WO2004037373A2

WO2004037373A2 - CHEMICAL CHAPERONES AND THEIR EFFECT UPON THE CELLULAR ACTIVITY OF β-GLUCOSIDASE

Info

Publication number: WO2004037373A2
Application number: PCT/US2003/033670
Authority: WO
Inventors: Jeffrey W. Kelly; Anu R. Sawkar; Ernest Beutler; Chi-Heuy Wong; William E. Balch
Original assignee: The Scripps Research Institute
Priority date: 2002-10-21
Filing date: 2003-10-21
Publication date: 2004-05-06
Also published as: AU2003284886A1; AU2003284886A8; WO2004037373A3

Abstract

A process of increasing the beta-glucosidase activity associated with Gaucher disease is provided. The process includes exposing the glucosidase to a chemical chaperone that has a high ratio of chaperone activity to inhibitor activity against glucosidase. Exemplary and preferred chaperones are a sterically bulky deoxynojirimycin compound and a sterically bulky 2,5-dideoxy-2,5-imino-D-mannitol compound. A process of stabilizing beta-glucosidase is also provided.

Description

Chemical Chaperones And Their Effect Upon The Cellular Activity of β-Glucosidase

Technical Field of the Invention

The field of this invention concerns activation of beta-glucosidase by certain chemical chaperones that possess a beneficial ratio between their activation concentration and their inhibition concentration. More particularly, this invention provides a process for activating a beta-glucosidase associated with Gaucher Disease using bulky alkyl substituted iminoglucitol and iminomannitol compounds.

Background of the Invention

The lysosome is an important organelle for the catabolism and recycling ofmacromolecules within a cell. Defects in lysosomal enzymes lead to accumulation of their substrates, resulting in a lysosomal storage disease. Over 40 lysosomal storage diseases have been characterized in humans (1). Lysosomal storage of non-degraded substrates leads to a range of phenotypes including enlargement of affected organs, neurologic abnormalities, skeletal lesions, and premature death (2). The missense mutations that cause lysosomal storage diseases manifest themselves through active site impairment, pH dependent protein instability, protein degradation mediated by the cellular quality control machinery, improper trafficking, or disruption of protein-protein interactions required for enzyme activation (3). Current therapeutic strategies include inhibition of substrate production using small molecule enzyme inhibitors and replacement of the defective enzyme (4). Enzyme replacement can be accomplished by protein infusions. Enzyme replacement for the most prevalent lysosomal storage disease, Gaucher disease, costs between $100,000 and $750,000 per year and is not very effective for the treatment of CNS involvement. While the enzyme has been modified to take advantage of mannose receptor-mediated endocytosis by macrophages, studies in rats suggest that less than 7% of the enzyme is taken up into liver macrophages (5), and the uptake in human bone marrow macrophages is so low that it is difficult to detect (6). Transplantation of hematopoietic stem cells can also reverse the disease, but thus far, attempts at gene transfer have been unsuccessful.

Some lysosomal storage diseases appear to be caused by lysosomal enzyme variants that retain catalytic activity but are predisposed to misfolding or mistraffϊcking in the cell (7). The use of chemical chaperones to template proper folding within the secretory pathway, to prevent post-secretory misfolding, or to stabilize proteins with a predilection to misfold is well-documented (8). Antagonists have been shown to increase cell-surface expression of vasopressin N₂ receptor variants (9). Ligands of δ opioid receptors also promote receptor maturation (10). Of particular interest, the inhibitor 1-deoxy-galactonojirimycin has been shown to chaperone the enzyme α-galactosidase in a rare variant of Fabry disease, another lysosomal storage disease (11, 12). Fabry lymphocytes cultured with inhibitor for 4 days exhibited a seven-fold increase in α- galactosidase activity that persisted 5 days after removal of drug. Transgenic mice to which 1-deoxy-galactonojirimycin was administered orally exhibited elevated α-galactosidase activity in major organs. These results suggest that small molecule binding may stabilize the mutant enzyme at neutral pH allowing it to be transported to the lysosome, where it remains stable due to the high substrate concentrations and low pH environment. Gaucher disease is the most prevalent lysosomal storage disorder with an estimated incidence of 1 in 40,000 to 60,000 in the general population (13) and 1 :800 among the Ashkenazi Jewish population (14). Five mutant alleles of β- glucosidase (β-Glu, glucocerebrosidase) account for the majority of reported cases (15). Accumulation of the substrate (glucosylceramide) leads to hepatomegaly, splenomegaly, bone crisis, anemia, and central nervous system (CΝS) involvement. Clinically, Gaucher disease is subdivided into three types: Type 1 is nonneuronopathic with adult onset, Type 2 is infantile onset with severe CΝS involvement, and Type 3 is typically early adult onset with milder CΝS involvement. β-Glucosidase is a 497 residue membrane-associated lysosomal glycoprotein whose activity is enhanced by negatively charged phospholipids and the activator protein saposin C (16). There is broad phenotypic diversity in Gaucher disease that cannot be explained by enzyme activity measurements in vitro (16, 17). In fact, there is significant disagreement regarding the activity associated with numerous β-Glu variants in the literature, many of which are known to be unstable (18, 19). We hypothesize that certain catalytically active variants of β-Glu may be identified by the quality control machinery of the cell as deficient in folding during translocation into the endoplasmic reticulum (ER) and degraded by the ER-associated proteosome pathway, precluding sufficient trafficking to the lysosome. This class of β-Glu variants should be amenable to chemical chaperoning. The c.1226 A_G (N370S) Gaucher disease mutation is found in over 98% of Jewish patients and about one-half of non- Jewish patients (20).

