TW201620517A - Dosing regimens for treating and/or preventing cerebral amyloidoses - Google Patents

Abstract

Described herein are dosing regimens and kits for the treatment and/or prevention of cerebral amyloidoses such as Alzheimer's disease (AD) and/or cerebral amyloid angiopathy (CAA).

Description

For the treatment and/or prevention of the administration of Cerebral Amyloidoses Program

The invention is generally directed to the treatment of human brain amyloidosis (e.g., Alzheimer's disease) and/or cerebral amyloid angiopathy.

Alzheimer's disease is one of the biggest social and economic health burdens. Alzheimer's disease is characterized by the presence of progressive dementia and histopathology of neurofibrillary tangles (NFTs) and neuritis (senile) plaques. The plaque is composed of a protein called β-amyloid (Aβ), which is composed of a protein called tau.

Amyloid plaques and NFTs are two hallmarks of Alzheimer's disease (AD). Mutations in amyloid precursor protein (APP) and premature aging lead to early-onset Alzheimer's disease, and support the hypothesis that APP treatment may also play an important role in the pathogenesis of sporadic AD. In addition, the "amyloid hypothesis" predicts that the accumulation of some toxic forms of Aβ is detrimental to the brain. APP can be treated by the alpha- and beta secretase pathways. To date, most studies have focused on the development of AD therapy, in which the progression of the disease is concentrated in inhibiting the metabolism of γ-secretase and β-secretase and APP to form Aβ peptide or activating α-secretase to increase the production of neuroprotective sAPPα peptide. While reducing Aβ The production. The development of specific beta secretase inhibitors is difficult, in part because there appears to be a non-linear relationship between a decrease in beta secretase activity in vivo and a decrease in A[beta] peptide in the brain. A further difficulty is the low brain penetration of most inhibitors, and gamma secretase inhibitors are further plagued by severe GI side effects associated with scoring inhibition, since gamma secretase processes many other receptors in addition to APP. Includes a score receptor. In addition, defects in gamma secretase activity have been shown to cause neurodegeneration, and may be autosomal dominant with mutations in presenilin 1 (a component of the gamma secretase complex, which contains the active site of the gamma secretase complex). Related to Alzheimer's disease.

Most efforts to treat Alzheimer's disease are focused on reducing the symptoms of AD. In particular, it was confirmed that a lower concentration of choline acetyltransferase in the affected neurons of the forebrain of AD patients was produced by inhibiting the hydrolysis of acetylcholine in the synaptic cleft and prolonging the acetylcholine at the synapse. Level of treatment. Although this strategy has led to at least partial correction of neurotransmitter levels, the therapeutic benefit has been small.

In addition, AD is classified as tauopathy. Tau protein lesions are caused by abnormal hyperphosphorylation of tau protein to promote aggregation of tau protein and formation of NFTs. Since both tau and APP mutations cause dementia, one or both of them may contribute to the progression of AD disease. It is clearly understood that mutations that result in APP mutation processing can cause AD. Currently, there are no licensed therapies for slowing the progression of Alzheimer's disease. therefore, There is still a need for more effective AD treatment. While most of the evolving therapies focus on altering the metabolism of APP (such as beta-secretase and gamma secretase inhibition) or blocking tau aggregation, the present invention provides treatment using pharmacological chaperones, the pharmacology The chaperone protein binds to one or more gangliosidases and/or sialidases, thereby increasing the production of sAPPα and reducing the production of Aβ and hyperphosphorylated tau.

Similarly, cerebral amyloid angiopathy (CAA) is a disease characterized by amyloid deposition in the vascular wall of the central nervous system, particularly in the pia mater and cortical arteries. CAA mostly occurs in the elderly with sporadic symptoms, and the incidence of CAA is related to the increase in age. These sporadic cases of CAA are due to A[beta] deposition of proteolytic cleavage from APP. The genetic form of CAA is generally familial, more severe and earlier than incidental CAA. CAA has also recently been recognized as a potential contributor to the development of AD.

All citations herein are incorporated by reference in their entirety.

It has been discovered that pharmacological escort proteins targeted to beta-hexosaminidase ([beta]-hex) can have many benefits associated with the treatment and/or prevention of cerebral amyloidosis. Certain words, it has been found that pharmacological chaperone proteins 2- acetylglucosamine 1,2-deoxynojirimycin (AdDNJ) in the Netherlands ^APP E693Q colonized gene transfected mouse body can be reduced GAβ lesions, and in a dose dependent manner Correct the behavioral phenotype of those mice. AdDNJ has good pharmaceutical qualities including good oral bioavailability, brain penetration, tolerance, selectivity and low toxicity. Therefore, it is expected that AdDNJ and other strategies for increasing beta-hex activity will help manage human brain amyloidosis, such as Alzheimer's disease (AD) and/or cerebral amyloid angiopathy (CAA).

Thus, one aspect of the present invention relates to the development of Alzheimer's disease and/or brain starch by administering an effective amount of 2-acetamido-1,2-dideoxynojirimycin (AdDNJ). A method of preventing and/or treating Alzheimer's disease and/or cerebral amyloid angiopathy in a patient with a risk of angiopathy or a patient diagnosed with Alzheimer's disease and/or cerebral amyloid angiopathy. In one or more embodiments of this aspect, the method comprises administering to the patient an effective amount of AdDNJ during the first enzyme enhancement; not administering AdDNJ during the substrate conversion; and then during the second enzyme enhancement The patient is administered an effective amount of AdDNJ.

The first enzyme enhancement period and the second enzyme enhancement period may have the same duration, or the first enzyme enhancement period and the second enzyme enhancement period may have different durations. In various embodiments, one or more of the first enzyme enhancement period and the second enzyme enhancement period are for a period of from about 1 day to about 8 days, such as from about 4 days to about 6 days. AdDNJ can be administered daily during one or more of the first enzyme enhancement period and the second enzyme enhancement period.

In one or more embodiments, AdDNJ is administered orally. Other routes of administration include, but are not limited to, intranasal administration.

According to one or more embodiments, the dosage of AdDNJ is in the range of from about 3 mg/kg/day to about 300 mg/kg/day, such as about 100 mg/kg/day.

In one or more embodiments, administration of AdDNJ during the mass transfer does not involve administration of AdDNJ during a period of from about 48 hours to about 96 hours, such as for about 72 hours. Other exemplary maturity transition periods include no administration of AdDNJ for about 24 hours or no administration of AdDNJ for about 48 hours.

Various combinations during enzyme enhancement and during mass transfer are possible. In an exemplary schedule, the first enzyme enhancement period is about 5 days, and administration of AdDNJ during the matrix transition does not involve administration of AdDNJ for a period of about 72 hours.

Another exemplary schedule includes administering an effective amount of AdDNJ to the patient on Day 1; not administering AdDNJ on Day 2; administering an effective amount of AdDNJ to the patient on Day 3; AdDNJ was administered; the patient was dosed with an effective amount of AdDNJ on day 5; then AdDNJ was not administered over a period of about 72 hours.

In yet another exemplary schedule, 1 is included, the first enzyme enhancement period is about 3 days, and no administration of AdDNJ during the matrix transition includes no administration of AdDNJ for a period of about 120 hours.

The method can include additional substrate conversion periods and/or additional enzyme enhancement periods. In one or more embodiments, the method comprises alternating between enzymatic enhancement and substrate conversion Perform a certain total treatment time, such as a treatment time of at least 1 month. The total treatment time may be any appropriate period of time for treatment, such as at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or 1 year, 2 years, 3 years or 4 years. In various embodiments, AdDNJ is administered for an undefined period of time. Each enzyme enhancement period or each substrate conversion period may be the same or different from another enzyme enhancement period or a substrate conversion period, respectively.

Another aspect of the invention encompasses a kit for treating and/or preventing Alzheimer's disease and/or CAA. In one or more embodiments, the kit comprises one or more dosage forms comprising an effective amount of AdDNJ and instructions for administering the dosage form in accordance with any of the methods described herein. For example, the kit can include an indication to administer the dosage form during the first enzyme enhancement; the dosage form is not administered during the substrate conversion; and then the dosage form is administered during the second enzyme enhancement.

The kit may comprise one or one or more inactive dosage forms that do not comprise an effective amount of AdDNJ. The kit can include instructions for administering the active dosage form during the first enzyme enhancement; administering the inactive dosage form during the substrate conversion; and then administering the active dosage form during the second enzyme enhancement.

Any of the features described herein can be possessed during enzyme enhancement and during substrate reduction.

Figures 1a through 1g illustrate a summary of the efficacy, selectivity, and cytotoxicity summary for the beta-hex pharmacological protein AdDNJ. AdDNJ is a specific inhibitor of β-hex (a), and at 100 μM or lysosomal enzyme β-glucocerebrosidase (GCase), α-galactosidase (α-Gal), or β- Galactosidase (β-Gal) does not inhibit O-GlcNAcase (cd) at 10 μM, the highest concentration tested (e). In addition, AdDNJ had little or no effect on human skin fibroblasts (f) and cell viability of SH-SY5Y (g) treated with AdDNJ at concentrations up to 1 mM for up to 120 hours. AdDNJ increased beta-Hex levels by 3 fold in fibroblasts. Treatment of healthy human-derived fibroblasts with AdDNJ (95-780 nM) for 5 days produced a dose-dependent increase of up to 3-fold in total wild-type β-hex levels (b). Data are expressed as mean ± standard deviation.

Figures 2a through 2b illustrate single dose AdDNJ pharmacokinetic studies in C57BL/J6 mice. A single 100 mg/kg dose of β-hex-targeted pharmacological protein AdDNJ was administered to male C57BL/J6 mice at 5 weeks of age. Plasma (a) and brain (b) AdDNJ curves. 100 mg/kg of AdDNJ crosses the blood-brain barrier and reaches levels in the brain that are expected to be sufficient for binding and increases the level of β-hex (>342 nM), and falls below β-H for less than Ki within 16 hours (at pH 5) The level was 253 nM) (n=5 mice per time point). Data are expressed as mean ± standard deviation.

Figures 3a through 3b illustrate AdDNJ repeated dose and dose-response studies in C57BL/J6 mice. Five-week-old male C57BL/6 mice were orally administered with a vehicle, 30, 100 or 300 mg/kg of β-hex-targeted pharmacological protein AdDNJ daily for up to 14 days. Whole brain Hex A&S (a) and Hex B (b) activity (n=5 mice per time point). Data are expressed as mean ± standard deviation.

Figures 4a to 4c show increased levels of brain β-hex after treatment with AdDNJ in Dutch APP ^E693Q gene-transferred mice. The 3-month-old male Dutch APP ^E693Q gene-transferred mice were either left untreated or orally administered with vehicle or AdDNJ for three months. Treatment with AdDNJ increased β-hex B(a), β-hex A&S(b) and total β-hex levels in a dose-dependent manner (untreated; n=3, vehicle; n=6, 3 and 10 mg/kg) AdDNJ; n = 7, 30 and 100 mg/kg of AdDNJ; n = 6). Data are expressed as mean ± standard deviation. One-way ANOVA had Bonferroni causality analysis, *p<0.05, **P<0.01, ***P<0.001.

Figures 5a to 5b illustrate the reduction in anxiety and prevention of learning behavioral disorder after treatment with AdDNJ in Dutch APP ^E693Q gene-transferred mice. Male large Dutch APP ^E693Q gene transgenic mice were not treated (n=15), or given vehicle (n=15) or AdDNJ (3, 10, 30 or 100 mg/kg AdDNJ; n=13/). The treatment group) was carried out for three months. In the elevated plus maze, Netherlands ^APP E693Q large transgenic mice treated with vehicle for 6 months showed a more than three-month-old mice without increasing anxiety treatment, which is in use at all doses tested following treatment AdDNJ In contrast (a). Large untreated mice in March showed complete memory, exploring new (new) objects more than familiar (old) objects, and 6-month-old vector-treated Dutch gene-transforming mice in novelty In the object recognition (NOR), learning behavior disorder was observed. In the NOR test, AdDNJ prevented the onset of learning disabilities in a dose-dependent manner ( b ). Data are expressed as mean ± standard deviation. *P<0.05, ***P<0.001.

