abuse therapy includes (but is not limited to) naltrexone, disulfiram, acamprosate, topiramate, nicotine replacement therapy, nicotinic receptor antagonists, nicotinic receptor partial agonists, sulben, levomethadol, dihydrocodeine, buprenorphine, ketamine, methadone, and dihydroetorphine.
Compositions comprising a combination of inhibitors comprise the individual inhibitors in any suitable ratio. For example, in one embodiment, the composition comprises two separate inhibitors in a 1:1 ratio. However, the combination is not limited to any particular ratio. Rather, any ratio that proves effective is contemplated.
Method of producing a composite material
In some embodiments, the present invention provides methods of inhibiting ACSS2 in a subject in need thereof. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor.
In one embodiment, the invention provides a method of modulating chromatin acetylation in a subject. In one embodiment, the chromatin acetylation is histone acetylation. In one embodiment, the chromatin is neurochromatin. In one embodiment, the methods of the invention modulate neuronal plasticity in a subject. In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor. In one embodiment, the inhibitor of ACSS2 reduces histone acetylation.
In one aspect, the invention provides methods for treating a neurological or cognitive disease or disorder in a subject. In one embodiment, the neurological or cognitive disease or disorder is a memory-related disease or disorder. In one embodiment, the neurological or cognitive disease or disorder is a neuropsychiatric disorder. For example, in one embodiment, the neuropsychiatric disorder includes, but is not limited to, anxiety disorders, psychotic disorders, mood disorders (mood disorders), and somatoform disorders.
Exemplary neurological or cognitive diseases or conditions include, but are not limited to, Post Traumatic Stress Disorder (PTSD), bipolar disorder (bipolar disorder), depression, Tourette's Syndrome, schizophrenia, obsessive compulsive disorder, generalized anxiety disorder, panic disorder, phobias, personality disorders, including antisocial personality disorders, and other conditions involving memory difficulties. In one embodiment, the neurological or cognitive disease or disorder is PTSD.
In another embodiment, the invention provides a method for treating an addiction or addiction related disease or disorder in a subject. In one embodiment, addiction includes (but is not limited to) addiction to: alcohol, tobacco, opioids, sedatives, hypnotics, anxiolytics, ***e, cannabis, amphetamines, hallucinogens, inhalants, phencyclidine, impulse control disorders, and behavioral addiction.
In one embodiment, the addiction is an alcohol addiction. In one embodiment, the methods of the invention treat acute and/or chronic alcohol-induced memory decline.
In one embodiment, the present invention provides a method for treating alcohol-related memory and environment-induced craving in intensive psychotherapy (intensive psychotherapy). In one embodiment, the method comprises administering to the subject an effective amount of a composition comprising an ACSS2 inhibitor. In one embodiment, the inhibitor of ACSS2 reduces histone acetylation.
In one embodiment, the method comprises administering to the subject an effective amount of a composition that reduces or inhibits the expression or activity of ACSS2.
One skilled in the art will appreciate that the inhibitors described herein can be administered alone or in any combination. Furthermore, the inhibitors according to the invention can be administered in a temporal sense individually or in any combination, where they can be administered simultaneously or before and/or after each other. One skilled in the art will appreciate, based on the disclosure of the invention provided herein, that the inhibitor compositions described herein can be used to prevent or treat an autoimmune disease or disorder, or the inhibitor compositions can be used alone or in any combination with another modulator to affect the therapeutic outcome. In various embodiments, any of the inhibitor compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with autoimmune diseases.
In one embodiment, the invention includes a method comprising administering a combination of inhibitors described herein. In certain embodiments, the methods have additive effects, wherein the overall effect of the combination of administered inhibitors is approximately equal to the sum of the effects of each individual inhibitor administered. In other embodiments, the methods have a synergistic effect, wherein the overall effect of the combination of administered inhibitors is greater than the sum of the effects of each individual inhibitor administered.
The method comprises administering a combination of inhibitors in any suitable ratio. For example, in one embodiment, the method comprises administering two separate inhibitors at a ratio of 1: 1. However, the method is not limited to any particular ratio. Rather, any ratio that proves effective is contemplated.
