Epitestosterone sulfate and / or steroid sulfatase inhibitors used to treat or improve age-related cognitive impairment.

Abstract

This invention belongs to the medical field and provides a composition for a method of improving age-related cognitive function and / or treating conditions involving age-related cognitive dysfunction or age-related cognitive impairment in subjects in need.

Description

Technical Field

[0001] This invention belongs to the medical field and provides a compound for preventing age-related protein toxicity caused by protein aggregation diseases and / or increasing the lifespan of eukaryotes. Background Technology

[0002] Animals can extend their lifespan by activating different genetic pathways. This lifespan increase is a regulated process transmitted through the coordination of various tissue and environmental signals. Hormones are key players in tissue and cell communication. Therefore, they participate in different pathways regulating lifespan, including insulin and insulin-like growth factor, TGF, or dafachronic acid, described as affecting lifespan at least in the model organism *Caenorhabditis elegans*. The gonad is an endocrine tissue that produces steroid hormones to regulate various physiological aspects of an organism, including lifespan. In *C. elegans*, germline ablation extends lifespan through mechanisms not fully understood. Increased lifespan requires several factors, including the synthesis of dafachronic acid by somatic gonads and the transcription factor encoded by daf-16, which is homologous to human FOXO, as well as the nuclear receptor-encoding genes daf-12, nhr-80, and nhr-492.

[0003] The classic function of steroid hormones is thought to be activating hormone receptors to transcribe their target genes. Steroid hormones are produced not only in the gonads but also in other tissues. Those produced in the nervous system are called neurosteroids. Neurosteroids regulate neurotransmission through direct interaction with neurotransmitter receptors or other mechanisms, in addition to binding to hormone receptors. Steroid hormones can be sulfated by sulfotransferases, profoundly altering their chemical characterization and impairing their function as hormone receptor activators. These sulfated hormones are considered an inactive reservoir of hormones that can be activated by removing the sulfated portion through hormone sulfatase activity. Sulfated steroid hormones can also function as neurosteroids, thereby regulating neurotransmission.

[0004] Some sulfated steroid hormones, such as dehydroepiandrosterone sulfate (DHEAS), have long been associated with aging. The decline in levels of this hormone with age and in age-related diseases such as sarcopenia or Alzheimer's disease has led to speculation about its causes.

[0005] Here, we demonstrate that inhibition of steroid sulfatase increases the percentage of sulfated hormones, thereby increasing lifespan and improving symptoms associated with protein aggregation diseases. This increase in lifespan is primarily attributable to factors identical to those described for longevity induced by germline ablation. Treatment with the specific inhibitor of steroid sulfatase, STX64, mimics the beneficial effects on lifespan and protein aggregation diseases observed in mutants. Interestingly, treatment with STX64 also alleviated cognitive symptoms and plaque formation in a mammalian model of Alzheimer's disease. Finally, the observed phenotypes, summarized by treatment with sulfated C19 androgen steroids rather than with non-sulfated forms or sulfated C21 pregnenolone steroids, suggest that the beneficial effects resulting from sul-2 inhibition are due to an increase in sulfated C19 steroids rather than a decrease in non-sulfated forms. Therefore, this invention suggests that STX64 or specific sulfated C19 steroids may be a treatment option for aging and / or age-related diseases, and more specifically, specific sulfated C19 steroids prolong lifespan and prevent age-related protein toxicity. Attached Figure Description

[0006] Figure 1 Decreased sul-2 activity increases lifespan and affects the levels of sulfated steroid hormones. A) The sul-2 (pv17) point-mutant allele has a longer lifespan than the wild type. B) The null allele sul-2 (gk187) also increases lifespan. C) Phylogenetic tree of mammalian sulfatases and three *C. elegans* sulfatases. Note that SUL-2 is clustered with steroid sulfatases (type C) and is shown in blue. Phylogenetic relationships with other *C. elegans* sulfatases are shown in gray. D) Inhibition of steroid hormone sulfatases by STX64 (1 μg / ml) increases lifespan in wild-type animals, but not in the sul-2 (gk187) background. Worms were cultured in UV-killed *E. coli*. E) sul-2 deficiency increases the percentage of steroid hormones in the sulfated stage. Data from three independent assays are shown. Single-tailed Mann-Whitney t-test.

[0007] Figure 2Genetic interactions and cellular localization of sul-2 expression. Genetic analysis showed that sul-2 mimics most of the genetic interactions described for germline-agnostic animals, but does not affect fertility. a. Sul-2 increases lifespan in the daf-2 (e1370) background. b. The Daf-16 transcription factor is essential for sul-2 longevity. c, d, e. Sul-2 longevity requires the essential factors for germline-agnostic longevity, kri-1 (ok1251), tcer-1 (tm1452), and, in part, nhr-80 (tm1011). f. Nhr-49 (nr2041) does not inhibit sul-2 lifespan extension. g, h. Sul-2 deletion does not significantly increase the lifespan of the germline-agnostic mutants glp-1 (e2141) and mes-1 (bn7). i. The number of progeny of sul-2 mutants is not significantly different from that of wild-type. Mean ± SEM; one-way ANOVA test; ns. j. The reproductive lifespan of the sul-2 mutant is unaffected (25°C). k. Dietary restriction (DR) conditions show increased lifespan in the sul-2 deficiency background. l. The Daf-12 transcription factor is essential for sul-2 longevity. m. The Rieske-like oxygenase daf-36 is essential for increased lifespan through inhibition of the steroid hormone sulfatase (STX64 (1 μg / ml)). n. sul-2 is transcribed in sensory neurons, with ADF and ASE predominating in the head region and PHA and PHB predominating in the tail region. Scale bar 10 μm.

[0008] Figure 3 Decreased steroid hormone sulfatase activity alleviated symptoms in a protein toxicity model in *C. elegans* and a mouse model. a. In the sul-2 (gk187) background (n≥17 / day), the NL5901 strain expressing α-synuclein in muscle cells showed a slower rate of decrease in activity with age. Statistical tests were performed compared with the α-synuclein control. b. Or under treatment with STX64 (1 μg / ml) (n≥9 / day). Other biological replication analyses showed... Figure 12a and c. c, d. Reduced neurodegeneration of dopaminergic (DA) neurons in the UA44 strain expressing human α-synuclein against a sul-2 (gk187) background. Representative images and quantifications of surviving neurons on day 9 for each condition. Scale bar 50 μm. Data from two biological replicates are shown, n = 37. e. Reduced Q35 aggregates in 8-day-old worms against a sul-2 (gk187) background (n ≥ 6). f. Or in 5-day-old worms treated with STX64 (1 μg / ml) against a Q40 background (n ≥ 12). Strains AM140 and AM141, respectively. g. Expression of human β-amyloid in muscle caused heat-dependent paralysis in L4 juvenile adults of the GMC101 strain, which was alleviated in the sul-2 deletion mutant. Data from four independent biological replicates, n = 125. h. Or treatment with STX64 (1 μg / ml). Data were obtained from six independent biological replicates, n ≥ 215. i, j. Effects of intra-hippocampal injection and oral administration of STX64 on the passive avoidance test in wild-type mice injected with β-amyloid oligomers in the hippocampus. Number of mice per group > 5. k. Images of representative β-amyloid immunoreactivity assessed in the frontal cortex and hippocampus of APP-PS1 mice older than 15 months of age 3–4 weeks after ingestion of the solvent or STX64 (0.005 mg / ml in drinking water). ln. Quantification of the percentage of β-amyloid area (c), deposition density (d), and mean plaque size (e) in the frontal cortex and hippocampus of APP-PS1 mice older than 15 months of age 3–4 weeks after oral administration of STX64 or the solvent. n = 4 mice per group. o. The time course of β-amyloid deposition in APP-PS1 mice and the effect of 3–4 weeks of oral STX64 treatment on the area of ​​β-amyloid in the frontal cortex (top) and hippocampus (bottom). More than 3 photomicrographs were used in all mice used. p. Effects of oral STX64 administration on APP-PS1 mice older than 15 months, and comparisons between APP-PS1 mice older than 15 months and wild-type mice older than 15 months in passive avoidance tests. Number of mice per group > 5. In histological analyses, * indicates a significant difference between solvent-treated and STX64-treated APP-PS1 mice. In behavioral tests, * indicates a significant difference between short-term and long-term memory stages (STM and LTM, respectively) and training stages within the same experimental group; and + indicates a significant difference between STM and LTM stages between each experimental group and the β-amyloid group. One sign, p < 0.05; two signs, p < 0.01; three signs, p < 0.001.

[0009] Figure 4Sulfated C19 androgen steroid hormone mimics sul-2 inactivation. a. The NL5901 strain expressing α-synuclein in muscle cells showed decreased activity with age. Treatment with DHEAS, TS, or ES improved activity (1 μg / ml). Data from three biological replicates are shown, n = 30. b. Paralysis in the GMC101 Alzheimer's disease model was alleviated by TS and ES. Data from six biological replicates are shown, n ≥ 200. Other assays in normal NGM plates are as follows. Figure 13 a. As shown in the image. c, d. In the Parkinson's model NL5901 strain, no effects were observed from non-sulfated steroid hormones DHEA or testosterone (T), as well as sulfated C21 steroid hormones, pregnenolone sulfate (PregS) (1 μg / ml). Data from three biological replicates are shown, n = 30. e. Treatment with abiraterone (Abi) (1 μg / ml) did not affect the activity phenotype of the NL5901 strain, but suppressed the beneficial effects of the sul-2 deletion allele. Data from three biological replicates are shown, n ≥ 30. f. Treatment with ES (1 μg / ml) increased lifespan in the wild-type background, but did not increase it further in sul-2 (gk187). g. DHEAS or TS (1 μg / ml) did not affect lifespan. Figure 13 Other biological repeat assays are shown in c and 13d.

[0010] Figure 5Lifespan determination of sul-2 and its genetic interaction with daf-2. The sul-2 mutant did not exhibit a visible phenotype but had a longer lifespan and enhanced the developmental phenotype of the daf-2 mutant. sul-2 (pv17) was long-lived, while fer-15 (b26) did not affect its lifespan. b. The sul-2 mutant was long-lived at 20°C. c. The sul-2 mutant was long-lived at 25°C. d. The pumping rate of the sul-2 mutant was similar to that of the wild type on adult day 1, while the pv17 allele maintained a higher pumping rate than the wild type on adult day 6. The worms were grown at 16°C until L4 and then transferred to 25°C, considered day 0 of adulthood. Pumping was counted over 30 seconds at 100X magnification under a Leica stereomicroscope. Data were obtained from two independent bioassays (n = 30 / day). Mann-Whitney t-test for two tails, ***p = 0.0006. The wriggling in e. sul-2 (gk187) was similar to the wild type, but slightly lower in sul-2 (pv17) at 25°C on day 1 of adult stage. On day 6 of adult stage at 25°C, the wriggling in sul-2 (gk187) was lower than in the wild type and sul-2 (pv17), with no difference between these final strains. The sul-2 (gk17) population on day 6 appeared to be distributed in two distinct groups, one with almost no wriggling and the other with values ​​similar to the wild-type sul-2 (pv17). Data were obtained from two independent bioassays (n = 20 / day). f. Sul-1 (gk151) and sul-3 (tm6179) did not increase lifespan. g. A small percentage of daf-2(e979) animals exhibit developmental arrest of the dauer larvae at 16°C. The sul-2(pv17) mutant does not show any larval arrest but enhances the daf-2(e979) larval arrest. h. Most daf-2(e1370) animals arrest at the dauer stage at 25°C, but the percentage arresting at the L1 stage is small. In this case, all animals from the daf-2(e1370); sul-2(pv17) double mutant arrest at the L1 stage. Similarly, in the daf-2(m577); sul-2(pv17) background, over 50% of animals arrest at the L1 stage, while single mutants do not show this phenotype. I. Examples of L1 arrest in sul-2(pv17) larvae, dormant larvae of daf-2(e1370) at 25°C, or L1 arrest in daf-2(e1370); sul-2(pv17), and daf-2(e1370); sul-2(gk187). Animals were grown to L4 at 16°C and then transferred to 25°C, where offspring were scored or imaged after 72 hours. Photographs were taken through a Leica microscope.