Several researchers have reported that N-substituted deoxynojirimycins (l,5-dideoxy-l,5-imino-D-glucitols) have inhibitory activity toward alpha and beta glucosidase. Since these two glucosidases are interspecies enzymes involved in glucose metabolism as well as gluco- and galacto-lipid metabolism, these reports have led to their investigation as effective antimicrobial and antiviral compounds. This inhibitory activity has also led to their study as candidates for treatment of human disease arising from glucosidase action. For example, Lodish et al. reported in 1984 that 1 -deoxynojirimycin is an inhibitor of glocosidase I and II, J. Cell Biology, 98, 1720 (1984). Partis et al. reported that N-alkyl derivatives of deoxynojirimycins have antiviral activity through their inhibitory activity toward glucosidases. Stutz and his colleagues indicate that sugar shaped glycosidase inhibitors with nitrogen instead of oxygen in the ring are tools for glycobiological research, www.ch.ac.uk/ectoc/echet96/papers/050.html and www.ch.ac.uk/ectoc/echet/papers/19/arn2.html October 15, 2003, published 1996, copies attached. Platt and Jacob reported that certain N-alkyl derivatives of deoxyglactonojirimycin are useful for inhibiting glycolipid synthesis U.S. Patent No's 5,786,386; 5,801,185; 6,495,570. Overkleeft et al. reported further studies of N-substituted deoxynojirimycins as tools for the study of ceramide messenger cascade. Ceramide is released by glucosidase hydrolysis of lipids. J. Biol. Chemistry, 273, 26522 (1998). Asano et al. also reported on a chaparone therapy for Fabry disease caused by alpha galactosidase deficiency, Eur. J. Biochem. 267, 4179 (2000). The compounds examined were deoxygalactonojirimycin derivatives. In a similar study, Fan et al. reported the same kind of galactosidase and glucosidase chaparone activity for deoxynojirimycin, isofagamine and calystegine derivatives in therapy for Fabry and other diseases, U.S. Patent No's. 6,274,597 and 6,599,919.

These authors demonstrate that sugar shaped nitrogen heterocycles have significant inhibitory activity against glucosidases. In some instances, their use at sub-inhibitory concentrations has been shown to chaparone glucosidases and related enzymes. However, the concentration of heterocycle needed to cause chaparone activity is close to the inhibitory concentration. Therefore, there is a need to develop sugar shaped nitrogen heterocycles that have a higher ratio of chaparone to inhibitor activity.

Summary of the Invention

These and other needs are met by the present invention. The present invention includes methods for treatment of Gaucher disease. These methods include a method for activating beta-glucosidase, a method for activating N370S beta-glucosidase and a method for treating a patient suffering from Gaucher disease. The method according to the invention calls for exposing beta- glucosidase to an effective activating amount of a chemical chaperone for beta- glucosidase wherein the chemical chaparone displays a high ratio of chaparone to inhibitor activity. In particular, the present invention for chaperoning beta- glucosidase associated with Gaucher disease uses sugar-mimic heterocycles that display a high ratio of chaparone to inhibitor activity.