Pharmaceutical compositions and formulations
The invention also encompasses the use of a pharmaceutical composition of the invention or a salt thereof for practicing the methods of the invention. Such a pharmaceutical composition may consist of at least one modulator (e.g., inhibitor) composition of the invention or a salt thereof in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one modulator (e.g., inhibitor) composition of the invention or a salt thereof and one or more pharmaceutically acceptable carriers, one or more other ingredients, or some combination of these. The compounds of the present invention may be present in the pharmaceutical compositions in the form of physiologically acceptable salts, e.g. in combination with physiologically acceptable cations or anions well known in the art.
In one embodiment, a pharmaceutical composition for practicing the methods described herein may be administered to deliver a dose of 1 ng/kg/day to 100 mg/kg/day. In another embodiment, a pharmaceutical composition for practicing the invention may be administered to deliver a dose of 1 ng/kg/day to 500 mg/kg/day.
In the pharmaceutical compositions of the invention, the relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any other ingredients will vary based on the identity, size, and condition of the subject being treated and also based on the route of administration of the composition. For example, the composition may comprise between 0.1% and 100% (w/w) of active ingredient.
Pharmaceutical compositions useful in the methods of the invention may be suitably developed for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ocular administration or another route of administration. The compositions useful in the methods of the present invention may be applied directly to the skin or any other tissue of a mammal. Other contemplated formulations include liposomal formulations, re-encapsulated red blood cells containing the active ingredient, and immunologically based formulations. The route of administration will be apparent to the skilled artisan and will depend on a variety of factors including the type and severity of the disease to be treated, the type and age of the animal or human subject to be treated, and the like.
The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacy or hereafter developed. Generally, these methods of preparation include the step of bringing into association the active ingredient with the carrier or one or more other auxiliary ingredients and then, if necessary or desired, shaping or packaging the product into the desired single-or multi-dose units.
As used herein, a "unit dose" is an individual amount of a pharmaceutical composition that contains a predetermined amount of active ingredient. The amount of the active ingredient is typically equal to the dose of active ingredient to be administered to the subject or a convenient fraction of the dose (a concurrent fraction), such as, for example, one half or one third of the dose. The unit dosage form can be used in a single daily dose or in one of a plurality of daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are employed, the unit dosage form may be the same or different for each dose.
In one embodiment, the compositions of the present invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical composition of the present invention comprises a therapeutically effective amount of a compound or combination of the present invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphate and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication co., new jersey).
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), suitable mixtures thereof, and vegetable oils. 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens (parabens), chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In most cases, isotonic agents, for example, sugars, sodium chloride or polyols, such as mannitol and sorbitol, are included in the compositions. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. In one embodiment, the pharmaceutically acceptable carrier is not DMSO alone.
The formulations may be employed in admixture with conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier materials known in the art to be suitable for oral, vaginal, parenteral, nasal, intravenous, subcutaneous, enteral or any other suitable form of administration. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliaries, such as, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, colorants, flavors and/or aromatic substances and the like. They may also be mixed with other active agents, e.g., other analgesics, as desired.
As used herein, "other ingredients" include (but are not limited to) one or more of the following: an excipient; a surfactant; a dispersant; an inert diluent; granulating and disintegrating agents; a binder; a lubricant; a sweetener; a flavoring agent; a colorant; a preservative; physiologically degradable compositions such as gelatin; an aqueous vehicle and a solvent; oily vehicles and solvents; a suspending agent; a dispersing or wetting agent; emulsifiers, analgesics; a buffering agent; salt; a thickener; a filler; an emulsifier; an antioxidant; (ii) an antibiotic; an antifungal agent; a stabilizer; and pharmaceutically acceptable polymers or hydrophobic materials. Other "other ingredients" that may be included in the Pharmaceutical compositions of the present invention are known in the art and described, for example, in the Genaro eds (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.
The compositions of the present invention may comprise from about 0.005% to 2.0% by total weight of the composition of a preservative. Preservatives are used to prevent spoilage upon exposure to contaminants in the environment. Examples of preservatives useful according to the present invention include, but are not limited to, those selected from benzyl alcohol, sorbic acid, parabens, imidazolidinyl urea (imidaurea), and combinations thereof. An exemplary preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.