Figure 6 is a graphical representation of environmentally relevant and clue-fear conditional learning behavior following treatment of Dutch APP ^E693Q gene-transferred mice with AdDNJ. 3 month old male Dutch APP ^E693Q gene transgenic mice were not treated (n=15), or given vehicle (n=15) or AdDNJ (3, 10, 30 or 100 mg/kg AdDNJ; treated) n=13/group) for three months. AdDNJ has no impact on learning behaviors related to the environment or clues. Data are expressed as mean ± standard deviation.

Figures 7a through 7o illustrate the levels of Aβ and oligomers after 3 months of treatment of Dutch APP ^E693Q gene-transferred mice with AdDNJ. The 3-month-old male Dutch APP ^E693Q gene-transferred mice were not treated or given orally with vehicle or AdDNJ (3, 10, 30 or 100 mg/kg AdDNJ; n=13/group after treatment) for 3 months. . AdDNJ has no effect on oligomers of Aβ40(ad), Aβ42 (eh), Aβ42/40 ratio (il) or A11 pre-fibril Aβ(m) or using antibodies (OC or NU-4, no) (n=5) /group). Data are expressed as mean ± standard deviation.

Figures 8a through 8f illustrate a reduction in A[beta] immune response following treatment of Dutch APP ^E693Q gene-transferred mice with AdDNJ. Sagittal brain sections of male Dutch APP ^E693Q gene-transferred mice from oral administration vehicle (a, c, e) or 100 mg/kg AdDNJ (b, d, f) for three months were stained with antibody 6E10 (vector; n = 4, 100 mg / kg AdDNJ; n = 4). The Aβ immunoreactivity was less intense in the lower leg (a, b), but the visual cortex (c, d) or the hippocampal CA1 region (e, f) of the Dutch APP ^E693Q gene-transferred mice treated with ^AdDNJ did not. Scale bars a-f 200 μM; and Figures 9a to 9p show a reduction in Aβ (GAβ) immunoreactivity in combination with gangliosides after treatment of Dutch APP ^E693Q gene-transferred mice with AdDNJ. Sagittal slices from the brain of male Dutch APP ^E693Q gene-transferred mice administered with vehicle (a, c, e, g, i) or AdDNJ (b, d, f, h, j) for three months with GAβ (43696C) Staining (vehicle; n = 4, 100 mg/kg AdDNJ; n = 4). In the lower and olfactory cortex (k, i), the GAβ immunoreactivity detected by the 43696C antibody was reduced. GAβ in the visual cortex, lateral olfactory cortex and hippocampal CA1 region was unchanged (m, n, o). Scale bar aj 200μM. Staining with Gaβ in blood vessels in the cortical region is detectable (p). Data are expressed as mean ± standard deviation. *P<0.05.

Figures 10a through 10d illustrate that AdDNJ-treated Dutch APP ^E693Q gene-transgenic mice have no detectable effect on TLC-assessed ganglioside distribution. Three-month-old male Dutch gene-transgenic mice were not treated or given vehicle or AdDNJ orally for three months. Thin-layer chromatography analysis of the entire forebrain showed that AdDNJ treatment did not significantly alter GM1, GM2, and GM3 ganglioside levels (ad) (vehicle; n=6, 100 mg/kg AdDNJ; n=7).

Aβ is a hydrophobic 38- to 43-amino acid peptide that is found in all biological fluids and is derived from the enzymatic cleavage of the larger type I membrane protein-amyloid precursor protein (APP). A linkage study of familial AD patients identified several mutations in two genes (APP and preseninia) associated with abnormal metabolism of APP and increased aggregate form of Aβ production. It is believed that toxic oligomers formed by Aβ may play an important role in the pathogenesis of Alzheimer's disease (Shankar et al., 2008).

Gangliosides promote a neurotoxic form of A[beta] produced in the brain (i.e., oligomers). Gangliosides are sialic acid glycosphingolipids found in the outer leaflets of cell membranes and are particularly abundant on the cell surface of neurons. Conventional gangliosides exist as clusters and form microdomains on the surface of the plasma membrane. Specificity of gangliosides They enable them to interact with a variety of biological effectors, including glycoproteins, peptide hormones, and growth factors. In addition, gangliosides (such as ganglioside GM1) can promote cell differentiation, prevent loss of neurogenesis, and play a neuroprotective role in in vitro and in vivo models of neuronal damage.

Gangliosides are the most abundant in the nervous system and participate in a variety of functions, including signal transduction mediation, cell adhesion, and cell differentiation. Although more than 200 gangliosides have been identified, most of the gangliosides in neurons are catabolized by one or more gangliosidases and/or sialidases.

The difference between α-secretase activity, soluble APPα (sAPPα) and β-secretase activity, and the product of soluble APPβ (βAPPβ) is that the first 16 residues of Aβ are included in sAPPa. Since APP cleavage by α-secretase cleaves the Aβ domain, no reaction product can produce amyloid. Therefore, it is hypothesized that activation or up-regulation of α-secretase activity prevents or reduces the formation of toxic Aβ oligomers and amyloid plaques, while increasing the shedding of neurotrophic and neuroprotective sAPPa. Interestingly, inhibition of the synthesis of glycosphingolipids and gangliosides has been shown to activate the shedding of sAPPα (Sawamura et al., 2004).

Mutations in APP also result in familial Alzheimer's disease and/or cerebral amyloid angiopathy (CAA). Gangliosides GM2, GM3, and GD3 regulate regional Aβ deposition because these gangliosides are expressed in a region-specific manner in the brain. (Yamamoto et al., 2006). The genetically altered combination of Dutch and Italian Aββ and Flanders-type Aβ are accelerated by ganglioside GM3 and ganglioside GD3, respectively. It is worth noting that cerebral vascular smooth muscle cells that make up the cerebral vascular wall (where the Dutch and Italian Aββ deposits) express only GM2 and GM3 (Yamamoto et al., 2006). Thus, the combination of hereditary A[beta] variants can be accelerated by local environmental factors, such as the presence of specific gangliosides in the brain.

Recently, glycosphingolipids and gangliosides have been implicated in misfolding and aggregation of neurodegenerative-related proteins (for example, the linkage of Parkinson's disease to glucocerebrosidase mutations) (Alcalay et al., 2012; Daniele et al., 2012). A growing body of evidence suggests that abnormalities in ganglioside metabolism may contribute to the pathogenesis of Alzheimer's disease (AD) and cerebral amyloid angiopathy by accelerating the production of neurotoxic forms of Aβ in the brain and blood vessels. (CAA). During aging and neurodegeneration, the physicochemical properties of the membrane change, resulting in an imbalance in the proportion of lipids in the membrane and/or a change in the ratio of membrane lipids, which may contribute to the pathogenesis of AD (Yanagisawa, 2007; Mutoh et al. , 2006; Hayashi et al., 2004; Karcun et al., 1991; Brooksbank et al., 1989; Matsuzaki, 2007). Consistent with this idea, elevated levels of monosialogangliosides (GM1, GM2, and GM3) have been reported in the cortex of AD brain (Gylys et al., 2007), where The sialic gangliosides are apparently localized to the membrane microdomains (resistance membranes) in the frontal, temporal and parietal cortex of the affected brain (Kracun et al., 1991; Molander-Melin et al., 2005). . At the same time, Aβ(GAβ) peptides that have been detected in the brain to bind gangliosides exhibit only very early stages of AD lesions (Yanagisawa et al., 1995; Yanagisawa et al., 1997; Yanagisawa et al., 1998). It is suggested that gangliosides may play some role in the early pathogenesis of AD, such as seeding of Aβ fibrils (Yanagisawa et al., 2007; Yamamoto et al., 2005; Selkoe, 1995). It is worth noting that some mutant forms of Aβ (especially those that favor oligomerization) exhibit specific susceptibility to the pro-aggregation properties of GM2 and GM3 (Yamamoto et al., 2005; Yamamoto et al., 2006). ).

Β-hexosaminidase (β-Hex) catabolizes GM2 gangliosides, and the lack of β-hexosaminidase leads to autosomal recessive lysosomal storage disorders, Tay-Sachs disease and mulberry Sandhoff (Mahuran, 1999). It has been found that anti-Aβ immunoreactivity accumulated in neurons uses antibody 4G8, anti-α-synuclein immunoreactivity, and anti-pTau immunoreactivity in the brain of HEXB KO mice (Keilani et al., 2012). Biochemical and immunohistochemical analysis confirmed that 4G8 immunoreactivity in neurons represents APP-CTFs and/or Aβ but not full-length APP. In addition, in Increased levels of Aβ40 and Aβ42 peptides were found in lipid-related fractions (compared to those recovered from wild-type brain). Aβ immunoreactive accumulation of gangliosides with GM1 and GM2 gangliosidase was also observed in the brains of brains and human subjects of HEXB KO mice. Taken together, these results depict a correlation between ganglioside accumulation and A[beta] accumulation.

GM2 and GM3 gangliosides have been shown to promote the assembly of Dutch, Iowa and Italian-type mutant A[beta] peptides that are particularly susceptible to oligomerization in vitro (Yamamoto et al., 2005; Yamamoto et al., 2006). It has been shown that the generation and characterization of the Dutch APPE693Q gene-transferred mouse is the accumulation of Aβ oligomers in the brain (especially in the hippocampus, the visual cortex and the neurons in the hippocampal CA1 region) and develops anxiety and learning behavioral deficits. But they have never developed amyloid plaques (Gandi et al., 2010). It has been speculated that the use of a "pharmacologically-associated protein" against β-hex to increase the activity of β-hex to reduce the level of GM2 will reduce the oligomerization of Aβ and prevent behavioral changes in the Dutch APPE693Q gene-transferred mouse. Pharmacological-associated proteins indicate that new therapeutic strategies are small molecules that act as selective binding and stable target proteins in behavior to promote proper folding and reduce premature degradation (Tropak et al., 2007; Maegawa et al., 2007; Tropak et al., 2004; Rountree et al., 2009). These pharmacological-associated proteins are small molecules that reversibly bind proteins, thereby stabilizing protein targets and increasing half-life. Pharmacological protein therapy for cystic fibrosis and several lysosomal storage diseases, including Fabry, Gaucher, Pompe, Sandhoff and Tay-Sax have been or are being evaluated in clinical trials (Boyd et al., 2013). Year; Wustman et al., 2012; Osher et al., 2011; Clarke et al., 2011).

One aspect of the invention provides a dosing regimen for the treatment and/or prevention of brain amyloidosis, such as Alzheimer's disease and/or cerebral amyloid angiopathy, using a pharmacological protein for beta-Hex. In one or more embodiments of this aspect, the pharmacological escort protein is 2-ethylamino-1,2-dideoxynojirimycin (AdDNJ).

AdDNJ, also known as 2-acetamido-1,2,5-trideoxy-1,5-imino-D-sorbitol, is a compound having the following structure:

AdDNJ can be administered in free form as indicated above, or can be administered in the form of a pharmaceutically acceptable salt, solvate or prodrug.

In one or more embodiments, AdDNJ is administered in a unique dosing regimen that balances the companion and inhibition of AdDNJ. For example, AdDNJ can be administered during enzymatic enhancement, wherein AdDNJ enhances β-hex activity by promoting proper folding, delivery, non-aggregation, and the like. After the enzyme enhancement period, it can be in the transition period AdDNJ was not administered to allow AdDNJ concentration to fall below the inhibitory concentration, allowing AdDNJ to dissociate from β-hex. The dosing regimen can include a second enzyme enhancement period that can be the same or different than the first enzyme enhancement period. The dosing regimen can include a second matrix transition period that can be the same or different than the first matrix transition period. The dosing regimen can also include cycling between enzyme enhancement and substrate conversion.