The sugar-mimic heterocycles include those having five or six membered rings wherein one or two of the members are heteroatoms. Preferred heteroatoms are oxygen (O) and nitrogen (N). Thus, the heterocycle may be a pyrrolidine ring, an oxazolidine ring, a piperazine ring, a piperidine ring, a tetrahydropyran ring, a morpholine ring, an isofagamine ring, or a similar ring. The heterocycle ring carbons may be substituted by hydroxyl groups so as to closely mimic a sugar structure or may be devoid of hydroxyls. The heterocycle ring may also be substituted at the carbons alpha to the nitrogen by a hydroxymethylene group so as also to mimic a sugar structure. Preferred nitrogen heterocycles according to the invention include a deoxynojirimycin (i.e., l,5-dideoxy-l,5-D-glucitol) compound and a 2,5-dideoxy-2,5-imino-D- mannitol compound. The deoxynojirimycin compound is substituted at the nitrogen or the 2 position carbon by the R group -(CH.2)_π-XY, wherein n is an integer from 2 to 10, X is -CH₂-or -CONH- and Y is adamantyl, cyclohexyl or bicyclo[2,l,2]heptane, provided that only one of the nitrogen and the 2 position carbon is substituted by this group and the other is substituted by hydrogen. The 2,5-dideoxy-2,5-imino-D-mannitol compound (i.e., a pyrrolidine ring) is substituted at the nitrogen by the R group-(CH₂)_n-XY as defined above, or its 2- hydroxymethyl group is converted either to -CH2-NHCO-(CH₂)_n -XY wherein X and Y are defined above, or is converted to -CH2-OCO-(CH₂)_n -XY wherein X and Y are defined above. Additional deoxynojirimycin compounds and additional 2,5-dideoxy-2,5-imino-D-mannitol compounds include those wherein one or more of the hydroxyl groups are acylated by acetyl groups.

The deoxynojirimycin compound and the 2,5-dideoxy-2,5-imino-D- mannitol compound have the following formulas IA and LB and II respectively.

II

Also included within the invention as less preferred chaperones are the deoxynojirimycin compounds of formula IA and B wherein the R substituent is a linear alkyl or linear ether group of 6 to 14 carbons, and the 2,5-dideoxy-2,5- imino-D-mannitol compound of formula II wherein the R substituent is a linear alkyl or linear ether group of 6 to 14 carbons.

In one embodiment, the beta-glucosidase is N370S beta-glucosidase. Thus, in a preferred embodiment, a present process includes the step of exposing N370S beta-glucosidase to an effective activating amount of the deoxynojirimycin compound or the 2,5-dideoxy-2,5-imino-D-mannitol compound.

In another aspect, the invention provides a method of stabilizing a beta- glucosidase. This method involves exposing the beta-glucosidase to an effective stabilizing amount of the deoxynojirimycin compound of the 2,5-dideoxy-2,5- imino-D-mannitol compound. The method is particularly useful in extending the effective half-life of a beta-glucosidase administered to a subject such as a patient having Gaucher disease. Thus, a method of this invention can be used to treat a patient having Gaucher disease. In accordance with the method of the invention, the patient is administered an effective beta-glucosidase activating amount of a suitable deoxynojirimycin compound of the 2,5-dideoxy-2,5-imino- D-mannitol compound.

Brief Description of the Drawings In the drawings that form a portion of the specification

Figure 1 shows the influence of selected alkylated deoxynojirimycins (DNJ) on β-Glu activity in N370S fibroblasts cultured for 5 days with butyl (♦), octyl (■), 7-oxadecyl (O), nonyl (σ), and dodecyl (D) DNJ. β-Glu activity was evaluated at pH 4.0 using 4-methyllumbelliferyl β-D glucoside as a substrate in intact cells. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. Mean values ± standard deviation are shown for quadruplicate experiments.

Figure 2 shows the dependence of time of incubation with NN-DNJ on the activity of N370S β-Glu in intact fibroblasts. (A) N370S fibroblasts were cultured for one (σ), two (D), or four (•) days with NN-DNJ. (B) N370S fibroblasts were pulsed with NN-DNJ for 4 days and then chased with fresh media for zero (•), four (D), or six (σ) days. The intact cells were assayed at pH 4.0 using 4-methyllumbelliferyl β-D glucoside as a substrate. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. Mean values ± standard deviation are shown for quadruplicate experiments.

Figure 3 shows the effect of NN-DNJ on β-Glu activity in three different cell lines. NN-DNJ was added to the culture medium of N370S (•), wt (D), or L444P (σ) fibroblasts for four days. The intact cells were assayed at pH 4.0 using 4-methyllumbelliferyl β-D glucoside as a substrate. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. The actual activity of L444P β-Glu « N370S β-Glu « wt β-Glu. Numbers were measured in triplicate, and standard deviations were less than 10%.