In one embodiment, the composition includes an anti-oxidant that inhibits degradation of the compound and a chelating agent. Exemplary antioxidants for some compounds include BHT, BHA, alpha-tocopherol, and ascorbic acid in a preferred range of about 0.01% to 0.3%. In one embodiment, the antioxidant is BHT in the range of 0.03% to 0.1% by weight of the total weight of the composition. In one embodiment, the chelating agent is present in an amount of 0.01% to 0.5% by weight of the total weight of the composition. Exemplary chelating agents include edetate (e.g., disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%. In one embodiment, the chelating agent is in the range of 0.02% to 0.10% by weight of the total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. Although BHT and disodium edetate are exemplary antioxidants and chelating agents, respectively, for some compounds other suitable and equivalent antioxidants and chelating agents may therefore be substituted, as will be known to those skilled in the art.
Liquid suspensions may be prepared using conventional methods to achieve a suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethanol, vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils, such as liquid paraffin. Liquid suspensions may also contain one or more other ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, analgesic agents, preserving agents, buffering agents, salts, flavoring agents, coloring agents, and sweetening agents. The oily suspensions may also contain a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phosphatides, such as lecithin, alkylene oxides and fatty acids, condensation products with long chain aliphatic alcohols, with partial esters derived from fatty acids and hexitol alcohols, or with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene glycol stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitan monooleate, and polyoxyethylenesorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia gum. Known preservatives include, but are not limited to, methyl, ethyl or n-propyl p-hydroxybenzoate, ascorbic acid and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.
Liquid solutions of the active ingredient may be prepared in aqueous or oily solvents in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. As used herein, an "oily" liquid is a liquid that contains carbon-containing liquid molecules and exhibits less polar properties than water. The liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described for liquid suspensions, it being understood that the suspending agent will not necessarily aid in the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil, fractionated vegetable oils and mineral oils, such as liquid paraffin.
Powder and granule formulations of the pharmaceutical formulations of the present invention may be prepared using known methods. These formulations can be administered directly to a subject for, for example, forming tablets, filling capsules, or preparing aqueous or oily suspensions or solutions by adding aqueous or oily vehicles thereto. Each of these formulations may also contain one or more dispersing or wetting agents, suspending agents and preservatives. Other excipients, such as fillers and sweeteners, flavoring or coloring agents may also be included in these formulations.
The pharmaceutical compositions of the present invention may also be prepared, packaged or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, a mineral oil, for example liquid paraffin, or a combination of these. These compositions may also contain one or more emulsifying agents, such as naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean or lecithin phosphatides, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. These emulsions may also contain other ingredients including, for example, sweetening or flavoring agents.
Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods of depositing or binding chemical compositions onto surfaces, methods of incorporating chemical compositions into material structures during material synthesis (i.e., such as with physiologically degradable materials), and methods of absorbing aqueous or oily solutions or suspensions into absorbent materials with or without subsequent drying.
The administration regimen may affect the constitution of the effective amount. The therapeutic formulation can be administered to the subject before or after disease diagnosis. Furthermore, several divided doses and staggered doses may be administered daily or sequentially, or the doses may be continuously infused, or the doses may be bolus injected. In addition, the dosage of the therapeutic agent can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic condition.
Administration of the compositions of the present invention to a subject, e.g., a mammal, including a human, can be carried out using known procedures, at dosages and for periods of time effective for preventing or treating the disease. The effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary depending on factors such as the activity of the particular compound used; the time of administration; the rate of excretion of the compound; the duration of treatment; other drugs, compounds or materials used in combination with the compound; the condition, age, sex, weight, condition, general health and prior medical history of the disease or disorder in the subject to be treated and similar factors well known in the medical arts. The dosage regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. A non-limiting example of an effective dosage range of a therapeutic compound of the invention is about 1 to 5,000mg/kg body weight/day. One skilled in the art will be able to study the relevant factors and make decisions regarding effective amounts of therapeutic compounds without undue experimentation.
The compound may be administered to the subject at a frequency of several times per day, or it may be administered less frequently, such as once per day, once per week, once every two weeks, once per month, or less frequently, such as once every few months or even once per year or less. It is understood that in non-limiting examples, the amount of compound administered in a daily dose may be administered daily, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, by administering every other day, a first 5mg daily dose may be administered on Monday, a first subsequent 5mg daily dose on Wednesday, a second subsequent 5mg daily dose on Friday, and so on. The frequency of the dosage will be apparent to the skilled person and will depend on a number of factors such as, but not limited to, the type and severity of the disease to be treated, the type and age of the animal, etc.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and administration form, but is not toxic to the subject.
