DESCRIPTION OF THE DRAWINGS
The invention will be better understood with reference to the drawings in which:
FIG. 1. Spatial reference memory test in six month old mice following 28 days of treatment, beginning at five months of age (n=10 mice per treatment arm) was performed. The performance of epi-cyclohexanehexol treated TgCRND8 mice was not different from untreated TgCRND8 littermates (p=0.27; FIG. 1A) and remained impaired with respect to non-Tg littermates (F1,14=11.7, p=0.004; FIG. 1C). In contrast, scyllo-cyclohexanehexol treated TgCRND8 mice were significantly better than untreated TgCRND8 littermates (p=0.01; FIG. 1B) and were indistinguishable from non-Tg littermates (F1,13=2.9, p=0.11; FIG. 1D). The probe trial, using annulus crossing index, demonstrated that scyllo-cyclohexanehexol treated mice were not statistically different from non-Tg littermates (p=0.64; FIG. 1E). Vertical bars represent s.e.m. After one month of scyllo-cyclohexanehexol treatment, mice had a lower plaque burden compared to control animals with a high plaque burden in the hippocampus (FIG. 1F, FIG. 1G). Plaque burden was identified using anti-Aβ antibody (brown) and astrocytes are labeled using anti-GFAP antibody (red). Scale bar 300 μm.
FIG. 2. Dot blot analyses of soluble oligomeric Aβ in scyllo-cyclohexanehexol and epi-cyclohexanehexol treated and untreated TgCRND8 mice (FIG. 2A). Soluble proteins isolated from 4 representative four and six month old untreated and treated TgCRND8 mice from the prophylactic study, and from the five month old treatment groups, untreated and treated were applied to nitrocellulose and probed with oligomer-specific antibody followed by re-probing with 6E10. Synthetic Aβ42, monomeric (bottom row: lane 1 and 2) and fibrillar (lane 3 and 4) were used as negative controls for the oligomer-specific antibody, which only recognizes soluble aggregates. 6E10 recognises all Aβ species (bottom lane, right four lanes). Long-term potentiation is blocked by soluble Aβ oligomers (FIG. 2B; green squares) and rescued by scyllo-cyclohexanehexol treatment (FIG. 2B; blue circles). LTP is unaffected by scyllo-cyclohexanehexol treated 7PA2 culture medium which contains Aβ oligomers (FIG. 2C; red squares; same data as in FIG. 2B) and plain CHO medium which lacks oligomers (FIG. 2C; blue circles).
FIG. 3. Cyclohexanehexols improve behaviour in TgCRND8 mice. Spatial reference memory version of the Morris Water Maze test in TgCRND8 mice (n=8-10 per treatment arm) was used as a measure of cognition. At four months of age, non-treated TgCRND8 mice show cognitive impairment relative to AZD-102 (A) and AZD-103(B) treated mice (F2,26=3.99, p=0.03). AZD-102 treated mice (C) were significantly different from treated and untreated non-Tg mice (F1,18=11.7, p=0.004), whereas AZD-103 treated mice (D) approached that of non-Tg mice (F1,17=2.89, p=0.97). At six months of age, non-treated TgCRND8 show cognitive impairment relative to non-Tg controls (F1,30=31.16, p<0.001) and AZD-102 (E) and AZD-103 (F) treated mice (F2,36=4.1, p<0.02). The performance of both AZD-102 treated TgCRND8 mice (F1,21=2.35, p=0.14; G) and AZD-102 approached that of non-Tg littermates (F1,22=3.26, p=0.44; D). Non-Tg littermate behaviour was not affected by either AZD-102 (G) or AZD-103 (H) treatment (F2,37=0.83, p=0.45).