Figure 4 shows the Stabilization of isolated β-Glu by NN-DNJ evaluated in vitro using heat inactivation. Ceredase® (alglucerace injection) aliquots were incubated with 0 μM (•), 50 μM (D), or 100 μM (σ) NN-DNJ at pH 7.4. The samples were heated at 48°C for the indicated amount of time and then assayed for activity at pH 5.0 with 0.1% Triton X- 100 and 0.2% TDC. Enzyme activity is reported relative to unheated enzyme. Mean values ± standard deviation are shown for duplicate experiments.

Figure 5 shows the activities of certain embodiments of the deoxynojirimycin compounds, the 2,5-dideoxy-2,5-imino-D-mannitol compounds, the morpholine compounds, the piperazine compounds and the isofagamine compounds. The percentages indicate the increase of activity of glucosidase N370S in the in vitro assay. The micromolar value indicates the concentration of compound needed to achieve fifty percent of the maximum increase of glucosidase activity.

Detailed Description of the Invention Gaucher disease is a lysosomal storage disorder caused by deficient lysosomal β-glucosidase (β-Glu) activity. A marked decrease in enzyme activity results in progressive accumulation of the substrate (glucosylceramide) in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, and sometimes central nervous system involvement. Enzyme replacement therapy for Gaucher disease is costly and relatively ineffective for central nervous system involvement. Deoxynojirimycins (DNJ) are known to inhibit several enzymes including the ER oligosaccharide processing enzymes α-glucosidase I/II, ceramide glucosyl transferase, and both non-lysosomal and lysosomal β- glucosidase (β-Glu), the latter enzyme being deficient in Gaucher disease. Alkylation of DNJ is not required to inhibit α-glucosidases, as DNJ presumably acts as a transition state mimetic (28, these numbers represent cites to references at the end of the Examples). DNJ alkylation is required for glucosyl transferase inhibition, with increasing chain length modestly increasing potency (29). The proposed mechanism of inhibition is through ceramide mimicry. The non-polar side chain may help target the inhibitor to the membrane-localized enzyme. N- butyl-deoxynojirimycin has been used in cell lines and tested in clinical trials to inhibit the formation of the substrate (glucosylceramide) that accumulates in Gaucher disease (30, 31).

Chemical chaperones have been shown to stabilize various proteins against misfolding, increasing proper trafficking from the endoplasmic reticulum. Inhibitors of beta glucosidase also act chaperones. However, their chaperoning activity is often close to their minimum inhibitory activity so that difficulties arise.

According to the invention, use of a sterically bulky heterocycle sugar mimic provides a chaperone activity that is not close in concentration requirement to that needed for inhibition activity. In particular, the present invention involves the discovery that certain sterically hindered derivatives of beta glucosidase inhibitors have a high ratio of chaperone activity to inhibitor activity. Addition of sub-inhibitory concentrations (0.2 to 10 μM) of the deoxynojirimycin compounds and the 2,5-dideoxy-2,5-imino-D-mannitol compounds of the invention to a fibroblast culture medium for nine days leads to a multifold- fold increase in the activity of Ν370S β-Glu, the most common mutation causing Gaucher disease. Moreover, the increased activity persists for several days after the withdrawal of the chaperone compound. Incubation of isolated soluble wt enzyme with NN-DNJ reveals that β-Glu is stabilized against heat denaturation in a dose-dependent fashion. These data indicate that the deoxynojirimycin compounds and the 2,5-dideoxy-2,5-imino-D-mannitol compounds chaperone β-Glu folding at neutral pH, thus allowing the stabilized enzyme to transit from the endoplasmic reticulum to the Golgi, enabling proper trafficking to the lysosome.

The present invention provides a method of increasing the activity of a beta-glucosidase associated with Gaucher disease. A method of stabilizing a beta-glucosidase and a method of treating Gaucher disease are also provided.

The method according to the invention includes exposing a beta-glucosidase to an effective activating or stabilizing dose of a deoxynojirimycin compound or a

2,5-dideoxy-2,5-imino-D-mannitol compound that displays a high ratio of chaperone activity to inhibitor activity.

I. Beta-Glucosidase Associated with Gaucher Disease

Gaucher disease is associated with a variety of beta-glucosidases.