[0011] Figure 6 Identification of the pv17 allele and the curated sequence of SUL-2. A. The pv17 allele is a missense mutation that replaces the asterisked glycine residue with an aspartic acid residue. The mutation is located near an evolution-restricted region (ECR) (indicated by a bar at the bottom) and also close to the catalytic core of the sulfatase (green). B. The DNA sequence we identified in wild-type SUL-2 differs from the DNA sequence published in the nematode database (SUL-2_wormbase) and is identified as a homologous sequence from ARSA (http: / / www.wormbase.org) with a 459 bp deletion, which was recently described by Li et al. (2015) and predicted as part of a new exon [wormbase_170818gw3] based on RNAseq data (SUL-2_modified). The protein sequence obtained by cDNA sequencing of the yk387h10 clone (SUL-2_curated) differs from the predicted sequence in three amino acids (bold). Please note that the three amino acids identified in this sequence are also present in other species (CRE: C. remanei, CBR: C. brigssae). c. Exon and intron composition of wild-type sul-2 and the region missing in the gk187 allele (red). Mutant lesions can be found in the nematode database ( http: / / www.worm-base.org / species / c_elegans / variation / WBVar00145594#02- 45-3) Obtained. The deletion of this allele encodes the sequence (green) of the sulfatase catalytic core and causes a frameshift, thus retaining only the first four amino acids of the original sequence; therefore, we consider gk187 to be a null allele. The position of the pv17 allele is indicated by an asterisk, the new DNA fragment is indicated by yellow, and the new exon is indicated by purple.

[0012] Figure 7 Treatment with STX64 phenotypes simulated the longevity and genetic interactions of the sul-2 mutant. a. STX64 in non-UV E. coli increased the lifespan of the wild type. b, c. STX64 dose curves in non-UV E. coli showed significant effects at 1 μg / ml and 5 μg / ml. d. Photographs of wild type and daf-2(e1370) at 25°C. Treatment with DMSO (STX64 solvent) or STX64 did not affect the development of the wild type. daf-2(e1370) mainly arrested in the dormant stage at 25°C, but mainly arrested at L1 when treated with STX64. Similar interactions were observed in the sul-2 mutant. Photographs were taken under a Leica microscope. Arrow: Dormancy, Arrow: L1.

[0013] Figure 8Genetic interactions of the sul-2 (pv17) alleles. Except in the glp-1 background, the sul-2 point-mutant allele pv17 showed a similar phenotype in lifespan interactions, determined to be sul-2 deletion. a, b. Sul-2 (pv17) increased the lifespan of two daf-2 alleles, e1370 and m577. c, d. Longevity of sul-2 (pv17) was suppressed by two daf-16 alleles, mu86 and m26. e. Longevity of sul-2 (pv17) was suppressed by tcer-1 (tm1452). f. Longevity of sul-2 (pv17) was partially suppressed by nhr-80 (tm1011). g. Longevity of sul-2 (pv17) was not suppressed by nhr-49 (nr2041). h. Sul-2 (pv17) increases lifespan in the glp-1 background. i. Sul-2 (pv17) longevity is suppressed by daf-12 (m20).

[0014] Figure 9 The sul-2 mutants affect the position of DAF-16 in intestinal cells, but they do not affect reproduction or gonadal morphology. a. Photographs show representative images of the position of Pdaf-16::gfp::daf-16 in wild type (left) and sul-2 mutants (middle and right). Both sul-2 mutants increase the nuclear position of DAF-16 in intestinal cells, as in ageroteric animals. Scale bar 20 μm. b. Quantification of nuclear fluorescence in foregut cells. Data from two independent assays, n ≥ 34 nuclei for each condition. One-way ANOVA test. c. The sul-2 mutants have similar progeny numbers at 20 °C. One-way ANOVA test; ns. e. The reproductive period of the sul-2 mutants is unaffected at 20 °C. e. The sul-2 mutants show normal gonadal morphology. Photographs of a representative gonadal arm of each strain at late L4 are shown.

[0015] Figure 10sul-2 is expressed in allelopathic and phasmid sensory neurons. In extrachromosomal transgenic worms, mCherry is expressed only in a few sensory neurons at the sul-2 promoter and its 3'UTR. a. The transcribed reporter gene for sul-2 is expressed in sensory neurons. Imaged in fluorescence and fused with light. b. Representative image of extrachromosomal expression of sul-2 in allelopathic neurons. Most transgenic animals express sul-2 in two pairs of allelopathic neurons (left panel), with a portion showing expression in other neurons besides those (right panel). n=64. c. Colocation of sul-2 neurons with FiTC staining. The top panel shows colocation of the head with the foremost paired allelopathic neurons, possibly ASK, ADF, or ADL neurons, but not the last paired neurons, ASG, or ASE. In the tail (bottom panel), four neurons expressing sul-2 are colocationed with FiTC staining in PHA and PHB phasmid neurons. d. Identification of two major pairs of sul-2-expressing chemosensors was performed by colocalization with the tph-1 neuron-specific promoter (for ADF) (top panel) and with the postneuronal ASE chemosensor expressed by the flp-6 promoter (bottom panel). Scale bar: 20 μm. In the gut, autofluorescence signals were nonspecific under imaging conditions (blue-green arrows).

[0016] Figure 11 sul-2 is not expressed in gonadal tissue and is unaffected in neuronal function. a. The integrated mCHERRY reporter of the sul-2 transcription unit is expressed only in sensory neurons and not in other tissues. Fluorescence imaging was performed and merged with bright-field images. b. Inset of the integrated worm, i) In the head, sul-2 is expressed in a few chemoreceptor neurons (white arrows). In the gut, the autofluorescence signal is nonspecific under imaging conditions (blue-green arrows). ii) No specific signal in the vulva, embryo, or reproductive area. iii) No specific signal in the gonads or mature oocytes. iv) No obvious signal in the tail sensilla. Scale bar 50 μm. c. Except for expression in Clˉ and Na+ sensing neurons (ASE), the sul-2 mutant responds to Clˉ similarly to the wild type. Data from three independent replicates. d. The sul-2 mutant responds to Na+ similarly to the wild type. tax-4 (p678) is a negative control. Data from three independent replicates. Mean ± SEM is shown in all charts. The e. sul-2 deletion increases the lifespan of the long-lived daf-10(m79) mutant, which is affected in sensory neurons.

[0017] Figure 12Neurodegenerative phenotypes are alleviated during aging when steroid sulfatase function is reduced and α-synuclein aggregation is decreased. a. Sul-2 has a beneficial effect on a muscular Parkinson's disease model in the adult stage. Data are from two independent biological replicates, n≥31 per day and per condition. b. Sul-2 also resulted in less body curvature compared to wild-type controls under the same experimental conditions, n≥15 per day and per condition (20°C). c. The protective effect of STX64 in a muscular Parkinson's disease model persists throughout aging. Data are from two independent biological replicates, n≥12 / day and condition. d, e. Sul-2 significantly reduced the number of α-synuclein aggregates in muscle at 7 days of age. Animal and quantitative examples are shown separately. n=18. Scale bar 25μm. f. Expression of human β-amyloid in muscle causes age-related paralysis (CL2006 strain), which is alleviated in the Sul-2 deletion mutant. g. Or alleviated by treatment with STX64. The Mantel-Cox test showed p = 0.0027 for sul-2 missing values ​​and p = 0.007 for STX treatment.

[0018] Figure 13 Phenotypic treatment with sulfated C19 steroid hormones simulated sul-2 inactivation. a. The paralytic phenotype in the GMC101 strain (Alzheimer's disease model) was reduced with TS and ES, but not with DHEAS (1 μg / ml). Assays were performed using normal NGM plates. Data were obtained from three independent biological replicates, n > 130. b. Similar to daf-2 (e1370); sul-2, the percentage of L1 arrest in daf-2 (e1370) increased with DHEAS, TS, or ES. c, d. Other biological replicates of the lifespan curves for sulfated C19 steroid hormones. ES (1 μg / ml) increased lifespan in the wild-type background, but did not increase it further in sul-2 (pv17), while DHEAS and TS (1 μg / ml) had no effect.

[0019] Figure 14 A lifespan regulation model based on SUL-2. Sulfatase SUL-2 and sulfotransferase (possibly SSU-1) regulate the levels of sulfated steroid hormones. High levels of sulfated steroid hormones lead to increased lifespan, dependent on the same factors contributing to longevity as germline reduction (DAF-16, DAF-12, KRI-1, TCERE-1). The fact that both sulfatase and sulfotransferase are expressed in sensory neurons suggests a coordination between the sulfation state of hormones and environmental signals.

[0020] Figure 15This figure shows that treatment with epitestosterone sulfate (ES) significantly increased lifespan (p < 0.05 log-rank (Mantel-Cox)). The number of nematodes was always over 100 for each strain and condition.

[0021] Figure 16 Three months of STX64 treatment improved cognition in 15-month-old mice. The 15-month-old mice retained cognitive abilities, but these abilities were lost over the following months. A: Motor indices were similar between the two groups (solvent group and STX group). B: Object recognition improved after three months of treatment. C: Short-term and long-term memory in passive avoidance tests. *p<0.05. **p<0.005. ***p>0.0005. One-way ANOVA.

[0022] Figure 17 Six months of STX64 treatment improved cognition in 15-month-old mice. The mice retained cognitive abilities at 15 months of age, but lost these abilities over the following months. Mice were 21 months old at the time of measurement. A: Motor indices were similar between the two groups (solvent group and STX group). B: Object recognition improved after three months of treatment. C: Short-term and long-term memory in passive avoidance tests. *p<0.05. **p<0.005. ***p>0.0005. One-way ANOVA.

[0023] Figure 18 One month of STX64 treatment improved cognition in 18-month-old mice. Cognitive impairment was present in 18-month-old mice. Mice were 19 months old at the time of measurement. A: Motor indices were similar in both groups (solvent group and STX group). B: Treated animals showed significant improvements in short-term and long-term memory in passive avoidance tests. *p<0.05. **p<0.005. ***p>0.0005. One-way ANOVA.

[0024] Figure 19 Three months of STX64 treatment improved cognition in 18-month-old mice. Cognitive impairment was observed in 18-month-old mice. Mice were 21 months old at the time of testing. A: Motor indices were similar between the two groups (solvent group and STX group). B: Object recognition improved after three months of treatment. C: Short-term and long-term memory in passive avoidance tests. *p<0.05. **p<0.005. ***p>0.0005. One-way ANOVA. B: *p<0.05. **p<0.005. ***p>0.0005. One-way ANOVA.