Exemplary enzyme enhancement periods can range from a few hours to several days. For example, AdDNJ can be administered once a day or multiple times a day (2 times, 3 times, 4 times, etc.) during a period of 1 day to 10 days. In various embodiments, the enzyme enhancement period is about 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 60, 72, 96, 120 hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

Exemplary yield transition periods can also range from hours to days. The substrate conversion period is defined as the time between successive enzyme enhancement periods. In various embodiments, the enzyme enhancement period is about 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 60, 72, 96, 120 hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. For example, if the duration of the substrate conversion is about 24 hours and the enzyme enhancement period consists of a single AdDNJ administration, the time between consecutive AdDNJ administrations is about 24 hours (ie, once a day). Similarly, if the substrate conversion period is about 48 hours and the enzyme enhancement period is composed of a single AdDNJ administration, the time between consecutive AdDNJ administrations is about 48 hours (ie every other one). day). Further examples include a 72-hour maturing transition period (eg, on Monday and Thursday but not on Tuesday and Wednesday). The terms "about 24 hours", "about 48 hours", and "about 72 hours" do not require that the drug be administered at the same time on each dosing day, but merely that the drug is administered on a different day.

Examples of suitable dosing regimens include, but are not limited to, the following administrations: a. five consecutive days per week (eg, Monday to Friday), one, two, or three times per day; b. seven days per week (eg, Monday to Sunday), 1 time, 2 times or 3 times a day; c. Three consecutive days per week (eg Monday to Wednesday), once, twice or three times a day; d. Three consecutive days per week (eg week 1. Wednesday, Friday, 1 time, 2 times or 3 times a day; and e. Every other day, once, 2 times or 3 times a day.

Each separate administration may include a therapeutically effective amount of a salt, solvate or prodrug of AdDNJ or AdDNJ. Each administration may include a salt, solvate or prodrug of AdDNJ or AdDNJ in an amount ranging from 1 mg/kg to about 1,000 mg/kg. Exemplary amounts include 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg. /kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, 65mg/kg, 70mg/kg, 75mg/kg, 80mg/kg, 85mg/kg, 90mg/kg, 95mg/kg, 100mg/ Kg, 125mg/kg, 150mg/kg, 175mg/kg, 200mg/kg, 225mg/kg, 250mg/kg, 275mg/kg, 300mg/kg, 325mg/kg, 350mg/kg, 375mg/kg, 400mg/kg, 425mg/kg, 450mg/kg, 475mg/kg, 500mg/kg, 550mg/kg, 600mg/kg, 650mg/kg, 700mg/kg, 750mg/kg, 800mg/kg, 850mg/kg, 900mg/kg, 950mg/ Kg and 1,000 mg/kg. Exemplary amounts also include 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,100 mg, 1,200 mg, 1,300 mg, 1,400 mg, 1,500 mg, 1,600 mg, 1,700 mg, 1,800 mg, 1,900 mg, 2,000 mg, 2,500 mg, 3,000 mg, 3,500 mg, 4,000 mg, 4,500 mg, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g and 20g. The amount of administration may be the same or the amount of administration may vary during different administrations.

Another aspect of the invention provides for the treatment and/or prevention of brain amyloidosis using a pharmacological protein for beta-Hex For example, a group of Alzheimer's and/or CAA. In one or more embodiments of this aspect, the pharmacological concomitant protein is 2-acetamido-1,2-dideoxynojirimycin (AdDNJ). The kit includes one or more dosage forms comprising an effective amount of a pharmacological adherent protein. The kit can include instructions for administering a pharmacological adherent protein in accordance with any of the dosing regimens described herein. The kit may also include an inactive dosage form that does not include a pharmacological-associated protein. Other therapeutic agents, such as those suitable for combination therapy, may be included in the active and/or inactive dosage form.

The pharmacological concomitant protein can be formulated for any route of administration, including, for example, in the form of a tablet or capsule or liquid, or as a sterile aqueous solution. When the pharmacological concomitant protein is formulated for oral administration, the tablet or capsule can be prepared in a conventional manner using a pharmaceutically acceptable excipient such as a binder (eg, pregelatinized corn starch, poly a vinylpyrrolidone or hydroxypropyl methylcellulose); a filler (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); a lubricant (such as magnesium stearate, talc or vermiculite); a disintegrating agent (for example) Potato starch or sodium starch glycolate; or a wetting agent (such as sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or can be presented as a dry product for use in combination with water or another suitable carrier before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (for example sorbitol sugars) Pulp, cellulose derivative or hydrogenated edible fat); emulsifier (such as lecithin or gum arabic); non-aqueous carrier (such as almond oil, oil ester, ethanol or fractionated vegetable oil); or preservative (such as methyl-pair) Hydroxybenzoate or propyl-p-hydroxybenzoate or sorbic acid). The liquid preparation may also suitably contain a buffer salt, a flavoring agent, a coloring agent or a sweetener. Formulations for oral administration can be suitably formulated to provide controlled or sustained release of the compound.

In one or more embodiments of the invention, the compound is administered in a dosage form that allows for systemic distribution or absorption such that the compound can cross the blood-brain barrier in order to function on the neuronal cells. Such dosage forms that allow for systemic distribution or absorption can be oral or parenteral. In some embodiments, the compound can be distributed throughout the body, including across the blood-brain barrier. For example, pharmaceutical preparations suitable for parenteral/injectable use usually comprise a sterile aqueous solution (where water soluble) or a dispersion and sterile powder for the preparation of a sterile injectable solution or dispersion. In all cases, the dosage form must be sterile and must flow to the extent that easy syringability exists. The dosage form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, polyethylene glycol, and the like), a suitable mixture of the above media, or a vegetable oil. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Pre The antimicrobial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sterol, sorbic acid, and the like. In many cases, it will be reasonable to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use of a delayed absorbent such as aluminum monostearate or gelatin in the composition.

Sterile injectable solutions are prepared by incorporating the compound in the required amount in a suitable solvent, if necessary, with various other ingredients enumerated above, followed by filtration or terminal sterilizing. In general, dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle which contains a base dispersion medium and other optional ingredients enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are the vacuum drying and lyophilization techniques of the active ingredient powder plus any additional ingredients from the prior sterile filtration solution of the active ingredient powder.

The formulation may contain excipients. Pharmaceutically acceptable excipients that may be included in the formulation are buffers such as citrate buffers, phosphate buffers, acetate buffers, and bicarbonate buffers, amino acids, urea, alcohols, ascorbic acid, Phospholipids; proteins such as serum albumin, collagen and gelatin; salts such as EDTA or EGTA, and sodium chloride; liposomes; polyvinylpyrrolidone; saccharides such as dextran, mannitol, sorbitol and glycerol; And polyethylene glycol (eg PEG-4000, PEG-6000); glycerol; glycine or other amino acids; and lipids. For the preparation of the slow The flushing system includes citrate; acetic acid; bicarbonate; and phosphate buffer. Phosphate buffer is a commonly used excipient.

The formulation may also contain a nonionic detergent. Examples of nonionic detergents include polysorbate 20, polysorbate 80, Triton X-100, Triton X-114, Nonidet P-40, octyl alpha glucoside, octyl beta glucoside, Brij 35, Pluronic, and Tween 20.

The therapeutic agent can be administered orally or parenterally, including intravenous, subcutaneous, intraarterial, intraperitoneal, intraocular, intramuscular, buccal, transrectal, transvaginal, intraorbital, intracerebral, intradermal, intracranial, spinal Internal, intraventricular, intrathecal, intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, or via inhalation. In a preferred embodiment, the therapeutic agent is administered orally.

The administration of the therapeutic agent can be by intravenous injection of the bolus of the preparation, or by intravenous or intraperitoneal administration from an external reservoir (e.g., an IV bag) or an internal reservoir (e.g., a biodegradable implant). See, for example, U.S. Patent Nos. 4,407,957 and 5,798,113 each incorporated herein by reference. Intrapulmonary delivery methods and devices are described, for example, in U.S. Patent Nos. 5,654,007, 5,780,014, and 5,814,607 each incorporated herein by reference. Other useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle delivery injection, needle-free Injection, nebulizer, aerosol, electroporation, and transdermal patches. The needle-free injector device is described in U.S. Patent Nos. 5,879,327, 5, 520, 639, 5, 846, 233, and 5, 704, 911, the disclosure of each of each of each Any of the above formulations can be administered using these methods.

Subcutaneous injection has the advantage of allowing self-administration, while also producing an extended plasma half-life compared to intravenous administration. In addition, various devices designed for patient convenience, such as fillable pens and needle-free injection devices, can be used with the formulations of the invention discussed herein.

Suitable pharmaceutical preparations are in unit dosage form. In such dosage forms, the preparation is subdivided into appropriately sized unit doses containing appropriate quantities (e.g., effective amount to achieve the desired purpose) of the active ingredient. In certain embodiments, the therapeutic agent is administered in one or more daily doses (eg, once a day, twice a day, three times a day). In certain embodiments, the therapeutic agent is administered intermittently.

According to one or more embodiments of the invention, the individual being treated may have any brain amyloidosis or be at risk of developing any brain amyloidosis. Such brain amyloidosis includes, but is not limited to, Alzheimer's disease and cerebral amyloid angiopathy (CAA). For example, cerebral amyloidosis can be any form of Alzheimer's disease, including early onset familial Alzheimer's disease. Cerebral amyloidosis can also be any form of CAA, including hereditary CAA and with Alzheimer's disease CAA. Individuals may have risk factors for brain amyloidosis or are at risk of developing cerebral amyloidosis. Such risk factors include, but are not limited to, the ApoE4 allele of lipoprotein E associated with increased risk of developing Alzheimer's disease and CAA. The individual may have been diagnosed with cerebral amyloidosis. In addition, the individual may not have been diagnosed with cerebral amyloidosis, but exhibits the characteristics of one of the diseases. In a further embodiment, Alzheimer's disease is caused by Down's syndrome or associated with Down's syndrome.

In addition, the treatment may include a combination of one or more pharmacological-associated proteins, but may also be combined with other conventional Alzheimer's disease treatments or other brain amyloidosis. Treatment may also include a combination of one, two, three or more pharmacological escort proteins or one, two, three or more escort protein molecules and one or more glucosylceramide synthetase inhibitors Combination of agents. Combination therapy comprising one or more chaserin molecules with one or more glucosylceramide synthase inhibitors can increase gangliosidase activity and reduce glucosylceramide synthesis enzyme activity .

definition

The terms used in the present specification generally have their ordinary meaning in the technical field of the invention and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the implementer in describing the invention and how to make and use the invention.

The term "pharmacological escort protein" as used herein refers to a molecule that specifically binds to one or more gangliosidases and/or sialidases or glucocerebrosidases and has one or more of the following. Effects: (i) enhance the formation of stable molecular conformation of the protein; (ii) enhance the normal delivery of the protein from the ER to another cellular location (preferably the location of the native cell), ie prevent ER-related degradation of the protein; Preventing the formation of unstable (ie, misfolded) proteins; (iv) restoring or enhancing at least part of the wild-type function, stability, and/or activity of the protein; and/or (v) modifying the phenotype of the cell or Features. Thus, a pharmacological-associated protein of gangliosidase or sialidase is a combination of one or more gangliosidases and/or sialidases to produce correct folding, transport, non-aggregation, and increased activity. A molecule of gangliosidase and/or sialidase. The molecule includes a specific binding molecule, such as an active site-specific accompanying protein molecule, an inhibitor, an ectopic binding agent, and an inactive site binding agent that enhances protein stability. In one or more embodiments, the pharmacological escort protein is specific for β-Hex, such as AdDNJ.

The term "pharmacologically-associated protein" (PC) refers to a small molecule that selectively binds to and stabilizes a target protein to promote proper folding, reduce premature degradation, and increase ER output efficiency. Small molecules are called "associated protein molecules" because small molecules help the protein to reach a predetermined location (such as a lysosomal or cell surface) from where it is synthesized (ER). Molecules are reversible bonds that bind and stabilize protein targets, help restore proper delivery, and then dissociate so that proteins can perform normal functions. Agent. "Pharmacological" modifiers refer to molecular specificity: molecules are designed to interact with only a single predetermined protein target and only stabilize a single predetermined protein target, and PCs typically do not affect multiple proteins or cell processing, such as protein delivery, ER quality Control, proteasome function, or activity of a bio-protection protein molecule (eg, a heat shock protein). This method is widely applicable to diseases in which it is expected to enhance the function of a particular protein (mutant or wild type) to provide a therapeutic benefit.