Disease associated beta-glucosidases include both wild type (wt) and mutant forms of the enzyme. In general, beta-glucosidase activity is markedly decreased in Gaucher disease. As described more particularly hereinafter, the present invention is directed to methods for increasing the activity of these mutant beta-glucosidases. An exemplary mutant beta-glucosidase whose activity is decreased in Gaucher disease is N370S.

II. Chemical Chaperone

The activity of Gaucher disease associated beta-glucosidase activity can be increased by exposing the glucosidase to an effective activating amount of a chemical chaperone specific for the glucosidase. As disclosed in detail hereinafter, disease associated glucosidase activity can be increased through the use of a sugar-mimic heterocycle. These sugar-mimic heterocycles include those having five or six membered rings wherein one or two of the members are heteroatoms. Preferred heteroatoms are oxygen (O) and nitrogen (N). Thus, the heterocycle may be a pyrrolidine ring, an oxazolidine ring, a piperazine ring, a piperidine ring, a tetrahydropyran ring, a morpholine ring, an isofagamine ring, or a similar 5 or 6 membered ring containing heteroatoms such as nitrogen and optional oxygen. The heterocycle ring carbons may be substituted by hydroxyl groups so as to closely mimic a sugar structure, such as for example hydroxylation and hydroxymethyl substitution of a piperidine ring will produce an isofagamine ring or a deoxynojirimycin ring. Alternatively, the heterocycle ring may be devoid of hydroxyls. The heterocycle ring may also be substituted at the carbons alpha to the nitrogen by a hydroxymethylene group also so as to mimic a sugar structure. In particular, the disease associated glucosidase activity can preferably be increased through the use of a deoxynojirimycin compound or a 2,5-dideoxy-2,5-imino-D-mannitol compound that displays a high ratio of chaperone activity to inhibitor activity.

The deoxynojirimycin compound is also known as a l,5-dideoxy-l,5-D- glucitol compound. The deoxynojirimycin compound has the following formula LA and formula LB

wherein the R group is -(CH₂)_n-XY, n is an integer from 2 to 10, X is -CH₂- or -CONH- and Y is adamantyl, cyclohexyl or bicyclo[2,l,2]heptane, provided that only one of the nitrogen and the 2 position carbon is substituted by the R group and the other is substituted by hydrogen. Additional deoxynojirimycin compounds also include those wherein one or more of the hydroxyl groups are acylated by acetyl groups. Less preferred deoxynojirimycin compounds include those of formula IA and B wherein the R group is a linear alkyl or linear ether group of 6 to 14 carbons.

The 2,5-dideoxy-2,5-imino-D-mannitol compound has the following formula II

II wherein the R group is -(CH₂)_n-XY as defined above, or the 2-hydroxymethyl group is converted either to -CH₂-NHCO-(CH )_n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH₂)n -XY wherein X and Y are defined above. Additional 2,5-dideoxy-2,5-imino-D-mannitol compounds include those wherein one or more of the hydroxyl groups are acylated by acetyl groups. Less preferred compounds include those wherein the R group is a linear alkyl or linear ether group of 6 to 14 carbons.

III. Methods

In one aspect, the present invention provides a method for increasing the beta-glucosidase activity associated with Gaucher disease. The method includes the step of exposing the beta-glucosidase to an effective glucosidase activating amount of a heterocycle compound sugar mimic, preferably a deoxynojirimycin compound or a 2,5-dideoxy-2,5-imino-D-mannitol compound having a high chaperone to inhibitor activity ratio in connection with beta-glucosidase. Suitable glucosidases and chaperones are set forth above and disclosed in detail hereinafter.

As used herein the term "exposing" means bring the chaperone and glucosidase into sufficient proximity to allow the chaperone to activate the glucosidase. Exposing can mean to perfuse cells or tissues containing the glucosidase with the chaperone. Exposing can also mean bringing the enzyme and chaperone into direct contact.