[0025] Figure 20This figure shows the results of the "Open Field" test and the "Y maze" test. The "Open Field" test requires mice to explore an unknown space for 15 minutes. This test allows for the study of activity and habituation (cognitive parameters measuring an individual's ability to recognize their environment). The "Y maze" test, on the other hand, requires mice to explore a maze for 6 minutes. This test assesses the distance traveled (a measure of activity) and the ability to visit all three arms without repeating any of them (a measure of immediate memory). The results showed that treatment with STX-64, ES, or both tended to improve motor skills in 16-month-old mice. Conversely, habituation and immediate memory in older animals were improved by treatment with STX, ES, and a combination of both.

[0026] Figure 21 In this figure, the assessment of learning and memory abilities in aged mice was determined in object recognition and passive avoidance tests. First, in object recognition, the ability to recognize different objects exposed alongside familiar objects was tested after training with the same objects. When this occurred, the object discrimination index was positive and above 0.1. In passive avoidance, the aim was to tell the mouse that its preferred location was not suitable for it; for them, this was associated with an aversive stimulus. To do this, during training, an entry latency to the preferred location was established and an aversive stimulus was applied. During short-term and long-term memory tests (STM and LTM), the entry latency increased with training if the mouse associated the preferred location with the aversive stimulus. Aged mice treated with solvent showed impairments in both learning and memory abilities. In both tests, mice treated with STX, ES, and a combination of both showed significant learning ability (STM) and the ability to store information (memory). Summary of the Invention

[0027] like Figure 20 As shown, treatment with STX-64, epitestosterone sulfate (ES), or both often improves motor skills, habituation, and immediate memory in older animals. Furthermore, as... Figure 21 As shown, the use of STX-64 and / or ES improved short-term and long-term memory tests (STM and LTM). Indeed, in this figure, solvent-treated older mice exhibited impairments in learning ability and memory. However, in both tests, mice treated with STX, ES, or a combination of both showed significant improvements in both learning (STM) and the ability to store information (memory). That is, treatment with STX-64, epitestosterone sulfate (ES), or both significantly improved age-related cognitive impairment, particularly age-related cognitive impairment associated with age-related memory impairment (AAMI) or age-related cognitive decline (ARCD).

[0028] Therefore, one aspect of the present invention relates to a composition in a method for improving age-related cognitive function and / or treating conditions involving age-related cognitive dysfunction or age-related cognitive impairment in subjects of need, preferably a pharmaceutical composition comprising epitestosterone sulfate (ES) or any salt or ester thereof, and / or a compound of formula (I) capable of inhibiting steroid sulfatase:

[0029]

[0030] in:

[0031] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0032] (b) At least one of R1-R6 is an aminosulfonate group, and two or more of R1-R6 are linked together to form an additional cyclic structure.

[0033] Specifically, this composition is used for age-related cognitive impairment associated with mild cognitive impairment (MCI), age-related memory impairment (AAMI), or age-related cognitive decline (ARCD). More specifically, this composition is used for age-related cognitive impairment associated with age-related memory impairment (AAMI).

[0034] In one implementation, age-related cognitive impairment, preferably age-related memory impairment (AAMI), is considered improved when compared to expected cognitive function in age-matched normal subjects. That is, cognitive function in the subject in need may be reduced by about 5%, about 10%, about 30%, or more compared to expected cognitive function in age-matched normal subjects, and treatment reduces this difference, preferably by at least 0.1%, 1%, 2%, 3%, 4%, 5%, or more.

[0035] In one embodiment, the composition is used in a method for treating age-related cognitive impairment in a subject in need, preferably in a method for improving and / or treating age-related short-term and / or long-term memory impairment in a subject in need.

[0036] In one embodiment, the composition is used in a method for treating age-related cognitive impairment in a subject in need, preferably in a method for improving and / or treating age-related short-term and / or long-term memory in a subject in need.

[0037] In one embodiment, the composition used comprises epitestosterone sulfate (ES) or any salt or ester thereof.

[0038] In one embodiment, the composition used comprises the sulfatase inhibitor STX64 of formula (II):

[0039]

[0040] Or any of its salts.

[0041] On the other hand, it relates to a non-treatment method for improving age-related cognitive function or age-related cognitive impairment in individuals in need; in particular, a non-treatment method for improving age-related cognitive impairment associated with mild cognitive impairment (MCI), age-related memory impairment (AAMI), or age-related cognitive decline (ARCD); more specifically, a non-treatment method for improving age-related cognitive impairment associated with age-related memory impairment (AAMI); the method comprising the steps of: providing a composition containing epitestosterone sulfate (ES) or any salt or ester thereof and / or a compound of formula (I) capable of inhibiting steroid sulfatase:

[0042]

[0043] in:

[0044] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0045] (b) at least one of R1-R6 is an aminosulfonate group, and two or more of R1-R6 are linked together to form an additional cyclic structure; and

[0046] Administer an effective amount of the compound to the subject in need. In particular, the subject's cognitive function may be reduced by approximately 5%, approximately 10%, approximately 30%, or more compared to the expected cognitive function in age-matched normal subjects.

[0047] In one embodiment, the method includes administering epitestosterone sulfate (ES) or any salt or ester thereof.

[0048] In one embodiment, the method includes administering the sulfatase inhibitor STX64 of formula (II):

[0049]

[0050] Or any of its salts. Detailed Implementation

[0051] definition

[0052] As used herein, “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable diluent” means any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The term “pharmaceuticalally acceptable excipient” refers to any substance formulated with the active pharmaceutical ingredient, including substances used for long-term stabilization, swelling of solid dosage forms containing small amounts of the active ingredient, or to impart therapeutic enhancements to the active ingredient in the final dosage form, such as promoting absorption, reducing viscosity, or increasing solubility. Excipients may also be used in the manufacturing process to aid in addressing issues related to the active substance, such as by promoting powder flowability or non-stick properties, and also to aid in in vitro stability, such as preventing denaturation or aggregation during the expected shelf life. The use of such media and agents for the active pharmaceutical ingredient is well known in the art. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used, and, without limiting the scope of the invention, include: additional buffers; preservatives; cosolvents; antioxidants, including ascorbic acid and methionine; chelating agents, such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counterions, such as sodium, polyols; amino acids, such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, and 2-phenylalanine. Acids, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbitol, xylose, ribose, ribitol, myoinisitose, inositol, galactose, galactitol, glycerol, cyclic alcohols (e.g., cellulose alcohol), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012), ISBN-13:9780857110626.

[0053] "Improving" cognitive function includes promoting and / or maintaining cognitive function.

[0054] “Cognitive function” or “cognitive state” refers to any higher-order intellectual brain process or state related to learning and / or memory, including but not limited to attention, information acquisition, information processing, working memory, short-term memory, long-term memory, anterograde memory, retrograde memory, memory retrieval, differential learning, decision-making, inhibitory response control, attentional set shifting, delayed reinforcement learning, backward learning, temporal integration of voluntary behavior, and expressing interest in the surrounding environment and self-care. In humans, cognitive function can be measured by, for example but not limited to, the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-cog), the Clinical Global Impression Scale (CGIC-plus), the Alzheimer’s Disease Collaborative Research Activities in Daily Living Scale (ADCS-ADL), the Clinical Global Impression Scale (CIBIC-plus); the Mini-Mental State Test (MMSE); the Neuropsychiatric Scale (NPI); the Clinical Dementia Scale (CDR); the Cambridge Automated Neuropsychological Tests (CANTAB); or the Sandow Clinical Assessment of Older Persons (SCAG). See Folstein et al., J Psychiatric Res 12:189-98, (1975); Robbins et al., Dementia 5:266-81, (1994); Rey, L'examen clinique en psychologie, (1964); Kluger et al., J Geriatr Psychiatry Neurol 12:168-79, (1999). Furthermore, cognitive function can be measured using imaging techniques such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single-photon emission computed tomography (SPECT), or any other imaging technique that allows for the measurement of brain activity. In animal model systems, cognitive function can be measured using a variety of conventional methods known in the art, including the Morris water maze (MWM), Barnes circular maze, elevated radial arm maze, T-maze, or any other maze in which the animal uses spatial information. Other tests known in the art can also be used to assess cognitive function, such as new object recognition and odor recognition tasks. Cognitive function can also be measured using imaging techniques such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), single-photon emission computed tomography (SPECT), or any other imaging technique that allows for the measurement of brain function. In animals, cognitive function can also be measured using electrophysiological techniques.

[0055] Age-related cognitive impairment, or "function," refers to cognitive function in older individuals that is less sound than expected in age-matched normal subjects (i.e., subjects with the average score on cognitive tests for a given age) or as sound as expected in younger adults. In some cases, cognitive function is reduced by approximately 5%, 10%, 30%, or more compared to expected cognitive function in age-matched normal subjects. In other cases, cognitive function is as expected in age-matched normal subjects, but is reduced by approximately 5%, 10%, 30%, 50%, or more compared to expected cognitive function in younger adults. Age-related cognitive impairment can be associated with mild cognitive impairment (MCI), age-related memory impairment (AAMI), and age-related cognitive decline (ARCD).

[0056] Mild cognitive impairment (MCI) is a condition characterized by isolated memory impairment without other cognitive abnormalities and relatively normal functional abilities. A set of criteria for clinical features of MCI specifies the following features: (1) a memory complaint (reported by the patient, informant, or physician); (2) normal activities of daily living (ADL); (3) normal overall cognitive function; (4) age-related memory impairment (defined as being more than 1.5 standard deviations below the mean for a given age); and (5) absence of dementia indicators (as defined in the DSM-IV guidelines; see also Petersen et al., Srch. Neurol. 56:303-308 (1999); Petersen, “Mildcognitive impairment: Aging to Alzheimer's Disease.” Oxford University Press, NY (2003)).

[0057] Age-related memory impairment (AAMI) refers to memory decline due to aging. A patient is considered to have AAMI if they are 50 years of age or older and meet all of the following criteria: a) the patient notices a decline in memory performance; b) the patient performs worse on standard memory tests compared to younger people; and c) all other obvious causes of memory decline besides normal aging have been ruled out (in other words, memory decline cannot be attributed to other causes such as a recent heart attack or head injury, depression, adverse drug reactions, Alzheimer's disease, etc.).

[0058] Age-related cognitive decline (ARCD) refers to a decline in memory and cognitive abilities, a normal consequence of aging (e.g., Craik & Salthouse, 1992). This is true for almost all mammalian species. Age-related memory impairment refers to a decline in objective memory relative to one's younger years, but cognitive function is normal relative to one's peers (Crook et al., 1986). Age-consistent memory decline is a less pejorative label, emphasizing that these are normal developmental changes (Crook, 1993; Larrabee, 1996), not pathophysiological (Smith et al., 1991), and rarely progress to obvious dementia (Youngjohn & Crook, 1993). The DSM-IV (1994) codified a diagnostic classification for ARCD.

[0059] As used in this application, the terms "treatment" and "therapy" refer to a group of hygienic, pharmacological, surgical, and / or physical means intended to treat and / or alleviate disease and / or symptoms with the aim of restoring health problems. The terms "treatment" and "therapy" include both preventative and curative methods, as both aim at maintaining and / or restoring the health of an individual or animal. Regardless of the origin of the symptoms, disease, and disability, in the context of this application, the administration of appropriate medicine to alleviate and / or treat health problems should be interpreted as a form of treatment or therapy.