The retention and premature degradation of incorrectly folded proteins is not limited to mutant proteins. It has been shown that the majority (up to 30%) of all newly synthesized proteins are premature degradation of the proteasome. Subsequent studies have shown that pharmacological-associated proteins can increase cellular levels of many wild-type proteins by promoting protein folding, stability, and ER export.

Molecular-associated proteins stabilize protein conformation. In the human body, proteins are involved in almost every aspect of cellular function. Certain human diseases are caused by mutations that cause changes in the amino acid sequence of the protein, which reduce the stability of the protein and may prevent proper folding of the protein. Most gene mutations that result in less stable or misfolded proteins are called missense mutations. These mutations result in the replacement of a single amino acid in the protein with another amino acid. Because of this error, missense mutations tend to produce proteins with reduced levels of biological activity. In addition to missense mutations, there are other types of mutations that produce proteins with reduced biological activity.

Proteins are typically folded in specific regions of the cell that are known to be endoplasmic reticulum or ER. Cells have a target quality control mechanism that ensures that proteins are folded into the correct three-dimensional shape before moving from the ER to the appropriate destination in the cell (often referred to as the process of protein delivery). After being initially retained in the ER, misfolded and/or unstable proteins are often eliminated by quality control mechanisms. In some cases, misfolded proteins accumulate in the ER before being eliminated.

Misfolded proteins that remain in the ER disrupt the normal delivery of misfolded proteins, and the resulting reduced biological activity leads to impaired cellular function and ultimately to disease. In addition, misfolded proteins accumulated in the ER may produce various types of stress on the cells, which may also cause cell dysfunction and disease.

Endogenous molecular chaperones are present in almost all cell types and in many cell compartments. Some endogenous molecular chaperones are involved in the delivery of proteins and allow cells to survive under stress such as heat shock and glucose starvation. Among the endogenous parenterative protein molecules (molecular accommodating proteins), BiP (immunoglobulin heavy chain binding protein, Grp78) is the best characterization of the ER protein. Like other escort protein molecules, BiP interacts with many secreted and membrane proteins throughout the ER. This interaction is usually weak and transient when the folding of the nascent protein progresses smoothly. Once the native protein conformation is achieved, the molecular chaperone protein no longer interacts with the protein. BiP that binds to proteins that cannot be folded, assembled, or properly glycosylated becomes stable and usually leads The protein is degraded by the ER-related degradation pathway. This process acts as a "quality control" system in the ER, ensuring that only those proteins that are properly folded and assembled are exported to the ER for further maturation, and that improperly folded proteins or unstable proteins are retained for subsequent degradation. Due to the combination of an inefficient thermodynamic protein folding process and an ER quality control system, only a portion of some wild-type proteins become folded into a stable configuration and successfully exit the ER.

Pharmacological-associated proteins derived from specific enzyme inhibitors rescue mutant enzymes and enhance wild-type enzymes. For example, binding of an enzyme associated with lysosomal storage disease (LSDs) to a small molecule inhibitor can increase the stability of both the variant enzyme and the corresponding wild-type enzyme (see U.S. Patent No. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829; and 7, 141, 582, all incorporated herein by reference. In particular, it has been found in vitro that small molecule derivatives of glucose and galactose (specific, selective competitive inhibitors of several target lysosomal enzymes) are effective in increasing the stability of enzymes in cells. Increase the delivery of enzymes to lysosomes. Therefore, by increasing the amount of enzyme in the lysosome, it is expected that the hydrolysis of the enzyme substrate will increase. Since the variant enzyme protein is unstable in the ER, the enzyme protein is hindered and prematurely degraded in the normal transport path (ER→high alkite→endosome→lysosomal). Therefore, certain compounds that bind to the variant enzyme and increase the stability of the variant enzyme can act as "associated protein molecules" of the enzyme and increase the amount that can leave the ER and move to the lysosome.

Since certain enzyme inhibitors are known to specifically bind to the catalytic center of the enzyme ("active site"), resulting in a stable enzyme configuration in vitro, these inhibitors are proposed and somewhat contradictory as can help recover The ER leaves and transports to the lysosome, an effective carrier protein for hydrolytic activity. These specific pharmacological proteins are referred to as "active site-specific accompanying proteins (ASSCs)" or "specific pharmacological partners" because these specific pharmacological proteins are bound in a specific way at the active site of the enzyme. . Pharmacological care protein therapy has potential advantages over enzyme replacement therapy (ERT) because small molecules can be administered orally and can have excellent biodistribution compared to protein-based therapies.

In addition to salvaging the variant enzyme, the pharmacological protein also enhances the activity of ER secretion and wild-type enzymes. Thus, a compound that induces a stable molecular conformation of the enzyme during folding acts as a "associated protein molecule" to stabilize the enzyme in an appropriate configuration to exit from the ER.

The term "ganglioside" or "sialoganglisides" refers to residues of N-mercaptosphingosine and one or more N-acetaminone (sialic acid, NeuAc) Glycosphingo lipids composed of oligosaccharide chains.

The term "asialogangliosides" means no N-acetylated nerves Amino acid (sialic acid, NeuAc) residues and include gangliosides of LacCer, GA2 and GA1 (Ariga et al.).

The term "ganglioside lipase" refers to the sequential removal of a single N-acetylneuraminic acid (sialic acid, NeuAc) and sugar residues from the non-reducing terminal units of gangliosides and asialo gangliosides. Sialidase and exoglycohydrolases. This degradation occurs primarily through the endocytosis-nuclear endosome-lysosomal pathway. Examples of gangliosidases include sialidase 2 (neuraminidase 2; NEU2), sialidase 3 (neuraminidase 3; NEU3), sialidase 4 (neuraminidase 4; NEU4), β-galactosidase (GLB1), β-hexosaminidase A ( HEXA / HEXB ), β-hexosaminidase B ( HEXB ), and β-hexosaminidase S (HEXS).

The term "sialidase" refers to an enzyme that removes a single N-acetaminoneminic acid (sialic acid, NeuAc) residue from the non-reducing terminal units of gangliosides, oligosaccharides and glycoproteins. The term "sialidase" includes sialidase 2 (neuraminidase 2), sialic acid, which removes a single N-acetinylamine residue from the ganglioside by endosomal-lysosomal pathway. Enzyme 3 (neuraminidase 3) and sialidase 4 (neuraminidase 4) and an enzyme such as sialidase 1 which removes a single N-acetaminoneminic acid residue from oligosaccharides and glycoproteins. N-acetamidine neuraminic residues on gangliosides have been shown to increase A[beta] binding affinity and increase the propensity to induce beta sheet conformation. According to recent reports, knocking in APP/PSEN mice (APPswe+PSEN1 AE9) The disialoganglioside synthase (GD3S) prevents Aβ accumulation and subsequent development of memory deficits, which are characteristic of APP/PSEN mice. Bernardo et al., Neurobiology of Aging. In Press, Corrected Proof, "Elimination of GD3 synthase improves memory and reduces amyloid-[beta]plaque in gene transfer mice Load in transgenic mice.)". GD3S is required for the synthesis of b and c series gangliosides via the alpha-2,8-link chain sialic acid to sialic acid. These results indicate that it may be advantageous to reduce the sialic acid content of the gangliosides in the treatment of AD.

The lysosomal enzyme "β-galactosidase" is an exohydrolase that removes β1,3-galactose from the non-reducing ends of asialo gangliosides and sialic gangliosides. Mutations in the gene GLB1 encoding β-galactosidase lead to lysosomal storage disease GM1 gangliosides, which result from the lack of β-galactosidase activity and the accumulation of GA1 and GM1 gangliosides of. Beutler, E., et al, Biol. Chem. 247 (22): 7195-200 (1972). GM1 gangliosides are the major components of microdomains that promote Aβ production and assembly in cell culture, and Aβ has been found to bind GM1 ( Aβ) exhibits an early stage of AD lesions in the brain.

Lysosomal enzymes β-hexosaminidase A and B hydrolyze β- from the non-reducing ends of asialo gangliosides and sialic gangliosides Linked N-acetyl galactosamine (GalNAc). Although GM1 gangliosides received the most attention, other a series of gangliosides (GD1A, GM2, and GM3) in vitro also combined and promoted the assembly of Aβ.

There are two isomerases of beta-hexosaminidase, HEXA and HEXB . HEXA consists of an alpha subunit and a beta subunit (αβ), while HEXB consists of two beta subunits (ββ). HEXA HEXA encoding the α subunit, and HEXA and HEXB HEXB encoding the β subunit. Mutations in HEXA lead to lysosomal storage disease, Tai-Sax, which is caused by a lack of HEXA activity and GM2 accumulation. Mutations in HEXB lead to lysosomal storage disease Sandhoff disease, which is caused by the lack of activity of HEXA and HEXB and accumulation of GM2 and GA2.

The term "specific binding" as used herein refers to the interaction of a pharmacological protein with gangliosidase and/or sialidase or glucocerebrosidase, in particular with direct involvement in pharmacological care. Interaction of gangliosidase and/or sialidase or amino acid residues of glucocerebrosidase. The pharmacological-associated protein specifically binds to a target protein, such as β-hexosaminidase B, to exert an accompanying protein action on the enzyme rather than a universal set of related or unrelated proteins. The β-hexosaminidase amino acid residue that interacts with any given pharmacological adhesion protein may or may not be within the "active site" of the protein. Specific binding can be performed by routine binding assays (eg, inhibition) System, thermal stability) to evaluate, or through structural studies, such as co-crystallization, nuclear magnetic resonance, etc. to evaluate.

The term "enhanced stability" or "increased stability" as used herein refers to a pharmacological parental protein that is specific for gangliosidase and/or sialidase, or glucocerebrosidase, in vitro or in combination with The gangliosidase and/or sialidase (preferably the same cell type or the same cell, for example at an earlier time), or the glucomannan, in contact with the cells, in contact with the pharmacologically-protected protein Glucosidase enhances the resistance of the enzyme to irreversible inactivation. Increasing protein stability increases the half-life of the protein in ER and the amount of functional protein delivered from the ER. In one aspect of the invention, the stability of wild-type gangliosidase or sialidase, or glucocerebrosidase, is enhanced or increased. In another aspect of the invention, the conformational stability of the mutant gangliosidase or sialidase or glucocerebrosidase is enhanced or increased.

The term "enhanced delivery" or "increased delivery" as used herein refers to a cell that is not in contact with a pharmacological protein that is specific for gangliosidase and/or sialidase or glucocerebrosidase. (preferably the same cell type or the same cell, for example at an earlier time) delivery efficiency of gangliosidase and/or sialidase or glucocerebrosidase, increased gangliosidase and / or sialidase or glucocerebrosidase to pharmacological partners specific for one or more gangliosidases and / or sialidase or glucocerebrosidase Transport efficiency of cytosol (sialidase 2) or endosomes and lysosomes of cells exposed to protein.

The term "enhancing activity" or "increasing activity" as used herein refers to pharmacological care with respect to one or more gangliosidase and/or sialidase or glucocerebrosidase. The activity of gangliosidase and/or sialidase or glucocerebrosidase in cells contacted with the protein (preferably the same cell type or the same cell, eg at an earlier time), as described herein Increased gangliosidase and/or sialidase in cells that are exposed to pharmacological-associated proteins specific for one or more gangliosidases and/or sialidase or glucocerebrosidase Or the activity of glucocerebrosidase. The pharmacological concomitant protein of the present invention can also increase the activity of the enzyme by increasing the total amount of enzymes in the cells and/or by increasing the specific activity of the enzyme.

The term "specific activity" refers to the quality of conversion per mg of proteinase per unit time in the preparation of the enzyme.