FIG. 4. Cyclohexanehexols improve pathological characteristics in TgCRND8 mice. Vascular Aβ burden was quantitated on serial sagittal sections in treated and untreated TgCRND8 mice. TgCRND8 mice have a significant vascular Ab burden that is associated with small and medium sized vessels; the load is decreased in AZD-103 treated TgCRND8 mice (A). AZD-103 treatment significantly decreased the total vascular load in comparison to untreated and epi-inositol treated TgCRND8 mice. Tg CRND8 mice demonstrate an astrogliotic response to increased Aβ levels within the CNS and treatment with AZD-102 decreased the percent brain area covered in astrogliosis at both 4- and 6-months of age (B). AZD-103 treatment decreased the astrogliotic response to a greater extent at both ages (B). Similarly, microgliosis has been correlated with plaque burden in the TgCRND8 mice (C). Treatment with both AZD-102 and to a greater extent with AZD-103 decreased the percent brain area covered with microgliosis. Kaplan Meier cumulative survival plot demonstrates the increased survival of TgCRND8 mice after treatment with AZD-103 (light gray circles; p=0.02) in comparison to untreated TgCRND8 mice (gray circles). AZD-102 did not significantly improve survival (black circles) (D). ANOVA *p<0.05, **p<0.001.
FIG. 5. At six months of age, the plaque burden and astrogliosis in TgCRND8 mice untreated, AZD-102 and AZD-103 treated mice were examined. Control animals have a high plaque load and astrogliosis in the hippocampus and cerebral cortex. Higher magnification demonstrates that astrocytic activation is not only associated with plaque load. AZD-102 treatment has a modest effect on amyloid burden with a decrease in astrogliosis. AZD-103 treatment significantly decreased amyloid burden and gliosis. Astrocytes labeled with anti-GFAP antibody (red) and plaque burden identified with anti-AP antibody (brown). Scale Bar 300 μm or 62.5 μm.
FIG. 6. Spatial reference memory test in six month old mice following 28 days of treatment, beginning at five months of age (n=10 mice per treatment arm) was performed. The performance of AZD-102 treated TgCRND8 mice was not different from untreated TgCRND8 littermates (p=0.27;A) and remained impaired with respect to non-Tg littermates (F1,14=11.7, p=0.004; C). In contrast, AZD-103 treated TgCRND8 mice were significantly better than untreated TgCRND8 littermates (p=0.01; B) and were indistinguishable from non-Tg littermates (F1,13=2.9, p=0.11; D).
FIG. 7. A, B, C and D. A cue test was performed at the end of the spatial memory version of the Morris Water Test. A flag was placed on the platform and the path length required to reach the platform is comparable for all treatment groups (p=0.78) indicating that treatment does not affect visual acuity. The open field test for duration of grooming (A), pausing (B) and walking (C) confirms that AZD-103 does not affect activity levels in the TgCRND8 mice or in their non-Tg littermates.
FIG. 8. Dot blot analyses of soluble oligomeric Aβ in AZD-102 and AZD-103 treated and untreated TgCRND8 mice (A,B). Soluble proteins isolated from all four and six month old untreated (solid bars) and treated (hatched bars) TgCRND8 mice from the prophylactic study (n=8-10 per experimental arm), and from the five month old treatment groups, untreated (solid bars) and treated (hatched bars) (n=8-10 per experimental arm) were applied to nitrocellulose and probed with oligomer-specific antibody followed by re-probing with 6E10. Synaptophysin reactivity was increased after AZD-103 treatment in both the prophylactic and treatment paradigms in the CA1 region (C) (n=3, for each experimental arm). In contrast, synaptophysin reactivity was unchanged in the TgTauP301L mice after treatment (n=3 for treated and untreated).
FIG. 9. In vitro γ-secretase assays in HEK293 cells transfected with human APPswe. After swAPP stable HEK293 cells were untreated or treated with AZD-103 or 10 nM compound E, cell membranes were used for APP-FL and —CTF detection and for e-stubs generation in vitro assays at 37° C. for 1 hr. Lanes 1&2.90 mg/ml AZD-103 treatment; lanes 3&4 900 mg/ml AZD-103 treatment; lanes 5&6 compound E treated and lanes 7&8 untreated HEK293 APPswe cells.