[0060] As used in this application, the term "prevention" refers to a group of sanitary, pharmacological, surgical, and / or physical means for preventing the onset and / or development of disease and / or symptoms. The term "prevention" encompasses preventive methods because these methods are used to maintain the health of an animal or individual.

[0061] The term "sulfatase inhibitor" refers to any substance capable of reducing the activity of esterases that catalyze the hydrolysis of sulfates. This substance can be a molecule that binds to any of the following elements and reduces or inhibits the expression and activity of the molecule it binds to, and / or its intracellular or extracellular signaling, thereby resulting in the complete or partial inhibition of sulfatase activity: a gene encoding a sulfatase, a transcription factor of said gene, any expression product of said gene, such as, but not limited to, messenger RNA, or sulfatase. Inhibitors can be selected from, but are not limited to, a list consisting of: sulfatase antagonists (preferably chemical antagonists), silencing RNA, or specific antibodies against sulfatase (preferably monoclonal antibodies); in this invention, the antibody can be defined as a neutralizing antibody against sulfatase action. Examples of chemical inhibitors of sulfatase activity include, but are not limited to: alternative substrates, such as those in the series 2-(hydroxyphenyl)indole sulfate; synthetic or natural steroids with inhibitory activity against STS, such as 5-androsten-3β,17β-diol-3 sulfate; competitive inhibitors, such as E1-MTP or EMATE; non-estrogen inhibitors, such as DU-14 (CAS NO: 186303-55-9), COUMATE (4-methylcoumarin-7-O-aminosulfonate) or STX64 (i.e., compound of formula (II)); or others, such as KW-2581 or STX213, whose IC50 against sulfatase has been determined in various studies (Purohit & Foster, 2012, J. Endocrinol., 212(2):99-110).

[0062] The term "steroid hormone sulfatase" ("STS") refers to any sulfatase involved in steroid metabolism. Specifically, the enzyme catalyzes the conversion of sulfated steroid precursors to free steroids. Exemplary STSs identified in humans have been sequenced, characterized, and the data are deposited in the UniProtKB database under accession number P08842. The term "steroid hormone sulfatase inhibitor" refers to any substance capable of reducing the activity of steroid hormone sulfatase. This substance can be a molecule that binds to any of the following elements and reduces or inhibits the expression and activity of the molecule it binds to, and / or its intracellular signaling, thereby resulting in the complete or partial inhibition of STS enzyme activity: a gene encoding the STS enzyme, a transcription factor of said gene, any expression product of said gene, such as, but not limited to, messenger RNA, or the STS enzyme. Inhibitors may be selected from, but are not limited to: STS enzyme antagonists (preferably chemical antagonists), silent RNA, or specific antibodies against the STS enzyme (preferably monoclonal antibodies; in this invention, the antibody may be defined as a neutralizing antibody against the action of the STS enzyme). Examples of chemical inhibitors of STS enzyme activity are, but are not limited to: alternative substrates, such as those in the series of 2-(hydroxyphenyl)indole sulfates; synthetic or natural steroids with inhibitory activity against STS, such as 5-androsten-3β,17β-diol-3-thiosulfate. Esters; competitive inhibitors such as E1-MTP or EMATE; non-estrogen inhibitors such as DU-14, COUMATE (4-methylcoumarin-7-O-aminosulfonate) or STX64 (i.e., compound of formula (II)); or others, such as KW-2581 or STX213, whose IC50 against sulfatase has been determined in various studies (Purohit & Foster, 2012, J. Endocrinol., 212(2):99-110).

[0063] The term "protein aggregation disease" refers to any disease in which certain proteins undergo structural abnormalities, thereby disrupting the function of the body's cells, tissues, and organs. These proteins typically fail to fold into their normal conformation; in this misfolded state, the proteins may become toxic in some way, or they may lose their normal function. Non-limiting examples of protein aggregation diseases include: systemic AL amyloidosis, Alzheimer's disease, type 2 diabetes, Parkinson's disease, infectious spongiform encephalopathy (e.g., bovine spongiform encephalopathy), fatal familial insomnia, Huntington's disease, medullary thyroid carcinoma, arrhythmias, atherosclerosis, rheumatoid arthritis, medial aortic amyloidosis, prolactinoma, familial amyloid polyneuropathy, hereditary non-neurotic systemic amyloidosis, dialysis-associated amyloidosis, and Finnish amyloidosis. Lattice corneal dystrophy, cerebral amyloid angiopathy, cerebral amyloid angiopathy (Icelandic type), sporadic inclusion body myositis, amyotrophic lateral sclerosis (ALS), prion-associated or spongiform encephalopathy (e.g., Kreutzfeldt-Jacob disease), Lewy body dementia, frontotemporal dementia with Parkinson's disease, spinocerebellar ataxia, spinocerebellar ataxia, spinobulbar muscular atrophy, hereditary dentate-pallidus atrophy, familial Alzheimer's disease, familial Danish dementia, non-neurotic focal disorders. (e.g., type II diabetes, medullary thyroid carcinoma, atrial amyloidosis, hereditary cerebral hemorrhage with amyloidosis, pituitary prolactinoma, injection-induced amyloidosis, medial aortic amyloidosis, hereditary corneal dystrophy, corneal amyloidosis with trichiasis, cataract, odontogenic calcified epithelial tumor, pulmonary alveolar proteinosis, inclusion body myositis, cutaneous lichen amyloidosis), and non-neurogenic systemic amyloidosis (e.g., AL amyloidosis, AA amyloidosis, familial Mediterranean fever, senile amyloidosis). Systemic amyloidosis, familial amyloid polyneuropathy, hemodialysis-associated amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Finnish hereditary amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, Icelandic hereditary cerebral amyloid angiopathy, familial amyloidosis, and systemic amyloidosis occurring in multiple tissues (e.g., light chain amyloidosis); as well as various other neurodegenerative diseases.

[0064] The term "protein aggregate" refers to any accumulation of abnormally folded proteins that cause protein aggregation disease and / or are associated with the negative progression of protein aggregation disease.

[0065] The term “amyloid” refers to a form of protein aggregate in which the aggregates form unbranched filaments bound to Congo red and then exhibit green birefringence when viewed between cross-polarizers (see, for example, Eisenberg & Jucker, 2012. Cell. 148(6): 1188-203 and Sipe et al., 2012. Amyloid. 19(4): 167-70).

[0066] The term “oligomery” refers to any accumulation of abnormally folded proteins that cause protein aggregation disorders and / or are associated with the negative progression of protein aggregation disorders, while not meeting the definition of amyloid. For example, polyglutamine oligomers can cause Huntington’s disease and / or are associated with the negative progression of Huntington’s disease (see Hoffner & Dijan, 2014. Brain Sci. 4(1): 91–122).

[0067] describe

[0068] The gonads are key tissues regulating lifespan. The germline regulates lifespan by inhibiting macroalkaline production in gonadal cells. Consistently, germline ablation or mutations that eliminate germline production increase lifespan by activating macroalkaline synthesis. The gonads are also classic tissues for producing sex steroids, although not the only ones. Our data suggest that inhibiting sulfatase activity through mutation or STX64 increases levels of a very specific group of sulfated steroid hormones, which actually increases lifespan. This increase in lifespan depends on common factors involved in the lifespan extension resulting from germline loss, suggesting that the two processes are indeed related. We cannot distinguish whether the lifespan-increasing effect of sulfated steroid hormones is involved in the same pathway or acts in parallel with germline lifespan, which shares certain elements of that pathway. In fact, sul-2 inhibition is independent of NHR-49, or only partially dependent on NHR-80, where both NHR-49 and NHR-80 are necessary for germline-mediated longevity, thus pointing to the second option.

[0069] We also investigated the levels of sulfated steroid hormones in the glp-1 mutant of the germline, and we did not observe an increase in sulfated hormones. These data support the idea that gonads produce steroid hormones through sulfate modification. These sulfated steroid hormones may alter neurotransmission and increase lifespan through factors common to agermic animals. The fact that enzymes involved in the sulfate modification of steroid hormones (sulfatase SUL-2 and sulfotransferase SSU-1) are expressed in sensory neurons suggests that altering the sulfated state of hormones can affect the integration of environmental cues (e.g., nutrient availability) with reproductive status—a biphasic cross-linking process.

[0070] In *C. elegans*, somatic cell proliferation occurs only during development, specifically in the larval stage and not in the adult stage; therefore, increased lifespan is attributed to post-mitotic cell maintenance. One of the stresses observed in *C. elegans* adult cells is the aggregation of endogenous proteins, which leads to cellular dysfunction. This age-related aggregate formation is also observed in the ectopic expression of easily aggregated proteins such as β-amyloid or α-synuclein. Longevity mutants such as daf-2 or glp-1 delay aggregation toxicity through various mechanisms, including chaperone expression and proteasome degradation or autophagy. Here we show that inhibiting sulfatase activity or treatment with certain sulfated C19 androgens not only affects lifespan but also reduces protein aggregation and its toxic consequences in a *C. elegans* model of protein aggregation disease.

[0071] Regulating steroid hormones via sulfation is a conserved process. In mammals, sulfotransferases and sulfatases are expressed in various tissues, including the nervous system, similar to what we have observed in *C. elegans*. In humans, C19 steroid hormones are also associated with lifespan. For example, dehydroepiandrosterone sulfate (DHEAS) declines with age and has been used as a marker of aging, leading to speculation about its causal role in sarcopenia, cognitive decline, and other age-related diseases, including Alzheimer's disease. Our data suggest that inhibiting steroid sulfatases through mutation or STX64 treatment prolongs the lifespan of *C. elegans* and protects against the toxicity of age-related proteins in the nematode. Interestingly, similar effects were observed after treatment with certain specific sulfated C19 steroid hormones.

[0072] Therefore, the present invention provides a method for increasing the lifespan of eukaryotic organisms, comprising the following steps: providing a sulfated C19 androgen compound, or a compound selected from the list of the following that is capable of inhibiting steroid sulfatase: 2-(hydroxyphenyl)indole sulfate, 5-androsten-3β,DU-14,17β-diol-3 sulfate, E1-MTP, EMATE, COUMATE, STX64 (compound of formula II), KW-2581, STX213, morpholine, silent RNA and a specific antibody against steroid sulfatase (STS), or steroid sulfatase (EC3.1.6.2) (formerly known as aryl sulfatase C); or a sulfatase inhibitor of formula (I):

[0073]

[0074] in:

[0075] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0076] (b) at least one of R1-R6 is an aminosulfonate group, and two or more of R1-R6 are linked together to form an additional cyclic structure; and

[0077] Applying an effective amount of the compound to eukaryotes can increase their lifespan.

[0078] In addition, such as Figures 16 to 19 As shown, the present invention also provides methods and compositions for improving age-related cognitive function and / or treating conditions involving age-related cognitive dysfunction, age-related cognitive impairment, or their risks by administering a sulfated C19 androgen compound or a compound selected from the list of compounds capable of inhibiting steroid sulfatases in subjects of need: 2-(hydroxyphenyl)indole sulfate, 5-androstene-3β,DU-14,17β-diol-3 sulfate, E1-MTP, EMATE, COUMATE, STX64 (compound of formula II), KW-2581, STX213, morpholine, silent RNA, and specific antibodies against steroid sulfatase (STS), or steroid sulfatase (EC3.1.6.2) (formerly known as aryl sulfatase C); or sulfatase inhibitors of formula (I):

[0079]

[0080] in:

[0081] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0082] (b) At least one of R1-R6 is an aminosulfonate group, and two or more of R1-R6 are linked together to form an additional cyclic structure.