A physician, e.g., a physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention used in the pharmaceutical composition at a level less than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.
In particular embodiments, it is particularly advantageous to formulate the compounds in dosage unit form to facilitate administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units (physically discrete units) suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of therapeutic compound calculated to produce the desired therapeutic effect in association with the required drug vehicle. The dosage unit forms of the invention are determined by and directly based on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of mixing/formulating such therapeutic compounds for the treatment of disease in a subject.
In one embodiment, the composition of the present invention is administered to a subject at a dose ranging from 1 to 5 times per day or more. In another embodiment, the compositions of the present invention are administered to a subject in a dosage range that includes, but is not limited to, daily, every other day, every three
The compounds of the invention for administration may be in the following ranges: from about 1mg to about 10,000mg, from about 20mg to about 9,500mg, from about 40mg to about 9,000mg, from about 75mg to about 8,500mg, from about 150mg to about 7,500mg, from about 200mg to about 7,000mg, from about 3050mg to about 6,000mg, from about 500mg to about 5,000mg, from about 750mg to about 4,000mg, from about 1mg to about 3,000mg, from about 10mg to about 2,500mg, from about 20mg to about 2,000mg, from about 25mg to about 1,500mg, from about 50mg to about 1,000mg, from about 75mg to about 900mg, from about 100mg to about 800mg, from about 250mg to about 750mg, from about 300mg to about 600mg, from about 400mg to about 500mg, and any and all whole or partial increments therebetween.
In some embodiments, the compound of the invention is administered in a dose of about 1mg to about 2,500 mg. In some embodiments, the dose of a compound of the invention used in the compositions described herein is less than about 10,000mg, or less than about 8,000mg, or less than about 6,000mg, or less than about 5,000mg, or less than about 3,000mg, or less than about 2,000mg, or less than about 1,000mg, or less than about 500mg, or less than about 200mg, or less than about 50 mg. Similarly, in some embodiments, the dose of the second compound as described herein (i.e., a drug for treating the same or another disease as the disease being treated by the composition of the invention) is less than about 1,000mg, or less than about 800mg, or less than about 600mg, or less than about 500mg, or less than about 400mg, or less than about 300mg, or less than about 200mg, or less than about 100mg, or less than about 50mg, or less than about 40mg, or less than about 30mg, or less than about 25mg, or less than about 20mg, or less than about 15mg, or less than about 10mg, or less than about 5mg, or less than about 2mg, or less than about 1mg, or less than about 0.5mg, and any and all whole or partial increments thereof.
In one embodiment, the present invention relates to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or combination of the present invention, alone or in combination with a second agent; and instructions for using the compound or combination to treat, prevent or reduce one or more symptoms of a disease in a subject.
The term "container" includes any receptacle for holding the pharmaceutical composition. For example, in one embodiment, the container is a package containing the pharmaceutical composition. In other embodiments, the container is not a package containing a pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial, containing a packaged or unpackaged pharmaceutical composition and instructions for use of the pharmaceutical composition. In addition, packaging techniques are well known in the art. It will be appreciated that instructions for use of the pharmaceutical composition may be contained on the package containing the pharmaceutical composition and as such the instructions form an enhanced functional relationship with the packaged product. However, it is to be understood that the instructions may contain information regarding the ability of the compound to perform its intended function, e.g., to treat or prevent a disease in a subject or to deliver an imaging or diagnostic agent to a subject.
The route of administration of any of the compositions of the present invention includes oral, nasal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (buccal) and nasal (intra)), intravesical, intraduodenal, intragastric, rectal, intraperitoneal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, or administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets (caplets), pills, soft capsules (gel caps), lozenges, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, granules, pastes, troches, creams, ointments, plasters, lotions, discs (discos), suppositories, liquid sprays for nasal or oral administration, dry or nebulized formulations for inhalation, compositions and formulations for intravesical administration, and the like. It is to be understood that the formulations and compositions that will be useful in the present invention are not limited to the particular formulations and compositions described herein.
Experimental examples
The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Thus, the present invention should in no way be construed as limited to the following examples, but rather should be construed to cover any and all modifications which become apparent as a result of the teachings provided herein.
Without further description, it is believed that one skilled in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the claimed methods. The following working examples should therefore not be construed as limiting the remainder of the disclosure in any way.
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