The term "enhanced level" or "increased level" as used herein refers to a pharmacology relative to one or more gangliosidase and/or sialidase or glucocerebrosidase. Levels of gangliosidase and/or sialidase or glucocerebrosidase in cells contacted with the protein (preferably the same cell type or the same cell, eg at an earlier time) One or more gangliosidases and/or saliva are added to cells that are specific for one or more gangliosidases and/or phytase-associated proteins of sialidase or glucocerebrosidase The level of acidase or glucocerebrosidase.

The term "stable and appropriate configuration" means that the gangliosidase and/or sialidase or glucocerebrosidase pharmacological protein is induced or stabilized and performs a predetermined function of wild-type gangliosidase and/or The ability of the sialidase or glucocerebrosidase to be functionally identical to the mutation or configuration of the wild-type gangliosidase and/or sialidase or glucocerebrosidase.

The term "functionally identical" means that although the configuration may vary slightly (almost all proteins exhibit some structural flexibility in a physiological state), the flexibility of configuration does not result in (1) protein aggregation, (2) Removal by endoplasmic reticulum-related degradation pathways, (3) impaired function of proteins such as APP metabolic activity, and/or (4) inappropriate delivery within cells, such as localization to the cytoplasm, to greater than wild-type proteins or To a lesser extent.

The term "stable molecular conformation" refers to a protein conformation induced by a pharmacological adhesion protein that provides at least partial wild-type function or enhances wild-type function in a cell, ie, gangliosidase and/or sialidase or glucose. The configuration of cerebrosidase. For example, the stable molecular configuration of gangliosidase and/or sialidase or glucocerebrosidase will be where gangliosidase and/or sialidase or glucocerebrosidase leaves ER and transport To the cytoplasmic configuration, rather than the configuration that is misfolded and degraded and/or does not perform its intended function. Furthermore, the stable molecular configuration of the mutated gangliosidase and/or sialidase or glucocerebrosidase may also have all or part of the activity. However, steady The molecular configuration does not necessarily have all of the wild-type protein functional properties.

The term "activity" refers to the normal predetermined physiological function of wild-type gangliosidase and/or sialidase or glucocerebrosidase in a cell. For example, gangliosidase and/or sialidase activity includes catabolism of gangliosides, while glucocerebrosidase activity includes catabolism of glycosylglycosylamine. Such functionality can be tested by any means known to establish functionality.

The term "ganglioside catabolism" refers to the removal of non-reducing end units of asialo gangliosides and sialic gangliosides by sequential formation of sialidase and exosaccharide hydrolase and ceramide. Sesic acid and sugar residues alone. This degradation occurs primarily through the endocytosis-nuclear endosome-lysosomal pathway, with the exception of sialidase 2 located in the cytosol.

In one non-limiting embodiment, any of the nucleic acid molecules encoding wild-type gangliosidase and/or sialidase or glucocerebrosidase may exhibit 50%, 60%, 70%>, A nucleic acid molecule of 80%> and up to 100% homology and any sequence that hybridizes under standard conditions encodes a gangliosidase and/or a sialidase or a glucocerebrosidase polypeptide. In another non-limiting embodiment, any other nucleotide encoding a gangliosidase and/or sialidase or glucocerebrosidase polypeptide (having the same functional properties and binding affinity as the aforementioned polypeptide sequence) Sequence, for example in a normal individual Dual variants have the ability to achieve functional conformation in the ER, achieve proper localization within the cell, and exhibit wild-type activity.

The term "mutation" gangliosidase and/or sialidase or glucocerebrosidase as used herein refers to gangliosidase and/or sialidase or glucose translated from a gene containing a gene mutation. A cerebrosidase polypeptide that mutates to produce altered gangliosidase and/or sialidase or glucocerebrosidase amino acid sequences. In one embodiment, the mutation produces a native conformation that is not achieved under conditions normally present in the ER when compared to wild-type gangliosidase and/or sialidase or glucocerebrosidase, or Ganglioside and/or sialidase or glucocerebrosidase protein exhibiting reduced stability or activity compared to wild-type gangliosidase and/or sialidase or glucocerebrosidase . This type of mutation is referred to herein as a "configuration mutation" and the protein with the mutation is referred to as a "configuration variant." Failure to achieve this configuration results in degradation or aggregation of gangliosidase and/or sialidase or glucocerebrosidase proteins, rather than being transported through the normal pathway to the native location in the cell or in the protein delivery system or Enter the extracellular environment. In some embodiments, mutations may occur in non-coding portions of genes encoding gangliosidase and/or sialidase or glucocerebrosidase, resulting in lower expression efficiency of the protein, eg, affecting transcription efficiency, Mutations such as splicing efficiency, mRNA stability, and the like. Administration of gangliosidase and/or by enhancing wild-type expression levels and conformational mutant variants of gangliosidase and/or sialidase and/or glucocerebrosidase The sialidase or glucocerebrosidase pharmacological protein can improve the defects caused by this inefficient protein expression.

Certain tests may assess the properties of proteins that may or may not be comparable to actual in vivo activities, but are still an appropriate substitute for protein functionality, and the wild-type behavior in this test demonstrates support for the proteins of the invention. Fold evidence of salvage or enhancement techniques. One such activity in accordance with the present invention is the proper delivery of functional gangliosidase and/or sialidase to the cytosol from the endoplasmic reticulum.

The terms "endogenous expression" and "endogenously expressed" refer to a lack of gangliosidase and/or sialidase or glucocerebrosidase deficiency, a dominant negative mutation in the absence or suspected of having Overexpression of the body, or other defects (eg, mutations in gangliosidase and/or sialidase or glucocerebrosidase nucleic acid or polypeptide sequences that alter (eg, inhibit) expression, activity, or stability) Normal physiological expression of gangliosidase and/or sialidase or glucocerebrosidase in cells in a related disease or disordered individual. The term also refers to the expression of gangliosidase and/or sialidase or glucocerebrosidase in cell types that are normally expressed in cells or healthy individuals, and is not included in cells or in healthy individuals that do not express gangliosides. Expression of gangliosidase and/or sialidase or glucocerebrosidase in cell types of lipase and/or sialidase or glucocerebrosidase, such as tumor cells.

The term "elevated gangliosides" as used herein refers to individuals, patients with increased levels of gangliosides in the brain. Or patient population. Ganglioside levels can be elevated in the membrane throughout the cell and also within the microdomain. The term "microdomain" or "lipid raft" refers to a detergent-resistant zone found in cell membranes that is rich in cholesterol, glycosphingolipids, and gangliosides. In one aspect of the invention, the pharmacological conserved protein is used to reduce ganglioside levels in the microdomains or lipid rafts by increasing the activity of enzymes that are known to catabolize gangliosides in the brain.

As used herein, the term "delivery efficiency" refers to the ability of a protein to be exported from the endoplasmic reticulum to a native location within the cell, to the cell membrane, or to the extracellular environment.

A "competitive inhibitor" of an enzyme may refer to a compound that is structurally similar to the chemical structure and molecular geometry of the enzyme substrate and binds to the enzyme at approximately the same position as the substrate. Thus, the inhibitor competes with the host molecule for the same active site, thereby increasing Km. If there are enough substrate molecules to replace the inhibitor, the competitive inhibition is usually reversible, ie the competitive inhibitor can reversibly bind. Thus, the amount of enzyme inhibition depends on the inhibitor concentration, the substrate concentration, and the relative affinity of the inhibitor and substrate for the active site.

Non-classical competitive inhibition occurs when the distal end of the inhibitor binds to the active site of the enzyme, creating a conformational change in the enzyme such that the receptor no longer binds to the enzyme. In non-classical competitive inhibition, binding at the active site prevents the inhibitor from binding at a single site and vice versa. This includes ectopic suppression.

The "linear mixed inhibitor" of the enzyme is a type of competitive inhibitor that allows binding of the substrate but reduces affinity, so Km increases and Vmax decreases.

A "non-competitive inhibitor" refers to a compound that forms a strong bond with an enzyme and that may not be replaced by the addition of an excess of a substrate, ie, a non-competitive inhibitor may be irreversible. Non-competitive inhibitors can bind at the active site, near or distal to the enzyme or protein, and have no effect on Km but reduce Vmax with the enzyme linkage. A non-competitive inhibitor refers to a situation in which an inhibitor binds only to an enzyme-substrate (ES) complex. When the inhibitor binds, the enzyme becomes inactive. This is different from non-classical competitive inhibitors, which can bind to enzymes in the absence of a substrate.

The term "Vmax" refers to the maximum initial rate at which an enzyme catalyzes a reaction, that is, at a saturated substrate level. The term "Km" is the concentration of the material required to achieve 1⁄2 Vmax.

An enzyme "enhancer" is a compound that binds to gangliosidase and/or sialidase and increases the rate of enzyme reaction.

The terms "therapeutically effective dose" and "effective amount" refer to a protein (in the case of an antagonist) sufficient to enhance protein processing (allowing a functional conformation) in ER without inhibiting expression at an appropriate cellular location, or without induction from The ligand of the protein at the appropriate cellular location mediates receptor internalization (in the case of an agonist) and enhances the activity of the target protein, thereby producing a therapeutic response in the subject. The treatment response can be identified by the user (eg, a clinician) as Any response to an effective response to the therapy, including the aforementioned symptoms and alternative clinical indicators. Thus, the therapeutic response will typically be an improvement or inhibition of one or more symptoms of a disease or disorder, such as Alzheimer's disease.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable when administered to humans and that generally do not produce an adverse reaction. Preferably, the term "pharmaceutically acceptable" as used herein means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized animal, and more particularly human. In the pharmacopoeia. The term "carrier" means a diluent, adjuvant, excipient, or any carrier used to administer a compound. For example, such pharmaceutical carriers can be sterile liquids such as water and oil. Water or aqueous salt solutions and aqueous dextrose and glycerol solutions are preferred as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences", 18th edition or other version by E. W. Martin.

The terms "about" and "approximately" shall generally refer to the degree of acceptable error of the quantity measured, given the nature or precision of the quantitative measurement. Typically, the degree of exemplification of the error is within 20 percent (%) of the range of given values or values, preferably within 10%, more preferably within 5%. Alternatively, and particularly in biological systems, the terms "about" and "about" may mean values within the order of magnitude, preferably within 5 times the given value, and more preferably within 2 times the given value. Unless otherwise Note that otherwise the numerical quantities given herein are approximate, which means that the term "about" or "approximately" can be inferred when not explicitly stated.

The term "isolated" as used herein means that the referenced material is removed from the environment in which the material is normally found. Thus, the isolated biological material may be free of cellular components, ie, components of cells in which the material is found or produced. In the case of a nucleic acid molecule, the isolated nucleic acid includes a PCR product, an mRNA band on a gel, cDNA, or a restriction fragment. In another embodiment, the isolated nucleic acid is preferably excised from the chromosome from which the nucleic acid is found, and more preferably no longer joined to a non-regulatory, non-coding region, or to other separates when found in a chromosome. A gene upstream or downstream of a gene contained in a nucleic acid molecule. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acids include sequences inserted into plastids, plastids, artificial chromosomes, and the like. Thus, in a specific embodiment, the recombinant nucleic acid is an isolated nucleic acid. The isolated protein can be associated with the cell membrane when it is associated with other proteins or nucleic acids, or proteins and nucleic acids, with the isolated protein in the cell, or if the isolated protein is a membrane-associated protein. The isolated organelles, cells, or tissues are removed from the anatomical sites found in the organism. The separated material can be, but need not be, purified.

The term "purified" as used herein refers to a material, such as a gangliosidase and/or sialidase nucleic acid or polypeptide that has been isolated under conditions that reduce or eliminate unrelated materials (ie, contaminants). For example, the purified protein is preferably substantially free of phase in the cell. Other proteins or nucleic acids that are associated. The term "substantially free" as used herein is used operatively in the context of analytical testing of materials. Preferably, the purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and even more preferably at least 99% pure. Purity can be assessed by conventional means such as chromatography, gel electrophoresis, immunoassays, compositional assays, bioassays, and other methods well known in the art.