[0083] In a preferred embodiment, the compound used in any of the above methods (for increasing the lifespan of eukaryotes and / or for improving age-related cognitive function and / or treating conditions or risks involving age-related cognitive impairment) is a sulfated C19 androgen selected from the list of the following: dehydroepiandrosterone sulfate (DHEAS), testosterone sulfate (TS), epitestosterone sulfate (ES), or androgenetic asterone sulfate (AS). In a more preferred embodiment, the sulfated C19 androgen is selected from the group consisting of testosterone sulfate (TS), epitestosterone sulfate (ES), or androgenetic asterone sulfate (AS).

[0084] Testosterone sulfate (TS) (https: / / pubchem.ncbi.nlm.nih.gov / compound / Testosterone-sulfate) is an endogenous, naturally occurring steroid and a minor urinary metabolite of testosterone. Its chemical name is hydrogen sulfate [(8R,9S,10R,13S,14S,17S)-10,13-dimethyl-3-oxo-1,2,6,7,8,9,11,12,14,15,16,17-dodecylhydrocyclopentan[a]phenanthrene-17-yl], and it has the formula:

[0085]

[0086] Other names: 17β-testosterone sulfate; testosterone 17β-sulfuric acid; 17β-(sulfonoxy)andros-4-en-3-one.

[0087] Epitestosterone differs from testosterone structurally only in the configuration at the hydroxyl carbon C17. Epitestosterone sulfate (ES), also known as 17α-testosterone sulfate, has the following chemical name: N,N-diethylethylamine; hydrogen sulfate [(8R,9S,10R,13S,14S,17R)-10,13-dimethyl-3-oxo-1,2,6,7,8,9,11,12,14,15,16,17-dodecylhydrocyclopentan[a]phenanthrene-17-yl], with the formula:

[0088]

[0089] Androsterone sulfate (AS), also known as 3α-hydroxy-5α-androst-17-one 3α-sulfate, is an endogenous, naturally occurring steroid and one of the main urinary metabolites of androgens. It is a sulfated steroid formed by the sulfation of androsterone by the steroid sulfotransferase SULT2A1, and can be desulfurized back to androsterone by steroid sulfatase. Its chemical name is hydrogen sulfate [(3R,5S,8R,9S,10S,13S,14S)-10,13-dimethyl-17-oxo-1,2,3,4,5,6,7,8,9,11,12,14,15,16-tetradecanoic-cyclopentan[a]phenanthrene-3-yl], with the formula:

[0090]

[0091] It should be noted here that any of the above-described sulfated C19 androgens includes their respective salts and esters. Preferably, any of the above-described sulfated C19 androgens includes their respective pharmaceutically acceptable salts, pharmaceutically acceptable solvates, isotopic variants (preferably containing deuterium atoms and / or one or more carbon atoms having 13C), different crystalline forms such as polymorphs, pharmaceutically acceptable esters, stereoisomers, tautomers, analogs, and derivatives.

[0092] Solvents, such as (A) or (B), where R = H, alkyl, or aryl, are formed by adding water or alcohol to the parent compound:

[0093]

[0094] Isotopic variants, such as one or more atoms being replaced by stable isotopes of the same atom, for example, replacing one or more hydrogen atoms with deuterium, e.g., (C), or replacing one or more carbon atoms with 13C, e.g., (D). It should be understood that this possibility applies to any atom within the structure, and combinations can also be considered:

[0095]

[0096] In another preferred embodiment, the compound capable of inhibiting any of the above-described methods (for increasing the lifespan of eukaryotes and / or for improving age-related cognitive function and / or for treating conditions involving age-related cognitive impairment, age-related cognitive disorders, or their risks) is a sulfatase inhibitor of formula (I):

[0097]

[0098] in:

[0099] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0100] (b) At least one of R1-R6 is an aminosulfonate group, and two or more of R1-R6 are linked together to form an additional cyclic structure.

[0101] Preferably, the compound capable of inhibiting steroid sulfatase is the sulfatase inhibitor STX64 represented by formula (II):

[0102]

[0103] Similar to the case of sulfated C19 androgens described above, STX64 of formula (II) above comprises its corresponding salt and ester. Preferably, STX64 of formula (II) above comprises its corresponding pharmaceutically acceptable salt, pharmaceutically acceptable solvate, isotopic variant (preferably containing deuterium atoms and / or one or more carbon atoms having 13C), different crystalline forms such as polymorphs, pharmaceutically acceptable esters, stereoisomers, tautomers, analogs, and derivatives.

[0104] In a preferred embodiment, the compound in any of the above methods (for increasing the lifespan of eukaryotes and / or for improving age-related cognitive function and / or for treating conditions involving age-related cognitive dysfunction, age-related cognitive impairment, or their risks) is a combination of sulfated C19 androgens (preferably testosterone sulfate (TS), epitestosterone sulfate (ES), or androsterone sulfate (AS)) and a sulfatase inhibitor selected from: 2-(hydroxyphenyl)indole sulfate, 5-androsten-3β,DU-14,17β-diol-3 sulfate, E1-MTP, EMATE, COUMATE, STX64, KW-2581, STX213, morpholine, silent RNA, and a specific antibody against the STS enzyme; or a sulfatase inhibitor of formula (I):

[0105]

[0106] in:

[0107] (a) R1-R6 are independently selected from hydrogen, halogens (fluorine, chlorine, bromine, iodine or astatine), hydroxyl groups, aminosulfonates (OSO2NH2), alkyl groups and their salts;

[0108] (b)(a) At least one of R1-R6 is an aminosulfonate group; and

[0109] Two or more of R1-R6 are connected together to form an additional ring structure.

[0110] In a preferred embodiment, the sulfatase inhibitor is STX64, and the sulfated C19 androgen is epitestosterone sulfate (ES).

[0111] From this point onward, when the above-mentioned compounds or combinations of compounds are used in methods for increasing the lifespan of eukaryotes, they shall be referred to as "lifespan-increasing compounds".

[0112] As described above, life-extending compounds are those that can be used to prevent age-related protein toxicity and / or increase the lifespan of eukaryotes. As used herein, lifespan refers to the length of time a cell or organism survives before death (e.g., days or years) (chronological lifespan). More specifically, life-extending compounds as described above are preferably compounds that prevent age-related protein toxicity caused by protein aggregation diseases and / or increase the lifespan of eukaryotes.

[0113] Protein aggregates, such as amyloid and oligomers, are associated with many diseases. In some cases, these protein aggregates can become toxic and cause severe damage to cells and tissues. This toxicity is considered one of the contributing factors to and / or the pathogenesis of protein aggregation diseases. The life-extending compounds of the present invention provide protection against and treatment for protein aggregation diseases. In a preferred embodiment, protein aggregation diseases are selected from the list of: systemic AL amyloidosis, Alzheimer's disease, type 2 diabetes, Parkinson's disease, infectious spongiform encephalopathy (e.g., bovine spongiform encephalopathy), fatal familial insomnia, Huntington's disease, medullary thyroid carcinoma, arrhythmia, atherosclerosis, rheumatoid arthritis, medial aortic amyloidosis, prolactinoma, familial amyloid polyneuropathy, hereditary nonneurogenic systemic amyloidosis, and dialysis-associated amyloidosis. Finnish amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy, cerebral amyloid angiopathy (Icelandic type), sporadic inclusion body myositis, amyotrophic lateral sclerosis (ALS), prion-associated or spongiform encephalopathy (e.g., Kreutzfeldt-Jacob disease), Lewy body dementia, frontotemporal dementia with Parkinson's disease, spinocerebellar ataxia, spinocerebellar ataxia, spinobulbar muscular atrophy, hereditary dentate-pallioidosis, familial Alzheimer's disease, familial Danish dementia, non-neurotic Localized diseases (such as type II diabetes, medullary thyroid carcinoma, atrial amyloidosis, hereditary cerebral hemorrhage with amyloidosis, pituitary prolactinoma, injection-induced amyloidosis, medial aortic amyloidosis, hereditary corneal dystrophy, corneal amyloidosis with trichiasis, cataracts, odontogenic calcified epithelial tumors, pulmonary alveolar proteinosis, inclusion body myositis, cutaneous lichen amyloidosis) and non-neurogenic systemic amyloidosis (such as AL amyloidosis, AA amyloidosis, familial Mediterranean fever) The list includes: age-related systemic amyloidosis, familial amyloid polyneuropathy, hemodialysis-associated amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Finnish hereditary amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, Icelandic hereditary cerebral amyloid angiopathy, familial amyloidosis, and systemic amyloidosis occurring in multiple tissues (e.g., light chain amyloidosis); and various other neurodegenerative diseases. Preferably, the protein aggregation disease is selected from the list consisting of Alzheimer's disease, Parkinson's disease, and Huntington's disease. In a preferred embodiment, the protein aggregation disease is not Alzheimer's disease and / or a type of cancer.

[0114] In a preferred embodiment, the protein aggregation disease is selected from the list consisting of Alzheimer's disease, Parkinson's disease, and Huntington's disease, and the sulfated C19 androgen of the present invention is preferably selected from the list consisting of testosterone sulfate (TS), epitestosterone sulfate (ES), or androgenetic acid sulfate (AS). More preferably, the sulfated C19 androgen is in the form of a pharmaceutical composition.

[0115] In a preferred embodiment, the protein aggregation disease is a localized protein aggregation disease of the central nervous system. In a preferred embodiment, the protein aggregation disease is also a neurodegenerative disease. The term "neurodegenerative disease" refers to any condition characterized by the progressive loss of neuronal structure or function, including neuronal death. For example, Alzheimer's disease is an example of both a protein aggregation disease and a neurodegenerative disease.

[0116] Therefore, effective amounts of the life-extending compound increase the lifespan of eukaryotes, preferably humans, and / or prevent age-related protein toxicity caused by protein aggregation diseases. In one embodiment, the effective amount of the life-extending compound increases the lifespan of eukaryotes by a statistically significant amount compared to the lifespan of untreated organisms. The lifespan of untreated organisms can be determined in parallel or obtained from a single study (control). In another embodiment, the effective amount of the life-extending compound increases the lifespan of eukaryotes by at least 5%. In other embodiments, the effective amount of LAC increases the lifespan of eukaryotes by at least 10%, 15%, 20%, 25%, 35%, 50%, or 100% compared to a control.

[0117] Examples of eukaryotes include single-celled and multicellular organisms, including higher organisms (such as mammals, including humans).

[0118] In one embodiment, the method of the present invention for increasing the lifespan of eukaryotic organisms can be used to generally increase the lifespan of eukaryotic cells and protect them from stress and / or apoptosis. In various other embodiments, the method can be used to: treat or prevent diseases or conditions induced or aggravated by cellular senescence in a subject; to prolong the lifespan of a subject; to treat or prevent diseases or conditions related to lifespan; to treat or prevent diseases or conditions related to cell proliferation capacity; and to treat or prevent diseases or conditions caused by cell damage or death.