FIG. 10. Cyclohexanehexols improve behavior in TgCRND8 mice. Spatial reference memory version of the Morris water maze test in TgCRND8 mice (n=8-10 per treatment arm) was used as a measure of cognition. At 4 months of age, nontreated TgCRND8 mice showed cognitive impairment relative to mice treated with epi-cyclohexanehexol (a) and scyllocyclohexanehexol (b; F2,26=3.99, P=0.03). Epi-cyclohexanehexol-treated mice (a) were significantly different from treated and untreated nontransgenic mice (F1,18=11.7, P=0.004), whereas scyllo-cyclohexanehexol-treated mice (b) approached that of nontransgenic mice (F1,17=2.89, P=0.97). At 6 months of age, nontreated TgCRND8 mice showed cognitive impairment relative to nontransgenic control mice (F1,30=31.16, P<0.001) and mice treated with epi-cyclohexanehexol (c) or scyllocyclohexanehexol (d; F2,36=4.1, P<0.02). The performance of both epi-cyclohexanehexol—treated TgCRND8 mice (F1,21=2.35, P=0.14; c) and scyllo-cyclohexanehexol-treated TgCRND8 mice approached that of nontransgenic littermates (F1,22=3.26, P=0.44; d). Nontransgenic littermate behavior was not affected by treatment with either epi-(c) or scyllo-cyclohexanehexol (d; F2,37=0.83, P=0.45). Vertical bars represent s.e.m.
FIG. 11. Cyclohexanehexols improve pathological characteristics in TgCRND8 mice. TgCRND8 mice have a considerable vascular Aβ burden that is associated with small and medium-sized vessels (a). Scyllocyclohexanehexol treatment significantly decreased the total vascular load in comparison to untreated and epi-inositol—treated TgCRND8 mice (a). TgCRND8 mice showed an astrogliotic response to increased Aβ levels, and treatment with epi-cyclohexanehexol decreased the percent brain area covered in astrogliosis at both 4 and 6 months of age (b). Scyllo-cyclohexanehexol treatment decreased the astrogliotic response to a greater extent than epi-cyclohexanehexol at both ages but had no effect in disease stage transgenic Tau (P301L) 23027 mice (b). Similarly, microgliosis has been correlated with plaque burden in the TgCRND8 mice (c). Treatment with both epi- and to a greater extent with scyllo-cyclohexanehexol decreased the percent brain area covered with microgliosis (c). Kaplan-Meier cumulative survival plot shows the increased survival of TgCRND8 mice after treatment with scyllo-cyclohexanehexol (P=0.02) in comparison to untreated TgCRND8 mice (d). Epi-cyclohexanehexol did not significantly improve survival. Synaptophysin immunoreactivity was used as a measure of synaptic density and was increased after scyllo-cyclohexanehexol treatment in both the prophylactic and treatment paradigms in the CA1 region (e). In contrast, synaptophysin reactivity was unchanged in TgTau (P301L) mice after treatment (e). *P<0.05, **P<0.001 by ANOVA.
FIG. 12. Spatial reference memory test was performed in 6-month-old mice after 28 d of treatment, beginning at 5 months of age (n=10 mice per treatment arm). The performance of epi-cyclohexanehexol-treated TgCRND8 mice was not different from untreated TgCRND8 littermates (P=0.27; a) and remained impaired with respect to nontransgenic littermates (F1,14=11.7, P=0.004). In contrast, scyllo-cyclohexanehexol-treated TgCRND8 mice were significantly better than untreated TgCRND8 littermates (P=0.01; b) and were indistinguishable from nontransgenic littermates (F1,13=2.9, P=0.11). The probe trial, using annulus-crossing index, showed that scyllo-cyclohexanehexol-treated TgCRND8 mice were not statistically different from nontransgenic littermates (P=0.64; c). nTg, nontransgenic mice; Tg, TgCRND8 mice. Vertical bars represent s.e.m.