The term "tau protein lesion" refers to any symptom caused by the pathological aggregation of tau protein that forms neurofibrillary tangles (NFT) in the human brain, including but not limited to, such as frontotemporal dementia, Alzheimer's disease, Progressive nuclear nerve palsy, cerebral cortex degeneration and frontotemporal dementia (Pick disease) and other diseases.

The term "brain amyloidosis" refers to a state in which amyloid is deposited or accumulated in the brain or in blood vessels of the brain. Examples of cerebral amyloidosis include, but are not limited to, Alzheimer's disease and cerebral amyloid angiopathy (CAA).

The term "Alzheimer's disease" or "AD" refers to a symptom characterized by slow progressive dementia and severe cerebral cortical atrophy. The presence of β-amyloid neuroplaques, neurofibrillary tangles of internal neurons, and amyloid angiopathy is characteristic of AD and was observed during autopsy. AD may be inherited as a family, or may be sporadic. Herein, AD includes family, sporadic, and above-described intermediate conditions and subgroups based on phenotypic expression. Familial AD usually has early onset (before age 65), while occasional AD is usually late (65 Years old and later). In a non-limiting embodiment, familial AD may be associated with a mutation in one or more genes selected from the group consisting of Presenilin 1 (Human Presenilin 1 , GenBank Accession Nos. NM_000021, NM_007318, And NM_007319; mouse presenilin 1, GenBank accession number NM 008943; and rat presenilin 1, GenBank accession number NM_019163), presenilin 2 (human presenilin 2, GenBank accession number NM_000447 and NM_012486; rat presenilin 2, GenBank login No. NM_011183; and rat presenilin 2, GenBank accession number NM 031087), and amyloid precursor protein (APP) (human APP, GenBank accession number NM_201414, NM_201413, and NM 000484; mouse APP, GenBank accession number NM 007471; Group of rat APP, GenBank accession number NM_019288). Sporadic AD cannot be tested directly, but certain risk factors may increase an individual's susceptibility to developing sporadic AD. In one non-limiting embodiment, there is at least one lipoprotein E (APOE) (human APOE, GenBank Accession No. NM 000041; murine APOE, GenBank Accession No. NM 009696; and rat APOE, GenBank Accession No. NM_138828) Individuals with e4 dual gene replication are at risk of developing late onset AD.

The term also includes individuals with trisomy 21 or Down's syndrome (DS) and develops dementia that is equivalent to the clinical and neuropathological features of AD (in their third or fourth decade). These include cerebral amyloid (Aβ) plaques and neurofibrillary tangles (NFTs), which are characteristic lesions of Alzheimer's disease (AD). Recent studies have shown that Aβ42 is the earliest form of this protein deposited in the brain of Down's syndrome and can be seen in subjects as young as 12 years old, while soluble Aβ can be affected by DS in only 21 weeks of gestational age. The tester's brain detected far earlier than the formation of Aβ plaques. Archives of Pathology and Laboratory Medicine, Gyure et al. 125: 489-492 (2000).

For the purposes of the present invention, "neurological disorder" refers to any central nervous system (CNS) or peripheral nervous system (PNS) disease associated with beta-amyloid treatment of amyloid precursor protein (APP). This may result in neuronal or glial cell defects including, but not limited to, neuronal loss, neuronal degeneration, neuronal demyelination, gliosis (ie, astrocyte hyperplasia), or abnormal proteins or toxins {eg amyloid The accumulation of neurons or extraneural neurons of protein β).

An exemplary neurological disorder is cerebral amyloid angiopathy (CAA), also known as arterial angiopathy. This disorder is a form of vascular disease in which the same amyloid protein associated with Alzheimer's disease, amyloid-β (Aβ) is deposited on the walls of the pia mater and superficial cortical blood vessels of the brain. The deposition of amyloid causes these blood vessels to be prone to failure, thereby increasing the risk of hemorrhagic stroke. because It is the same amyloid associated with Alzheimer's disease dementia, so such cerebral hemorrhage is more common in people with Alzheimer's disease, but it can also occur in people without a history of dementia. Bleeding in the brain is usually limited to specific brain lobe, which is slightly different from cerebral hemorrhage, which occurs because of the more common cause of hypertension-hemorrhagic stroke (or cerebral hemorrhage). CAA is also associated with transient ischemic attack, subarachnoid hemorrhage, Down's syndrome, post-irradiation necrosis, multiple sclerosis, white matter lesions, spongiform encephalopathy, and boxer-type dementia. Illustrative forms of CAA that can be treated in accordance with embodiments of the invention include hereditary CAA (also known as familial CAA) and hereditary cerebral hemorrhage associated with amyloidosis - Dutch type (HCHWA-D). In some embodiments, the hereditary CAA is caused by a mutation in the APP.

The term "individual", "patient", or "patient population" refers to a person (multiple persons) who is diagnosed with one of the various diseases described herein or who is at risk of developing one of the various diseases described herein. For example, an individual may be diagnosed with familial AD or at risk of developing familial AD. In another example, the individual is diagnosed with sporadic AD or at risk of developing sporadic AD. The diagnosis of AD can be based on the genotype or phenotypic characteristics of the individual's performance. For example, an individual with a mutant variant of presenilin 1, presenilin 2, or APP is at risk of developing familial AD. In another non-limiting example, an individual with an E4 variant of APOE is at risk of developing sporadic AD.

An individual can be diagnosed with AD or at risk of developing AD by exhibiting a phenotype associated with AD. The phenotype associated with AD may be cognitive or spiritual. Examples of cognitive phenotypes include, but are not limited to, amnesia, aphasia, apraxia, and agnosia. Examples of psychiatric symptoms include, but are not limited to, personality changes, depression, hallucinations, and delusions. As a non-limiting example, the Diagnostic and Statistical Manual of Mental disorders 4th Edition (DSM-IV-TR) (published by the American Psychiatric Association) contains the following Setting criteria for Alzheimer's type dementia: A. Development of multiple cognitive deficits manifested by one or more of memory impairment and aphasia, apraxia, agnosia, and executive dysfunction; B. Cognition Defects manifest as a significant impairment from previous functional decline and social or occupational function; C. The process is characterized by slow onset and persistent decline; D. Cognitive deficits are not due to other central nervous systems that lead to progressive deficits in memory and cognition, Systemic, or substance-inducing conditions; and E. Another mental disorder does not explain the disorder better.

Another non-limiting example is the National Association of Neurological and Linguistic Disorders and Stroke-Alzheimer's Disease and Related Disorders for Alzheimer's Disease (The National Institute of The Neurological and Communicative Disorders and Stroke-Alzheimer's disease and Related Disorder Association, NINDS-ADRDA) standards are as follows:

A. Definite Alzheimer's disease: A pathological evidence of Alzheimer's disease that meets the criteria for possible Alzheimer's disease and is examined by autopsy or biopsy.

B. Possible Alzheimer's disease: Dementia as determined by clinical and neuropsychological examinations, and involves (a) progressive defects in two or more cognitive areas, including memory, (b) at 40 Between the ages of 90 and 90 years old, and (c) no systemic or other brain diseases that can cause dementia, including delusions

C. Possible Alzheimer's disease: dementia with atypical morbidity, manifestations, or progression, and no known cause; any common pathological condition capable of producing dementia is not considered to be the cause

D. unlikely Alzheimer's disease: symptoms of dementia with either sudden onset, localized nervous system signs, or seizures or disordered steps in the early stages of the disease.

The indicated phenotypic expression can also be physical, for example by direct (imaging) or indirect (biochemical) detection of amyloid-beta plaques. Determination of amyloid-β (1-40) in peripheral blood using a high performance liquid chromatography with a linear ion trap connected to a mass spectrometer (Du Et al, J Biomol Tech. 16(4): 356-63 (2005)). Detection of single β-amyloid aggregates by fluorescence-related spectroscopy in cerebrospinal fluid of Alzheimer's patients has also been described (Pitschke et al, Nature Medicine 4: 832-834 (1998)). A method for detecting soluble amyloid-beta is described in U.S. Patent No. 5,593,846. Indirect detection of amyloid-beta peptides and receptors using an antibody to the final glycation end product (RAGE) has also been described. Recently, biochemical detection of increased BACE-1 activity in cerebrospinal fluid using chromogenic receptors has also been considered as a diagnostic or prognostic indicator of AD (Verheijen et al, Clin Chem. Apr 13 [Epub.] (2006)).

In vivo imaging of β-amyloid can be achieved using radioiodinated flavonoid derivatives as imaging agents, Ono et al, J Med Chem. 48(23): 7253-60 (2005), and using amyloid-binding dyes , such as binding to putrescine radioiodinated a 40 residue peptide (putrescein) (produced ^{125 I-PUT-a 1-40)} , and shown to cross the blood bound to the αβ and plaques. Wengenack et al, Nature Biotechnology. 18(8): 868-72 (2000).芪SB-13 and benzothiazole 6-OH-BTA-1 (also known as PIB) were also used to show imaging of β-amyloid. Nicholaas et al, Am J Geriatr Psychiatry, 12: 584-595 (2004).

Instance

The invention is further described by the examples set forth below. The use of such examples is illustrative only and in no way limits the scope and meaning of the invention or any exemplary terms. As such, the invention is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the present invention will be apparent to those skilled in the art. Accordingly, the invention is limited only by the scope of the appended claims and the scope of the claims.

Example 1: AdDNJ crosses the blood-brain barrier and increases beta-hex without affecting viability

To evaluate the β-hex target pharmacological protein AdDNJ as a therapeutic strategy for the treatment of Dutch APP ^E693Q gene-transferred mice, we first conducted a study to assess target engagement and effect on cell viability. At the same time, in order to determine the appropriate dose and dosing regimen used in our Dutch APP ^E693Q gene transfer mouse proof of concept (POC) study, we completed pharmacokinetic and dose-response/time-course studies for AdDNJ using C57B16 mice. . The purpose of pharmacokinetic and dose-response/time-course studies was to confirm that AdDNJ crosses the blood-brain barrier (BBB) and determines the minimum duration and minimum duration of daily dose to provide the most increase in brain levels in C57BL/J6 mice. The endogenous wild-type β-hex, while allowing AdDNJ to intermittently fall below the inhibitory concentration, in order to be able to dissociate from β-hex and convert to the substrate.

Materials and Method

The effect of AdDNJ on cell viability. The viability of human fibroblasts (CRL2076) and human neuroblastoma (SH-SY5Y) cells was tested after prolonged exposure to different concentrations of AdDNJ. Fibroblasts were incubated for 72 or 120 hours in a 96-well dish at 37 ° C in a complete medium containing AdDNJ at concentrations up to 1 mM in a humidified 8% CO ₂ incubator. Viable cells were quantified using the tetrazoliuim-formazan transformation method according to the manufacturer's instructions (Promega Non-Radioactive 96-well Titer assay, TB112). SH-SY5Y cells as fibroblasts were tested in the same manner but inoculated in a collagen-coated dish (Nunc # 152036). After 24 hours, 48 hours, and 120 hours of exposure to AdDNJ, live SH-SY5Y cells were quantified as described above. All dose concentrations were performed in triplicate.

Enzyme activity and selectivity of AdDNJ. By measuring hexosaminidase (Total Hex, Hex A/S and Hex B) and 3 other lysosomal hydrolases: α-galactosidase, β-galactosidase and glucosinolate The effect of lipase enzyme activity was evaluated to assess the selectivity of AdDNJ. For the enzyme assay, 200 μl of pH 5 lysis buffer (phosphate/citrate, 0.25% tauorocholate, 0.1% TX100) was used to dissolve wild-type fibroblast (CRL2076) particles (~2.5e6) Cells/pellets). The fibroblast lysate was diluted to 1:200 with a pH 5, reaction buffer containing 2 mM of appropriate 4-methylumbelliferyl (4-MU) (dissolution buffer without TX100). Incubate at 37 ° C for 1 hour and stop with 0.5 M sodium carbonate. The released 4MU was measured using a Perkin Elmer Victor 5x fluorescent disc reader (ex355/em460). Total Hex activity was measured using 4-methylumbellone β-N-ethionine glycoside (MUG); 4MU-β-N-acetylglucosamine-6-sulfate for Hex A/S activity (MUGS) Calculation of Hex B activity as a difference between Total Hex and Hex A/S activity; 4MU-α-D-glucopyranoside for α-galactosidase activity; 4-MU for β-galactosidase activity β-D-galactoside; 4 MU β-D-glucopyranoside for glucocerebrosidase activity. For enzyme inhibition, 100 μM of the following compounds were used as positive controls: 1-deoxygalactonojirimycin for α-galactosidase; N-butyl-1- for β-galactosidase Deoxygalactofuranomycin; for glucocerebrosidase, isofagomine. For the β-hexosaminidase enhancement assay, fibroblasts were exposed to AdDNJ at 0, 95, 390, and 780 nM for 5 days, after which β-hexosaminidase activity was measured. Enzyme activity measurements were performed in duplicate.