[0119] In various embodiments, this method can be used to prevent aging and its associated consequences, or diseases caused by protein aggregation disorders. In a preferred embodiment, protein aggregation disorders are selected from: systemic AL amyloidosis, Alzheimer's disease, type 2 diabetes, Parkinson's disease, infectious spongiform encephalopathy (e.g., bovine spongiform encephalopathy), fatal familial insomnia, Huntington's disease, medullary thyroid carcinoma, arrhythmia, atherosclerosis, rheumatoid arthritis, medial aortic amyloidosis, prolactinoma, familial amyloid polyneuropathy, hereditary non-neurotic systemic amyloidosis, dialysis-associated amyloidosis, and Finnish amyloidosis. Degeneration, lattice corneal dystrophy, cerebral amyloid angiopathy, cerebral amyloid angiopathy (Icelandic type), sporadic inclusion body myositis, amyotrophic lateral sclerosis (ALS), prion-associated or spongiform encephalopathy (e.g., Kreutzfeldt-Jacob disease), Lewy body dementia, frontotemporal dementia with Parkinson's disease, spinocerebellar ataxia, spinocerebellar ataxia, spinobulbar muscular atrophy, hereditary dentate red nucleus-pallidus atrophy, familial British dementia, familial Danish dementia, non-neurotic focal disorders Diseases such as type II diabetes, medullary thyroid carcinoma, atrial amyloidosis, hereditary cerebral hemorrhage with amyloidosis, pituitary prolactinoma, injection-induced amyloidosis, medial aortic amyloidosis, hereditary corneal dystrophy, corneal amyloidosis with trichiasis, cataracts, odontogenic calcified epithelial tumors, pulmonary alveolar proteinosis, inclusion body myositis, cutaneous lichen amyloidosis, and non-neurogenic systemic amyloidosis (such as AL amyloidosis, AA amyloidosis, familial Mediterranean fever, and old age). Systemic amyloidosis, familial amyloid polyneuropathy, hemodialysis-associated amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Finnish hereditary amyloidosis, lysozyme amyloidosis, fibrinogen amyloidosis, Icelandic hereditary cerebral amyloid angiopathy, familial amyloidosis, and systemic amyloidosis occurring in multiple tissues (e.g., light chain amyloidosis); and various other neurodegenerative diseases. Preferably, the protein aggregation disease is selected from the list consisting of Alzheimer's disease, Parkinson's disease, and Huntington's disease. In a preferred embodiment, the protein aggregation disease is not Alzheimer's disease and / or a type of cancer.

[0120] In a preferred embodiment, the protein aggregation disease is selected from the list consisting of Alzheimer's disease, Parkinson's disease, and Huntington's disease, and the sulfated C19 androgen of the present invention is preferably selected from the list consisting of testosterone sulfate (TS), epitestosterone sulfate (ES), or androgenetic acid sulfate (AS). More preferably, the sulfated C19 androgen is in the form of a pharmaceutical composition.

[0121] As another example, the method of the present invention can also be applied to the development and growth stages of mammals, preferably humans, in order to, for example, alter, delay or accelerate the development and / or growth process.

[0122] It should be noted that all compounds of the present invention, whether used to enhance cognitive abilities or to extend the lifespan of eukaryotes, may be in the form of pharmaceutical compositions.

[0123] In a preferred embodiment, the compound is in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier and / or diluent. Preferably, the pharmaceutical composition may also comprise a pharmaceutically acceptable excipient.

[0124] The pharmaceutical compositions described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, lyophilization protectants, surfactants, fillers, antioxidants, and stabilizers. In some embodiments, the pharmaceutical composition may be lyophilized.

[0125] As used herein, the term "cryoprotectant" includes agents that provide stability to a composition against freeze-induced stress. Cryoprotectants can also provide protection during primary and secondary drying as well as during long-term product storage. Non-limiting examples of cryoprotectants include: sugars such as sucrose, glucose, trehalose, mannitol, mannose, and lactose; polymers such as dextran, hydroxyethyl starch, and polyethylene glycol; surfactants such as polysorbates (e.g., PS-20 or PS-80); and amino acids such as glycine, arginine, leucine, and serine. Cryoprotectants that exhibit low toxicity in biological systems are generally used.

[0126] In one embodiment, a lyophilization protectant is added to the pharmaceutical composition described herein. As used herein, the term "lyophilization protectant" includes an agent that provides stability to the composition during a freeze-drying or dehydration process (one and two freeze-drying cycles). This helps to minimize product degradation during freeze-drying cycles and improves long-term product stability. Non-limiting examples of lyophilization protectants include: sugars, such as sucrose or trehalose; amino acids, such as monosodium glutamate, amorphous glycine, or histidine; methylamines, such as betaine; colloidal ion salts, such as magnesium sulfate; polyols, such as ternary or higher sugar alcohols, such as glycerin, erythritol, glycerol, arabinol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; Pluronic acid; and combinations thereof. The amount of lyophilization protectant added to the pharmaceutical composition is generally an amount that will not cause unacceptable degradation when the pharmaceutical composition is freeze-dried.

[0127] In some embodiments, a filler is included in the pharmaceutical composition. As used herein, the term "filler" includes agents that provide the structure of a freeze-dried product without directly interacting with the pharmaceutical product. In addition to providing a pharmaceutically desirable bulk, fillers can impart useful qualities in improving collapse temperature, providing freeze-thaw protection, and enhancing long-term storage stability. Non-limiting examples of fillers include mannitol, glycine, lactose, and sucrose. Fillers can be crystalline (e.g., glycine, mannitol, or sodium chloride) or amorphous (e.g., dextran, hydroxyethyl starch) and are typically used in formulations in amounts from 0.5% to 10%.

[0128] Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington Pharmaceutical Sciences (16th edition, Osol, A.Ed. (1980)) or Remington: The Science and Practice of Pharmacy (22). nd Those described in the edition of Pharmaceutical Press (2012), ISBN-13:9780857110626 may also be included in the pharmaceutical compositions described herein, provided that they do not adversely affect the desired properties of the pharmaceutical composition.

[0129] For solid pharmaceutical compositions, conventional non-toxic solid carriers can be used, including, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For injectable solutions, the pharmaceutical composition may further include cryoprotectants, lyophilization protectants, surfactants, fillers, antioxidants, stabilizers, and pharmaceutically acceptable carriers. For aerosol administration, the pharmaceutical composition is typically provided in a well-separated form with the surfactant and propellant. The surfactant must, of course, be non-toxic and is generally soluble in the propellant. Representative of such agents are esters or metaesters of fatty acids containing 6 to 22 carbon atoms (e.g., hexanoic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid) with aliphatic polyols or their cyclic anhydrides. Mixed esters, such as mixed glycerides or natural glycerides, can be used. If desired, a carrier, such as lecithin for intranasal delivery, may also be included. For suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides.

[0130] In a preferred embodiment, the compound is prepared for oral, sublingual, oral, intranasal, intravenous, intramuscular, intraperitoneal, and / or inhalation-mediated administration.

[0131] The compounds of the present invention are contemplated to be delivered to organisms using any available methods and routes suitable for compound delivery, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes. Those skilled in the art will recognize that the form and characteristics of the specific dosing regimen employed in the methods of the present invention will be determined by the route of administration and other well-known variables, such as the size and age of the eukaryote. The determination of such dosing regimens is within the capabilities of those skilled in the art. The administration of the compounds can be combined with any conventional therapy intended to treat age-related diseases or conditions, including topical, oral, or injectable methods.

[0132] Therefore, the compounds of the present invention can be administered using any route known to those skilled in the art. In preferred embodiments, the compounds of the present invention are administered transdermally, sublingually, intravenously, intranasally, intravenously, intra-arterially, intracerebrally, intramuscularly, intraperitoneally, orally, or by inhalation.

[0133] In a preferred embodiment, the compounds of the present invention are administered transdermally, sublingually, intravenously, intraperitoneally, orally, or by inhalation. When the compounds are administered by inhalation, the composition can be nebulized and administered, for example, via an anesthetic mask.

[0134] In a preferred embodiment, the compound of the present invention is administered transdermally, sublingually, intravenously, subcutaneously, or orally, or by inhalation. Preferably, the composition is administered orally or sublingually.

[0135] The compounds of the present invention can be administered once or multiple times. Those skilled in the art will be able to determine the most effective dosage regimen for the patient. For example, the most effective dosage regimen could be one in which the patient administers the compound twice daily, once daily, once every three days, once weekly, once monthly, once every three months, once every six months, or once a year.

[0136] The following non-limiting embodiments provide a further description of the invention.

[0137] Example

[0138] Sul-2 was identified as a lifespan regulator.

[0139] Uncovering new elements of the genetic control that governs aging is key to improving our understanding of this complex biological process and increasing healthy lifespan in humans. To this end, we isolated a heat-resistant mutant of *C. elegans* and identified the pv17 allele of the sul-2 gene, which encodes one of the three members of the *C. elegans* sulfatase family.

[0140] Worms carrying the segregating allele of sul-2 (pv17) or the null allele (gk187) lived longer than wild-type worms, although the gk187 allele showed a bimodal curve, and its subpopulations had earlier mortality. Figure 1a-1b and Figure 5 a). Overall, the pv17 allele showed a similar or slower decline in pumping frequency and activity during aging as the wild type, suggesting a healthier lifespan. Figure 5 d-5e). The deletion of the other two sulfatase genes, sul-1 and sul-3, did not increase lifespan, indicating that sulfatase sul-2 plays a major role in lifespan regulation. Figure 5 f). Mutations in sul-2 also enhance insulin / insulin-like growth factor (IGF) receptor daf-2 (f). Figure 5 The developmental phenotype of the g-5i mutant was used for gene mapping and identification. The introduction of a single amino acid substitution (G46D) in the pv17 allele resulted in a reduced functional phenotype. The planned sequence differs slightly from the published sequence. Figure 6 ).

[0141] sul-2 encodes sulfatases of steroid hormones.

[0142] Sulfatases are a large family of proteins involved in various biological processes and with a wide range of substrates. The position of the sul-2 cluster in the sulfatase phylogenetic tree is uncertain, but compared to mammalian sulfatases, the sul-2 cluster is closer to H, F, E, and D type aryl sulfatases and C type steroid sulfatases. Figure 1 c) They may have originated from a common ancestral gene. sul-1 and sul-3 belong to different sulfatase families ( Figure 1 c). We hypothesize that sul-2 can exert its activity by modifying sulfated steroid hormones. Steroid hormone sulfatases are conserved proteins involved in processes such as stimulating the proliferation of hormone-dependent cancers. Specific inhibitors of this enzyme have been developed, such as STX6410, which has been used to treat patients with hormone-dependent cancers. We treated wild-type animals with STX64 and observed an increase in lifespan ( Figure 1 d and Figure 7 a-7c). STX64 treatment also phenotypically mimicked other sul-2 mutant phenotypes ( Figure 7 d). STX64 does not further increase the lifespan of the sul-2 deletion mutant, indicating that STX64 increases lifespan by inhibiting the sulfatase activity of SUL-2. Figure 1 d).