FIG. 13. Dot-blot analyses of soluble oligomeric Aβ in TgCRND8 left untreated or treated with epi-cyclohexanehexol (a) or scyllo-cyclohexanehexol (b). Soluble proteins isolated from untreated (black bars) and treated (gray bars) TgCRND8 mice from the 4- and 6-month prophylactic study and from the 5-month-old treatment group (n=8-10 per experimental arm) were probed with oligomer-specific antibody followed by re-probing with 6E10 (c). Synthetic Aβ42, monomeric Aβ42 and fibrillar Aβ42 were used as negative controls and aggregated Aβ42 was used as a positive control for the oligomer-specific antibody 6E10 recognizes all Aβ species and was used as an Ab positive control. Westernblot analyses of soluble fractions from 4-month-old TgCRND8 mice untreated (n=4) or treated (n=4) with scyllo-cyclohexanehexol showed a decrease in high-molecular-weight Aβ species and a subsequent increase in smaller oligomers (d). The highmolecular-weight Aβ species does not comigrate with holo-APP, as reprobing the same blot with 22C11 recognizing an N-terminal epitope in the APP ectoderm identifies a faster migrating doublet (d). The gel was reprobed with GAPDH-specific antibody as a loading control. Quantification of changes in high-molecular-weight Ab oligomer, trimer and monomer bands using densitometry confirmed the western blot analyses (e).
FIG. 14. Dose-dependent effects of scyllo-cyclohexanehexol on 4-month-old TgCRND8 mice. Four-month-old TgCRND8 mice with signs of clinical disease were administered scyllo-cyclohexanehexol orally for 1 month (n=8-9 per group). Outcome measures indicated improvement in cognitive deficits using the Morris water maze test of spatial memory (a), plaque count using 6F3D immunostaining and image analyses (b), and soluble Ab oligomers using oligomerspecific antibody in a dot-blot assay (c, d). To show that the dose-response effect is not specific to younger mice, 5-month-old TgCRND8 mice were gavaged with 0-30 mg/kg/d scyllo-cyclohexanehexol. Brain Aβ42 levels were examined and a dose-dependent decrease in both soluble and insoluble Aβ42 levels was detected (e,f).
FIG. 15. Cyclohexanehexol stereoisomer structures. The positioning of the hydroxyl groups on the ring structure of myo (1), epi-(2) and scyllo-cyclohexanehexol (3) are shown.
FIG. 16. At six months of age, the plaque burden and astrogliosis in TgCRND8 mice untreated, epi- and scyllo-cyclohexanehexol treated mice were examined. Control animals have a high plaque load and astrogliosis in the hippocampus (a) and cerebral cortex (b). Higher magnification demonstrates that astrocytic activation is not only associated with plaque load (c). Epi-cyclohexanehexol treatment has a modest effect on amyloid burden with a decrease in astrogliosis (d, e, f). Scyllo-cyclohexanehexol treatment significantly decreased amyloid burden and gliosis (g.h.i). Higher magnification illustrates the smaller mean plaque size in scyllo-cyclohexanehexol treated mice (i). Astrocytes labeled with anti-GFAP antibody (red) and plaque burden identified with anti-Aβ antibody (brown). Scale Bar 300 μm (a, b, d, e, g, h) and 62.5 μm (c, f, j).
FIG. 17. Spatial reference memory version of the Morris Water Maze test in six month old TgCRND8 mice non-treated or treated with mannitol. Mannitol treated TgCRND8 mice (dashed line) were not significantly different from untreated TgCRND8 mice (solid line: P=0.89; a). The performance of mannitol treated TgCRND8 mice (dashed line) was significantly different from mannitol treated non-Tg littermates (solid line: P=0.05; b). Vertical Bars represent SEM. Plaque burden was analysed at six months of age by quantitative image analyses (c). Mannitol treated TgCRND8 mice were indistinguishable from untreated TgCRND8 mice when plaque count was used as a measure of total plaque burden (P=0.87). Vertical bars represent SEM. Kaplan-Meier Cumulative survival plots for TgCRND8 mice treated and untreated with mannitol (d). The two cohorts of animals, n=35 per group, were not significantly different as determined by the Tarone-Ware statistical test, P=0.87.
FIG. 18. A cue test was performed at the end of the spatial memory version of the Morris Water Test. A flag was placed on the platform and the path length required to reach the platform is comparable for all treatment groups (P=0.78) indicating that treatment does not affect visual acuity (a). The open field test for duration of grooming (b), pausing (c) and walking (d) confirms that scyllo-cyclohexanehexol does not affect activity levels in the TgCRND8 mice or in their non-Tg littermates.
Patent Application
20070197452