The effect of AdDNJ on O-GlcNAcase activity. The effect of AdDNJ on cellular O-GlcNAcase activity was tested in wild-type fibroblasts (AG07059, Coriell) and SH-SY5Y cells. , With or without 10μM AdDNJ Thiamet G or fibroblasts incubated for 24 hours in 8% CO ₂ humidified incubator at 37 [deg.] C in complete medium. For SH-SY5Y cells, various concentrations (0 μM to 100 μM) of AdDNJ and ThiametG were used. Cell pellets were lysed in RIPA buffer and protein-related total O-GlcNAc levels were measured using Western blotting. Briefly, a total of 40 μg of protein was isolated and transferred to PVDF and probed with an anti-O-GlcNAc antibody (18B10.C7, Pierce). Chemiluminescence was imaged using an Alpha Innotech FluoroChem Q camera and measured by density using AlphaView SA software (Alpha Innotech). The blot was stripped and re-probed using an anti-calcium-associated protein antibody (ab22595, Abcam). For O-GlcNAc quantification, the entire O-GlcNAc density measurement was subtracted as background and normalized to the calcium-linked protein-loaded control band.

AdDNJ repeated dose/dose-response and PK studies. Five-week-old C57BL/J6 mice (n=5 mice/group, Taconic Farm, Germantown, NY) were orally administered with vehicle (water) or AdDNJ (30, 100 or 300 mg/kg) daily. 14 days. Mice were euthanized on day 15 and brain/plasma collected for analysis. For a single-dose PK study, 5-week-old C57BL/J6 mice (n=5 mice/group, Taconic Farm, Germantown, NY) received a single dose of 100 mg/kg (oral gavage) AdDNJ and each The group was euthanized after 0.5, 1, 2, 4, 8, 16, 24 and 48 hours of administration for brain and plasma analysis. Whole brain tissue (10-15 mg) was homogenized in 1 mL of pH 5 lysis buffer to measure β-hexosaminidase activity as described above. AdDNJ levels were measured using LC-MS/MS in plasma and brain samples.

result

AdDNJ is a potent specific inhibitor of β-hex (Ki = 253.4 nM at pH 5; Ki = 342.3 nM at pH 7; Figure 1a), and does not inhibit O-GlcNAcase at concentrations up to 100 μM (p. 1c to 1d) or lysosomal enzyme β-glucocerebrosidase (GCase), α-galactosidase (α-Gal), or β-galactosidase (β-Gal) at 10 μM, tested The highest concentration (Fig. 1e). Furthermore, AdDNJ had little or no effect on the cell viability of SH-SY5Y and human skin fibroblasts treated with up to 1 mM AdDNJ concentration (Fig. 1f to Fig. 1g). Treatment of healthy human skin fibroblasts with 95, 390, or 780 nM of AdDNJ for 5 days increased the beta-hex level by up to 3 fold in a dose-dependent manner (Fig. 1b) confirming the ability of AdDNJ to engage the target β-hex.

In a single-dose (oral gavage) pharmacokinetic study, we found that a dose of 100 mg/kg of AdDNJ crosses the blood-brain barrier and is expected to be beneficial in the brain to favor β-hex in neutral. The level of binding in the pH ER environment (342 nM, AdDNJ is used for β-hex inhibition at pH 7), and falls below expectations within 16 hours to inhibit lysosomal β-hex levels (ie 253 nM, AdDNJ is used for β-hex inhibition at pH 5 (Figs. 2a to 2b). In addition, daily dose-dependently increased beta-hex levels (Figs. 3a-3b) were observed for 14 days via fertilized medications of 30, 100, and 300 mg/kg AdDNJ without increasing the total brain extract. GM2 level (Fig. 10a and Fig. 10c). At these doses, the increase in β-hex levels occurred at most ~5 days of treatment (Figs. 3a-3b). Because the half-life of elevated β-hex levels in the brain was ~2 days (data not shown), we chose to treat mice once a day for 5 days to maximize beta-Hex enhancement, after 2 days without Drugs (to maximize the quality conversion without AdDNJ. All doses of mice are well tolerated.

Example 2: Proof of Concept Proof of AdDNJ

Next, we conducted a POC study to assess whether ^AdDNJ reduced Aβ lesions in combination with gangliosides in Dutch APP ^E693Q gene-transforming mice.

Animal and research design. The production and characteristics of the Dutch APPE693Q mouse gene-transforming mouse are described in Gandy, S. et al. The number of days for the standard of toxicity associated with human Alzheimer's amyloid-β oligomer, neurology Yearbook 68 , 220-230 (2010) (Days to criterion as an indicator of toxicity associated with human Alzheimer amyloid-beta oligomers. Annals of neurology 68 , 220-230 (2010)). Oral gavage 3 months old male Dutch APP ^E693Q mouse transgenic mouse vector (water) (n=15) or 3, 10, 30 or 100 mg/kg AdDNJ for 3 months (n) =13). All animal studies are licensed and conducted in accordance with the Institutional Animal Care and Use Committee. Mice were maintained in a pathogen free environment for 12 hours of light/dark cycle and given ad libitum access to food and water. At 6 months of age, the treatment group and the 3-month old untreated baseline group (n=15) were assessed for cognitive assessment by NOR, EPM, and FC tests. After 48 hours of the last dose, the mice were perfused with ice-cold 1x PBS. Remove the brain and break it down into two hemispheres. Half was quickly frozen on dry ice and stored at -80 °C for biochemistry; the other half was fixed in 4% polyoxymethylene, then sagitally sliced into 30 μm sections on a microtome and stored in storage buffer at 4 °C. Until the histological analysis.

Behavioral testing. Mice were placed in the test chamber for 1 hour prior to testing to allow the mice to adapt to the room. All tests were completed Tuesday through Friday from 8 am to 3 pm two weeks prior to the final AdDNJ administration. Clean all equipment between animals.

New object recognition (NOR). On day 1, mice were acclimated in a NOR field (20 cm in diameter) for 10 minutes. On day 2, the mice were tested and consisted of two periods. In test phase 1, the mice were placed in the field and allowed to explore two identical strangers for 10 minutes. The mice were then returned to their home cage for an interval of 1 hour. During this time, one of the two objects previously allowed to be explored by the mouse is removed and replaced with a new one. In test phase 2, the mice were placed back into the field and the mice were allowed to explore familiar objects and new objects for 4 minutes. The test was recorded using an overhead camera. Then use ANY-maze (Stoelting, Wood Dale, IL) to measure the duration of the flower being explored.

Elevated Cross Maze (EPM). The labyrinth is in a + configuration and consists of two open arms (35 x 5 x 0cm) that pass through each other and perpendicular to the two closed arms (35 x 5 x 16cm) with a central neutral zone (Stoelting, Wood Dale, IL). The mice were placed in the neutral zone and allowed to explore for 8 minutes. Each trial was videotaped and the amount of time spent on open and closed arms was scored using ANY-maze (Stoelting, Wood Dale, IL). The entry arm is defined as the head and the two jaws entering the arm.

Fear conditions. Early fear memory deficits in the TgCRND8 Alzheimer's mouse model, as previously described in Steele, JW et al., are associated with altered synaptic plasticity and molecular structure, Comparative Neurology Journal (2014) (Early fear memory defects are associated) With altered synaptic plasticity and molecular architecture in the TgCRND8 Alzheimer's disease mouse model. The Journal of comparative neurology (2014)), using the ANY-Maze fear condition system (Stoelting Co., Wood Dale, IL, USA) to evaluate and environment Related and clues to the fear condition.

Aβ test. According Kawarabayashi, T. Et al. In the Tg2576 transgenic mouse model of Alzheimer's disease, brain, CSF, and plasma amyloid (beta]) protein age-related changes, Neuroscience 21, 372-381 ( 2001) (Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J Neurosci 21 , 372-381 (2001)) treated by differential detergent dissolution right Half brain. For analysis of the structure of the native oligomeric A[beta] protein, 2-4 [mu]L of native protein sample from TBS soluble was spotted onto an activated/pre-wetted PVDF membrane (0.22 [mu]m; microwell) and allowed to dry. After spotting the protein, the membrane was blocked at room temperature in TBS containing 0.1% v/v Tween-20 (Fisher Scientific; TBS-T) in 5% w/v skim milk (Santa Cruz). hour. The membrane was then incubated overnight at 4 °C in the indicated primary antibody (in 5% milk/TBS-T), 4 times in TBS-T, and species-specific HRP-binding secondary antibody at room temperature. Incubate for 1 hour (in 5% milk/TBS-T) and then wash 4 times in TBS-T. The film was then developed with an ECL Western blotting (Pierce) using a Fuji LAS-3000 developer. The membrane was then washed once in TBS-T and stripped with vigorous shaking in low pH stripping buffer [25 mM glycinate acid, pH 2.0 and 1% w/v SDS] to remove primary and secondary antibodies. Wash 3 times in TBS-T and block for 1 hour at room temperature (in 5% milk/TBS-T) and probe with the next major antibody. The integration density of immunoreactive spots was measured using MultiGauge software (Fuji film) and normalized to control % (vehicle). Rabbit pAβ A11 (anti-profibrillary oligomer, 0.5 μg/ml), rabbit pAβ OC (antigen fibrillar oligomer and fibril; 0.25 μg/ml) and mouse mAβ Nu-4 (anti-oligomer; 1 μg The production, purification and characterization of /ml) have been previously described (Tomic, JL, Pensalfini, A., Head, E. and Glabe, CG Alzheimer's disease brain soluble fibril oligomer level rise High and associated with cognitive dysfunction, neurobiology of the disease 35, 352-358 (2009) (Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction. Neurobiology of disease 35, 352-358 (2009)); Lambert, MP et al. Single-celled antibody for pathological assembly of Aβ, Journal of Neurochemistry 100, 23-35 (2007) (Lambert, MP, et al. Monoclonal antibodies that target pathological assemblies of Abeta. Journal of neurochemistry 100, 23 -35 (2007)) Standardization of total APP/Aβ signals was achieved by detecting human APP transgene metabolites using mouse mAb 6E10 (1:1000; Covance). Peroxidase-conjugated goat anti-rabbit IgG was used ( H+L; 1:20,000; vector laboratory) or goat anti-mouse IgG (H+L) 1:20,000; vector laboratory) for detection. To quantify the level of monomeric Aβ, use the human/rat Aβ 1-40/1-42 ELISA kit (Wako) according to the manufacturer's instructions. Monomer or oligomerization The absolute concentration of Aβ was normalized to the initial tissue weight before analysis.

Histology. Aβ was assessed via free-floating immunohistochemistry using mAb 6E10 (1:1000, Covance) as previously described. The GAβ was evaluated by free-floating immunohistochemistry using a mouse anti-GAβ clone 4396C (1:100) as described previously, from a gift from Dr. Katsuhiko Yanagisawa. Use the attached Olympus DP71 camera to capture images on the Olympus BX61 upright microscope. The integration density of GAβ stains was measured using Image J (National Institute of Health, Bethesda, Maryland).