[0143] We measured the levels of sulfated steroid hormones in the sul-2 mutant using high-resolution HPLC-TOF-MS and found that the proportion of sulfated hormones was higher in this strain compared to wild-type worms. Figure 1e). All these data suggest that SUL-2 can act as a steroid hormone sulfatase and regulate lifespan by altering the sulfation state of one or more steroid hormones. The increased lifespan due to SUL-2 depends on genes mediating gonadal lifespan; to investigate whether SUL-2 plays a role in known lifespan pathways, we conducted genetic interaction studies on known alleles affecting lifespan. Mutations in the IGF receptor daf-2 increase lifespan through the transcription factor DAF-16 / FOXO12. SUL-2 mutations further prolong daf-2 function and reduce the lifespan of mutants (…). Figure 2 a, Figure 8 (a-8b) This indicates that sul-2 plays a role in regulating longevity through different pathways. However, the increased longevity of sul-2 mutants is primarily suppressed by loss of DAF-16 function. Figure 2 b and Figure 8 (c-8d). The lack of germline-conferred longevity also necessitates DAF-1612,13, which primarily translocates to the nucleus of intestinal cells. However, in insulin signaling mutants, DAF-16 is localized to the nucleus of most cells. In the sul-2 mutant, DAF-16 is primarily localized to the intestinal cell nucleus ( Figure 9 (a-9b) This indicates that sul-2 plays a role in germline-mediated longevity. Other important factors in germline-mediated longevity, such as the intestinal ankylosing sul-2 repeat protein KRI-1 / KRIT-1 and the transcription elongation factor TCER-1 / TCERG1, are also essential for the full increase in longevity in sul-2 mutants, although a slight effect was observed on the nuclear hormone receptor NHR-80 and no effect was observed on NHR-49, both of which are essential for germline-mediated longevity. Figure 2 c-2f and Figure 8 e-8g). Furthermore, the deletion of sul-2 has little effect on the longevity of germline mutants glp-1 or mes-1.18 Figure 2 g-2h); however, we did observe a cumulative effect of reduced functional allele pv17 in the glp-1 background ( Figure 8 h), which may indicate some cumulative effects of mutant proteins.

[0144] All these data suggest that sul-2 mediates signaling from the gonads to regulate lifespan. Interestingly, sul-2 mutations do not affect fertility, reproductive age, or gonadal morphology. Figure 2 i-2j and Figure 9(c-9e). In summary, our results indicate that sul-2 influences signals that regulate lifespan, thereby modulating lifespan to a reproductive state without affecting gonadal function. Similar to germline ablation, we also observed a further increase in lifespan in sul-2 mutants under dietary restriction, suggesting that sul-2 is not implicitly involved in this lifespan-influencing intervention. Figure 2 In aregenerate animals, activation of the nuclear receptor DAF-12 by a bile acid-like steroid called daf-12 (DA) induces an increase in lifespan. We observed that daf-12 is also essential for the increased lifespan in the sul-2 mutant. Figure 2 l and Figure 8 i) This indicates that sul-2 inactivation leads to changes in the sulfated steroid hormone pool, thereby generating a signal upstream of DAF-12 that mimics the longevity signal in gonadless animals. DAF-36 converts cholesterol to 7-dehydrocholesterol in the first step of the Δ7-DA biosynthesis pathway.21,22 Therefore, DAF-36 is also essential for increased lifespan in aregenerative animals.23 Similarly, DAF-36 is essential for the longevity conferred by the steroid sulfatase inhibitor STX64. Figure 2 m), placing the signal generated by sulfated steroid hormones upstream or parallel to DA biosynthesis.

[0145] sul-2 is expressed in sensory neurons

[0146] We investigated the anatomical location of sul-2 expression in extrachromosomal arrays and in single-copy insertion transgenic lines. We found that sul-2 is expressed only in a few sensory neurons, primarily in the chemoreceptors ADF and ASE, and the tail sensors PHA and PHB. No detectable expression was found in the germline of any transgenic line. Figure 2 n and Figure 10 , 11 (a-11b). ASE neurons are responsible for the attraction of Na+ and Cl-, etc. Odor sensing defects can affect lifespan. Therefore, we tested the sul-2 mutant's responsiveness to Cl- or Na+ and found no difference compared to wild-type animals (a-11b). Figure 11 c-11d). Furthermore, the sul-2 mutation increases the lifespan of daf-10(m79), a long-lived mutant with a sensory ciliary formation defect.25 Figure 11e). These results indicate that the longevity phenotype observed in the sul-2 mutant is not due to impaired sensory neuron function. Reducing sul-2 improves age-related diseases in *C. elegans*. Aging is considered a major risk factor for the onset of neurodegenerative diseases such as Parkinson's disease, Huntington's disease, or Alzheimer's disease. These diseases are caused by a progressive decline in protein homeostasis, leading to protein aggregation that impairs cellular function and ultimately cell death. Germline-deficient *C. elegans* delays symptoms caused by the protein toxicity of ectopically expressed human β-amyloid (βA). We tested whether sul-2 mutation or STX64 treatment improved symptoms in a *C. elegans* neurodegenerative disease model. In a *C. elegans* Parkinson's disease model induced by age-dependent paralysis due to human α-synuclein expression in muscle cells, sul-2 mutation or STX64 treatment significantly improved activity (…). Figure 3 a-3b、 Figure 12 a-12c).

[0147] Loss of SUL-2 function reduces the number of α-synuclein aggregates. Figure 12 (d-12e), indicating that protein aggregates in worms with reduced steroid sulfatase activity were better manipulated. To further determine the neuroprotective effect of reduced sul-2 activity, we tested strains expressing α-synuclein in dopaminergic neurons. In this model, GFP-labeled dopaminergic neurons died due to α-synuclein toxicity. Consistently, compared to control worms, the sul-2 mutant showed increased neuronal survival, suggesting the neuroprotective effect of reduced steroid sulfatase activity (d-12e). Figure 3 c-3d). In a Huntington's neurodegenerative model expressing a polyglutamine repeat sequence fused with YFP (an construct that aggregates in adult worms), we found that both the sul-2 mutation and STX64 treatment reduced the number of aggregates. Figure 3 e-3f). We also tested two different Alzheimer's disease (AD) strains in worms caused by immobility resulting from the expression of βA protein in muscle cells. Consistently, the sul-2 mutation and STX64 treatment delayed paralysis ( Figure 3 g-3h and Figure 12 All these data indicate that inhibiting sul-2 protects nematodes from aging-related protein toxicity.

[0148] Reduced sul-2 activity improves Alzheimer's disease in mammalian models

[0149] Since STX64 alleviates neurodegeneration in a Caenorhabditis elegans model, we tested the effect of this drug on cognitive changes induced by βA infusion oligomers in the hippocampus (an acute AD mammalian model). Figure 3 i). Previously, it was reported that topical application of the steroid hormone sulfatase inhibitor DU-14 could alleviate memory loss induced by βA oligomers in the hippocampus in mammalian models. We observed that both topical and systemic STX64 treatment restored cognitive deficits induced by βA oligomers in the hippocampus (measured by a passive avoidance test). Figure 3 To evaluate the effects of oral STX64 treatment on amyloid pathology in a chronic AD mouse model, we assessed the effects of 3–4 weeks of oral STX64 treatment on amyloid deposition in the neocortex (cerebral cortex and hippocampus) of APP-PS1 mice older than 15 months. Figure 4 k). At this age, corresponding to the late stage of amyloid deposition in the neocortex of the APP218 PS1 model, analysis of βA plaque density and size showed a significant reduction in mice treated with STX64, except for plaque size in the hippocampus. Furthermore, the βA immunoreactive area was reduced in both tissues (k). Figure 4 Interestingly, when we compared the normal time course of βA deposition in STX64-treated aged (>15 months) APP-PS1 mice with that in untreated APP-PS1 mice, we observed that STX64 reduced βA deposition in APP-PS1 mice older than 15 months compared to that observed in 10–12 months of age. Figure 4 All these results indicate that STX64 treatment in APP-PS1 mice reduced βA deposition. We wanted to know if this histological improvement was associated with a reduction in cognitive and behavioral deficits. To this end, we compared the cognitive abilities of APP-PS1 mice older than 15 months after treatment with either solvent or STX64 for 3–4 weeks. Although solvent-treated APP-PS1 mice showed deficits in the passive avoidance test ( Figure 4 However, the cognitive deficits in mice treated with STX64 were fully recovered, reaching levels similar to those in wild-type mice <15 months of age. All these results indicate that STX64-induced changes in βA metabolism reduce cognitive and behavioral deficits caused by βA accumulation in both acute and chronic AD mouse models, suggesting that STX64 has the potential to be used as a therapeutic agent for neurodegenerative diseases.

[0150] Sulfated C19 androgen hormones summarize the beneficial effects of reducing sul-2 activity.

[0151] In mammals, sulfated hormones have long been considered inactive forms, primarily functioning as reservoirs and activated by steroid sulfatases.6 However, direct effects of sulfated hormones on the reproductive and nervous systems have been observed. In the latter tissues, these hormones are named neurosteroids, and their primary function is to regulate neurotransmission. To clarify whether the beneficial effects of sul-2 inhibition are due to a decrease in non-sulfated hormones or an increase in sulfated hormones, we tested commercially available sulfated steroid hormones that were highly present in mutants (Table 1). We observed that the C19 androgens dehydroepiandrosterone sulfate (DHEAS), testosterone sulfate (TS), and epitestosterone sulfate (ES) improved activity in a *Caenorhabditis elegans* Parkinson's disease model, with ES showing better results. Figure 4 a). Similar results were obtained in the Alzheimer's model using TS and ES, but not when using DHEAS. Figure 4 b and Figure 13 a). Neither non-sulfated DHEA nor non-sulfated testosterone, nor pregnenolone sulfate (belonging to the C21 group of steroid hormones) showed any effect. Figure 4 These results indicate that at least some sulfated C19 androgens are involved in the protective effect against protein aggregation diseases, and strongly suggest that the beneficial effect of sul-2 inhibition is due to increased levels of these hormones. Consistent with these results, treatment with the anti-androgen compound abiraterone (Abi)36 did not affect wild-type animals but inhibited the beneficial effect of sul-2 mutations. Figure 4 e).

[0152] We then tested whether these hormones were also involved in other phenotypes observed in the sul-2 mutant. As observed in animals treated with sul-2 or STX 64, treatment with any of these sulfated hormones resulted in an increase in L1 stagnation against a daf-2 (e1370) background. Figure 13 b). Interestingly, treatment with ES increased lifespan in the wild-type background, but did not further increase lifespan in the sul-2 mutant background, while TS or DHEAS did not increase lifespan ( Figure 4 F-4G and Figure 13 (c-13d) indicates that the two interventions share the same molecular mechanism. Therefore, the addition of ES summarizes all the phenotypes described by sul-2 inhibition and strongly suggests that the causal role of sul-2 mutations is due to the increase in C19 androgen sulfated hormone associated with ES.

[0153] Lifespan determination methods:

[0154] Finally, as Figure 15As shown, strain synchronization was achieved by treating pregnant adults with hypochlorite for two generations at 16°C. F2 L4 animals were transferred to 25°C (t=0). ES treatment began at the L4 stage, and both control and treated plates were freshly prepared. Animals were transferred every three days until death. Censorship was performed on animals lost or killed for non-physiological reasons. Survival curves were generated using the Kaplan and Meier product restriction method. Differences in survival were assessed using the Mantel–Cox log-rank test, with p-values ​​below 0.05 considered significant. All life-time measurements used a fer-15(b26) mutant background to avoid progeny generation. The fer-15(b26) mutation had no effect on lifespan.

[0155] Treatment with STX-64, ES, or both often improves motor skills, habituation, and immediate memory in older animals.

[0156] Figure 20 Results of the "open field" test and the "Y-maze" test are presented. The "open field" test requires mice to explore an unknown space for 15 minutes. This test allows for the investigation of activity and habituation (cognitive parameters measuring an individual's ability to recognize their environment). The "Y-maze" test, on the other hand, requires mice to explore a maze for 6 minutes. This test assesses the distance traveled (a measure of activity) and the ability to access all three arms without repeating any (a measure of immediate memory). The results showed that treatment with STX-64, ES, or both tended to improve motor skills in 16-month-old mice. Conversely, habituation and immediate memory in older animals were improved by treatment with STX, ES, and combinations thereof (these compounds improve age-related memory impairment).

[0157] Short-term and long-term memory tests (STM and LTM) were improved using STX-64 and / or ES.

[0158] Figure 21The assessment of learning and memory abilities in aged mice was demonstrated in object recognition and passive avoidance tests. First, in object recognition, the ability to recognize different objects exposed alongside familiar objects was tested after training with the same objects. When this occurred, the object discrimination index was positive and above 0.1. In passive avoidance, the aim was to tell the mouse that its preferred location was not suitable for it, which, for them, was associated with an aversive stimulus. To do this, during training, an entry latency to the preferred location was established and an aversive stimulus was applied. During short-term and long-term memory tests (STM and LTM), the entry latency increased with training if the mouse associated the preferred location with the aversive stimulus. Aged mice treated with solvents exhibited impairments in both learning and memory abilities. In both tests, mice treated with STX, ES, and a combination of both showed significant learning abilities (STM) and the ability to store information (memory) (these compounds improved age-related memory impairment). sequence list <110> Pablo Deolavida University <120> Sulfated C19 steroid hormones prevent age-related protein toxicity <130> 906 727 <140> <141> <160> 13 <170> BiSSAP 1.3.6 <210> 1 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Figure 6 Sequence SUL-2 <400> 1 Pro Asn Ile Val Ile Leu Met Ile Asp Asp Leu Gly Tyr Gly Asp Ile 1 5 10 15 Ala Ser Tyr Gly His Pro Thr Gln Glu Tyr Thr Gln Val Asp Arg Met 20 25 30 Ala Ala Glu Gly Thr Arg Phe Thr Gln Ala Tyr Ser Ala Asp Ser Met 35 40 45 Cys Ser Pro Ser Arg Ala Gly Phe Ile Thr Gly Arg 50 55 60 <210> 2 <211> 60 <212> PRT <213> Synthetic sequence <220> <223> Figure 6 Sequence ARSC <400> 2 Pro Asn Ile Ile Leu Val Met Ala Asp Asp Leu Gly Ile Gly Asp Pro 1 5 10 15 Gly Cys Tyr Gly Asn Lys Thr Ile Arg Thr Pro Asn Ile Asp Arg Leu 20 25 30 Ala Ser Gly Gly Val Lys Leu Thr Gln His Leu Ala Ala Ser Pro Leu 35 40 45 Cys Thr Pro Ser Arg Ala Ala Phe Met Thr Gly Arg 50 55 60 <210> 3 <211> 60 <212> PRT <213> Synthetic sequence <220> <223> Figure 6 Sequence ARSD <400> 3 Pro Asn Ile Leu Leu Ile Met Ala Asp Asp Leu Gly Thr Gly Asp Leu 1 5 10 15 Gly Cys Tyr Gly Asn Asn Thr Leu Arg Thr Pro Asn Ile Asp Gln Leu 20 25 30 Ala Glu Glu Gly Val Arg Leu Thr Gln His Leu Ala Ala Ala Pro Leu 35 40 45 Cys Thr Pro Ser Arg Ala Ala Phe Leu Thr Gly Arg 50 55 60 <210> 4 <211> 59 <212> PRT <213> artificial sequence <220> <223> Figure 6 sequenced <400> 4 Pro Asn Ile Leu Leu Leu Met Ala Asp Asp Leu Gly Ile Gly Asp Ile 1 5 10 15 Gly Cys Tyr Gly Asn Asn Thr Met Arg Thr Pro Asn Ile Arg Leu Ala 20 25 30 Glu Asp Gly Val Lys Leu Thr Gln His Ile Ser Ala Ala Ser Leu Cys 35 40 45 Thr Pro Ser Arg Ala Ala Phe Leu Thr Gly Arg 50 55 <210> 5 <211> 60 <212> PRT <213> artificial sequence <220> <223> Figure 6 ARSH sequence <400> 5 Pro Asn Ile Val Leu Leu Met Ala Asp Asp Leu Gly Val Gly Asp Leu 1 5 10 15 Cys Cys Tyr Gly Asn Asn Ser Val Ser Thr Pro Asn Ile Asp Arg Leu 20 25 30 Ala Ser Glu Gly Val Arg Leu Thr Gln His Leu Ala Ala Ala Ser Met 35 40 45 Cys Thr Pro Ser Arg Ala Ala Phe Leu Thr Gly Arg 50 55 60 <210> 6 <211> 60 <212> PRT <213> Synthetic sequence <220> <223> Figure 6 Sequence ARSF <400> 6 Pro Asn Ile Val Leu Ile Met Val Asp Asp Leu Gly Ile Gly Asp Leu 1 5 10 15 Gly Cys Tyr Gly Asn Asp Thr Met Arg Thr Pro His Ile Asp Arg Leu 20 25 30 Ala Arg Glu Gly Val Arg Leu Thr Gln His Ile Ser Ala Ala Ser Leu 35 40 45 Cys Ser Pro Ser Arg Ser Ala Phe Leu Thr Gly Arg 50 55 60 <210> 7 <211> 60 <212> PRT <213> Synthetic sequence <220> <223> Figure 6 Sequence ARSG <400> 7 Pro Asn Phe Val Ile Ile Leu Ala Asp Asp Met Gly Trp Gly Asp Leu 1 5 10 15 Gly Ala Asn Trp Ala Glu Thr Lys Asp Thr Ala Asn Leu Asp Lys Met 20 25 30 Ala Ser Glu Gly Met Arg Phe Val Asp Phe His Ala Ala Ala Ser Thr 35 40 45 Cys Ser Pro Ser Arg Ala Ser Leu Leu Thr Gly Arg 50 55 60 <210> 8 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> Figure 6 Sequence ARSA <400> 8 Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly Asp Leu 1 5 10 15 Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp Gln Leu 20 25 30 Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val Ser Leu 35 40 45 Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg 50 55 60 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Figure 6 Sequence SUL-2 nematode database <400> 9 Ser Lys Arg Phe Arg Gly Ser Ser Lys Glu Ala 1 5 10 <210> 10 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Figure 6 Sequence modified by SUL-2 <400> 10 Ser Lys Arg Phe Arg Gly Ser Ser Ile Tyr Gly Asp Ser Ile Asn Glu 1 5 10 15 Met Ser Trp Ala Val Gly Glu Val Leu Asp Ser Leu Val Asn Ala Gly 20 25 30 Ile Ala Glu Asn Thr Leu Val Ile Leu Met Ser Asp His Gly Pro His 35 40 45 Val Glu Leu Cys Leu Asn Gly Gly Ser Thr Ala Gly Leu Lys Gly Gly 50 55 60 Lys Ser Asn Ser Tyr Glu Gly Gly Phe Arg Ile Pro Phe Ile Ala Trp 65 70 75 80 Gln Pro Gly Thr Val Lys Pro Ser Arg Val Ser His Glu Val Ile Ser 85 90 95 Ser Met Asp Leu Phe Pro Thr Phe Arg Gly Met Gln Glu Gln Cys Leu 100 105 110 Phe Glu Lys Glu Ala 115 <210> 11 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Figure 6 Designed by sequence SUL-2 <400> 11 Ser Lys Arg Phe Arg Gly Ser Ser Val Arg Gly Ile Tyr Gly Asp Ser 1 5 10 15 Ile Asn Glu Met Ser Trp Ala Val Gly Glu Val Leu Asp Ser Leu Val 20 25 30 Asn Ala Gly Ile Ala Glu Asn Thr Leu Val Ile Leu Met Ser Asp His 35 40 45 Gly Pro His Val Glu Leu Cys Leu Asn Gly Gly Ser Thr Ala Gly Leu 50 55 60 Lys Gly Gly Lys Ser Asn Ser Tyr Glu Gly Gly Phe Arg Ile Pro Phe 65 70 75 80 Ile Ala Trp Gln Pro Gly Thr Val Lys Pro Ser Arg Val Ser His Glu 85 90 95 Val Ile Ser Ser Met Asp Leu Phe Pro Thr Phe Arg Gly Met Gln Glu 100 105 110 Gln Cys Leu Phe Glu Lys Glu Ala 115 120 <210> 12 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Figure 6 Sequence CRE‑SUL‑2 <400> 12 Ser Lys Arg Phe Arg Gly Ser Ser Val Arg Gly Ile Tyr Gly Asp Ser 1 5 10 15 Ile Asn Glu Met Ser Trp Ala Val Gly Glu Val Leu Asp Ser Leu Val 20 25 30 Asn Ala Gly Ile Ala Glu Asn Thr Leu Val Ile Leu Met Ser Asp His 35 40 45 Gly Pro His Val Glu Leu Cys Leu Asn Gly Gly Ser Thr Ala Gly Leu 50 55 60 Lys Gly Gly Lys Ser Asn Ser Tyr Glu Gly Gly Phe Arg Ile Pro Phe 65 70 75 80 Ile Ala Trp Gln Pro Gly Thr Val Lys Pro Ser Arg Val Ser His Glu 85 90 95 Val Val Ser Ser Met Asp Leu Phe Pro Thr Phe Arg Ala Met Asn Glu 100 105 110 Glu Cys Leu Phe Glu Lys Glu Ala 115 120 <210> 13 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Figure 6 Sequence CBR-SUL-2 <400> 13 Ser Lys Arg Phe Arg Gly Ser Ser Val Arg Gly Ile Tyr Gly Asp Ser 1 5 10 15 Ile Asn Glu Met Ser Trp Ala Val Gly Glu Val Leu Asp Ser Leu Val 20 25 30 Asn Ala Gly Ile Ala Glu Asn Thr Leu Val Ile Leu Met Ser Asp His 35 40 45 Gly Pro His Val Glu Leu Cys Leu Asn Gly Gly Ser Thr Ala Gly Leu 50 55 60 Lys Gly Gly Lys Ser Asn Ser Tyr Glu Gly Gly Phe Arg Ile Pro Phe 65 70 75 80 Ile Ala Trp Gln Pro Gly Thr Val Lys Pro Ser Arg Val Ser His Glu 85 90 95 Val Ile Ser Ser Met Asp Leu Phe Pro Thr Phe Arg Ala Met Asn Glu 100 105 110 Glu Cys Leu Phe Glu Lys Glu Ala 115 120

Claims

1. A composition comprising epistestosterone sulfate (ES) or any salt thereof, and a sulfatase inhibitor STX64 of formula (II): or any salt thereof.

2. The composition according to claim 1, wherein the composition is a pharmaceutical composition.

3. Use of the composition of claim 1 or 2 in the manufacture of a medicament for the treatment of an age-related cognitive impairment in a subject in need thereof, wherein the age-related cognitive impairment is associated with mild cognitive impairment, age-related memory impairment, or age-related cognitive decline.

4. Use of the composition of claim 1 or 2 in the manufacture of a medicament for the treatment of mild cognitive impairment, age-related memory impairment, or age-related cognitive decline in a subject in need thereof.

5. The use according to claim 4, wherein the composition is used in the manufacture of a medicament for the treatment of age-related memory impairment in a subject in need thereof.

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