Statistical analysis of behavioral, biochemical, and histological outcomes. All data are expressed as mean ± standard deviation. Statistical significance (P < 0.05) was determined using a Student Test or one-way ANOVA with Bonferroni causality analysis (GraphPad Prism, San Diego, CA).

result AdDNJ in the Netherlands APP ^E693Q Increased β-hex levels in genetically transformed mice

We wanted to assess whether ^AdDNJ binds the target β-hex in the Dutch APP ^E693Q gene-transferred mouse. We observed that the 3-month course of AdDNJ increased the total brain β-hex by a factor of three at the highest dose in a dose-dependent manner (Fig. 4c). AdDNJ also increased β-hex B (Fig. 4a) and β-hex A&S (Fig. 4b) in a dose-dependent manner. TLC analysis of whole brain extract did not show any change in ganglioside levels, indicating that β-hex is usually not inhibited throughout the brain, even after 3 months of administration of 100 mg/kg AdDNJ (10a) Figure to figure 10d). Based on the pharmacological escort protein mechanism proposed for the action of AdDNJ, we hope to observe a decrease in GM2 gangliosides. However, the TLC analysis from Figures 10a to 10d is not sensitive enough to show relatively small changes in ganglioside levels, although TLC analysis would expect an order of magnitude increase in ganglioside levels, which is sustained Inhibition of β-hex is expected. In addition, regional changes can be masked because only whole brain homogenates are evaluated. The most pronounced reduction in GAβ was observed in the hippocampal and olfactory cortical regions, indicating that a very small percentage of the total brain volume was used to generate homogenate.

AdDNJ Correction Netherlands APP ^E693Q Behavioral phenotype of gene-transferred mice

We evaluated anxiety with an elevated plus maze (EPM) and observed that the 6-month-old vector-treated Dutch APP ^E693Q gene-transferred mice were more anxious than the 3-month-old untreated mice (Fig. 5a). We observed that AdDNJ for the 3-month course was associated with reduced anxiety at all doses tested (Fig. 5a).

We evaluate learning behavior by using a new object recognition (NOR) test. The three-month-old untreated Dutch APP ^E693Q gene-transferred mice showed complete learning behavior, as they apparently spent more time exploring new objects than familiar objects, while the 6-month-old vector Treated mice exhibited learning behavioral disorders (Fig. 5b). In the 3-month course of AdDNJ, we observed in the high-dose NOR test that the same mice that showed reduced anxiety (now 6 months old) also showed protection from typical aging-related learning behavioral disorders ( Figure 5b). Six-month-old Dutch APP ^E693Q gene-transferred mice treated with AdDNJ at a dose of 30 or 100 mg/kg exhibited learning behavior at half age. Conversely, AdDNJ has no effect on environmentally relevant or clued fear conditions (Figure 6).

AdDNJ in the Netherlands APP ^E693Q Reduction of Aβ and Aβ lesions in combination with gangliosides in gene-transferred mice

Next, after 3 months of AdDNJ treatment, we evaluated Aβ and Aβ (GAβ) binding to gangliosides in Dutch APP ^E693Q gene-transferred mice. We found Aβ40 (Figs. 7a to 7d), Aβ42 (Fig. 7e to 7h), Aβ42/40 ratio (Fig. 7i to Fig. 71), pre-fibril Aβ at any AdDNJ test dose. (Level 7m), or the level of Aβ oligomer (Fig. 7n, Fig. 7o) did not change. Then, we continue to look for regional changes in Aβ and/or GAβ. Immunohistochemical analysis revealed a qualitatively specific reduction in Aβ accumulation, particularly in the lower leg (Fig. 8a, 8b), however, in the visual cortex of the Dutch APP ^E693Q gene-transferred mice taking 100 mg/kg AdDNJ (p. No difference was observed in the 8c map, the 8th map) or the CA1 hippocampus (Fig. 8e, Fig. 8f).

Regionally specific reduction of Aβ is evident in the lower leg (Fig. 9a, Fig. 9b, Fig. 9k) and the olfactory cortex (Fig. 9c, Fig. 9d, Fig. 9i) (regions involving NOR in the brain) Quantitatively reduce GAβ. In the outer olfactory cortex of the brain region involving NOR, a tendency toward a decrease in GAβ was observed (Fig. 9e, Fig. 9f, Fig. 9m). The decrease in GAβ in the visual cortex (9g, 9h, 9n) or in the CA1 region of the hippocampus (Fig. 9l, 9j, 9o) did not reach a significant extent. A GAβ-like immune response can also be easily detected in cortical blood vessels (Fig. 9p).

discuss

GM2 and GM3 gangliosides promote assembly of the mutant form Aβ (Dutch Aβ), which is highly sensitive to oligomerization in vitro (Yamamoto et al., 2005). GM2 and GM3 gangliosides are selectively expressed in cerebrovascular diseases and have been shown to promote not only the assembly of the Dutch mutant Aβ, but also the assembly of Iowa-type and Italian-type mutant Aβ peptides, all These are all under the family CAA (Yamamoto et al., 2005; Yamamoto et al., 2006). Convergence of localization of gangliosides and region-specific accumulation of mutant peptides susceptible to accelerated aggregation of gangliosides increase the likelihood that these data are specifically implicated for familial CAA and AD. The Dutch APP ^E693Q gene-transferred mouse accumulates intracellular Aβ, which can be observed in the early stages of AD.

In the present study, we demonstrated that the pharmacological-associated protein AdDNJ crosses the blood-brain barrier and causes an increase in the level of wild-type β-hex in the brain of wild-type C57BL/J6 mice. Then, we continued to evaluate the effects of AdDNJ on Aβ and GAβ deposition and anxiety onset and learning behavioral disorders in Dutch APP ^E693Q gene-transferred mice. We confirmed that the Dutch APP ^E693Q gene-transferred mice showed aging-related defects in anxiety and learning behavior, and the age of onset of memory impairment was 6 months of age. The 3-month-old Dutch APP ^E693Q gene-transferred mice had complete learning behavior in the NOR test, while the 6-month-old vehicle-treated mice showed impaired learning behavior. The dysfunction observed in the 6-month-old Dutch APP ^E693Q gene-transforming mice was associated with Aβ accumulation, particularly in the lower extremities, visual cortex, and neurons in the hippocampal CA1 region. These are brain regions that we previously demonstrated that the Dutch APP ^E693Q gene-transferred mice accumulate the most significant aging-associated Aβ deposits. The strongest Aβ-like immune response was observed in the lower leg. Recent studies have involved lower ^feet in memory, especially in the NOR test in mice, where we saw defects in the Dutch APP ^E693Q gene-transforming mice (Chang et al., 2012).

We also observed that the drug-associated protein against β-hex modifies two characteristics of the behavioral phenotype of the Dutch APP ^E693Q gene-transferred mouse in the association of significantly reduced Aβ and GAβ accumulation. The 3-month progression of AdDNJ was associated with a 3-fold increase in β-hexin levels in the brain of Dutch APP ^E693Q gene-transferred mice, indicating that AdDNJ binds to its target. A significant quantitative decrease in GAβ was observed in the lower and peripheral cortex (the brain region involving NOR). A decrease in GAβ was also observed in the lateral entorhinal cortex, visual cortex, and hippocampal CA1 region, but did not reach statistical significance.

Our study provides the primary evidence for a β-hex pharmacological protein to increase beta-hex levels, while reducing GAβ lesions in areas exhibiting the highest amount of Aβ immunoreactivity. . The occurrence of these molecular and organizational changes is associated with two aspects of correcting behavioral phenotypes (ie, anxiety, learning behavior). These data highlight the potential benefits of using targeted pharmacological-associated proteins to increase beta-hex activity. Such compounds show promise to reduce brain amyloidosis and are expected to be beneficial for the management of human brain amyloidosis, particularly those associated with amyloid angiopathy and APP/A[beta] mutations. AdDNJ has good bioavailability, BBB penetration, high selectivity to β-hex and low toxicity, making AdDNJ an excellent drug candidate, and may be rapidly developed for clinical trials of patient populations with Dutch APP mutations. The results observed in the Dutch APP ^E693Q gene-transferred mice are particularly relevant.

Claims (26)

2-Ethylamino-1,2-dideoxynojirimycin (AdDNJ) is used in the manufacture of a drug for the development of Alzheimer's disease and / or cerebral amyloid angiopathy or is diagnosed with Azhai Use of an agent for preventing and/or treating Alzheimer's disease and/or cerebral amyloid angiopathy in a patient with morbidity and/or cerebral amyloid angiopathy, wherein the patient is administered during a first enzyme enhancement period An effective amount of the agent; the agent is not administered to the patient during a matrix transition; and then the patient is administered an effective amount of the agent during a second enzyme boost. The use of claim 1, wherein the first enzyme enhancement period and the second enzyme enhancement period have the same duration. The use of claim 1, wherein one or more of the first enzyme enhancement period and the second enzyme enhancement period is a period of from about 1 day to about 8 days. The use of claim 2, wherein one or more of the first enzyme enhancement period and the second enzyme enhancement period is a period of from about 4 days to about 6 days. The use of claim 1 wherein the first The agent is administered daily during the enzyme enhancement period and during the second enzyme enhancement period. The use of claim 1, wherein the agent is administered orally. The use of claim 1, wherein the agent is administered at a dose ranging from about 3 mg/kg/day to about 300 mg/kg/day. The use of claim 7, wherein the agent is administered at a dose of about 100 mg/kg/day. The use of claim 1, wherein not administering the agent during the matrix conversion comprises not administering the agent for between about 48 hours and about 96 hours. The use of claim 9, wherein not administering the agent during the matrix conversion comprises not administering the agent for a period of about 72 hours. The use of claim 1, wherein the first enzyme enhancement period is about 5 days, and not administering the agent during the matrix conversion comprises not administering the agent for about 72 hours. The use of claim 1, comprising: administering to the patient an effective amount of the agent on day 1; not administering the agent on day 2; administering an effective amount of the agent to the patient on day 3 ; the agent is not administered on the fourth day; The patient is administered an effective amount of the agent on day 5; then the agent is not administered over a period of about 72 hours. The use of claim 1, wherein the injecting of the agent during a matrix conversion comprises not administering the agent for a period of about 24 hours. The use of claim 1, wherein not administering the agent during a matrix conversion comprises not administering the agent for a period of about 48 hours. The use of claim 1, wherein the first enzyme enhancement period is about 3 days, and not administering the agent during the matrix conversion comprises not administering the agent for about 120 hours. The use of claim 1 comprising alternately performing a total treatment time of at least one month between the enzyme enhancement period and the substrate conversion period. The use of claim 16, wherein the total treatment time is at least 3 months. The use of claim 16, wherein at least one of the substrate conversion periods has a duration that is different from at least one other period of the matrix transition. A kit comprising: one or more dosage forms comprising an effective amount of 2-acetamido-1,2-dideoxynojirimycin (AdDNJ); The indication is for: administering the dosage form during a first enzyme boost; not administering the dosage form during a substrate conversion; and then administering the dosage form during a second enzyme enhancement. The kit of claim 19, wherein the first enzyme enhancement period and the second enzyme enhancement period have the same duration. The kit of claim 19, wherein one or more of the first enzyme enhancement period and the second enzyme enhancement period is a period of from about 1 day to about 8 days. The kit of claim 19, wherein the non-administration of AdDNJ during the matrix transition comprises not administering AdDNJ for between about 48 hours and about 96 hours. A kit comprising: one or more active dosage forms comprising an effective amount of 2-acetamido-1,2-dideoxynojirimycin (AdDNJ); one or more excluding an effective amount An inactive dosage form of AdDNJ; and instructions for: administering the active dosage form during a first enzyme enhancement; administering the inactive dosage form during a loading transition; and then administering the active dosage form during a second enzyme enhancement. The set of claim 23, wherein the first The enzyme enhancement period and the second enzyme enhancement period have the same duration. The kit of claim 23, wherein one or more of the first enzyme enhancement period and the second enzyme enhancement period is a period of from about 1 day to about 8 days. The kit of claim 23, wherein the non-administration of AdDNJ during the matrix transition comprises not administering AdDNJ for between about 48 hours and about 96 hours.