As used herein, "an effective glucosidase activating amount" means a dose of chaperone that serves to increase the activity of the enzyme. As is well known in the art, certain chaperones at high concentrations act to inhibit enzyme activity. In general, a heterocycle compound sugar mimic, preferably a deoxynojirimycin compound or a 2,5-dideoxy-2,5-imino-D-mannitol compound will deliver a beta-glucosidase chaperoning concentration for activation that is significantly less that concentration needed for inhibition. Such concentrations can be readily determined by the protocols described herein. Specific activating amounts for particular enzymes and chaperones are set forth in detail hereinafter. In another aspect, the present invention provides a method that stabilizes beta-glucosidase. The method includes exposing the glucosidase to an effective stabilizing amount of a heterocycle compound sugar mimic, preferably a deoxynojirimycin compound or a 2,5-dideoxy-2,5-imino-D-mannitol compound that is a chaperone for that enzyme. An effective stabilizing amount is an amount of chaperone that will prevent the degradation of and/or increase the effective half- life of the glucosidase. Means for determining that amount are well known to a skilled artisan. Specific examples are given hereinafter. As especially useful application of this method is the treatment of Gaucher disease. As set forth hereinbefore, current treatment for Gaucher disease can involve the administration of beta-glucosidase (e.g., Ceredase®) to a patient having Gaucher disease. The effective half-life of the administered enzyme can be increased by administering to the patient an effective stabilizing amount of a heterocycle sugar mimic, preferably a deoxynojirimycin compound or a 2,5-dideoxy-2,5- imino-D-mannitol compound of the invention. The co-administration can occur in the same vehicle or sequentially in different vehicles. Suitable enzymes and chaperones are set forth above.

The data presented below show that the incubation of fibroblasts with μM concentrations of active site-directed molecules can lead to increased N370S β-Glu intracellular activity. The N370S mutation is the most common variant causing Gaucher disease. The activity of this variant increases with putative chaperone incubation time and persists for at least 6 days upon removal of the small molecule from the culture medium. The nature of the core structure and the length and steric bulk of the N-alkyl or substituted alkyl chain strongly influence the potency of the putative chaperone. DNJ alkylated with short chains (4 carbons) produced inactive compounds at low concentrations, while long alkyl chains (12 carbons) proved to be toxic. DNJs modified with alkyl side chains having 8-10 carbons are well-tolerated and enhance β-Glu activity significantly. DNJs modified with sterically bulky alkyl side chains such as alkyl adamantyl, cyclohexyl or bicycloheptyl bonded through amide bonds also will enhance β-Glu activity while being orders of magnitude lower that the inhibitory activity. While it is not to be considered a limitation of the invention, it is believed that these chaperones seem to function through both ceramide- mimicry and by targeting the small molecule to the membrane, increasing its local concentration. NN-DNJ has been shown to increase the activity of both N370S and wt β-Glu in fibroblasts, presumably through protein stabilization, as demonstrated in vitro. However, it does not enhance the intracellular activity of the L444P variant. Clinical data indicate that a small increase in enzyme activity may be effective in treating disease. Although patients receiving Cerezyme infusions experience reduced hepatosplenomegaly, improved blood counts, and amelioration of bone crises, the increase of enzyme activity in bone marrow due to treatment can be quite small. After infusion of either 1.15 U/kg or 60 U/kg of enzyme into five patients, a 1.7-9.6-fold increase of β-Glu activity was observed (6). This suggests that the 1.5-2-fold increase in activity due to DNJ or mannitol chemical chaperoning may be clinically useful. Since the intracellular half-life of much of the infused Cerezyme is only hours and enzyme is often administered only every two weeks, the average increase in patients is miniscule compared to the increases we observed in tissue culture with chaperone treatment.

IN. Synthetic Procedures

Following published procedures, 1 -deoxynojirimycin (DΝJ) can be prepared from 2,3,4,6-tetra-O-benzyl-α-glucopyranose, see reference 23. 2,5- Anhydro-imino-D-glucitol can be prepared from 5-keto-D-fructose according to Reitz's method, see references 24 and 25. Ν-alkyl compounds can prepared by reductive amination of the alkyl aldehyde with the corresponding nitrogen heterocycle such as 2,5-anhydro-imino-D-glucitol, morpholine, 1- (hydroxyethyl)piperazine, isofagamine, or trans-3-hydroxy-L-proline. Reductive amination procedures are well known, See for example J. March, "Advanced Organic Chemistry", 4^th Ed. John Wiley & Sons, New York, NY (1992). The adamantyl, cyclohexyl or bicycloheptyl moieties can be added by either of two methods. The first involves addition of an omega-haloalkanoic ester to the heterocycle nitrogen through an amine alkylation reaction. The ester is then deprotected to form the carboxylic acid and the acid group is condensed with the amine or hydroxy-adamantane, cyclohexane or bicycloheptane. The condensation can be accomplished by carbodiimide coupling, activated acyl coupling such as through an acyl azide or acid chloride and the like. The second method involves formation of the R side chain followed by its coupling to the heterocycle. The R side chain can be formed by amidating the corresponding amino or hydroxyl-adamantane, cyclohexane or bicycloheptane using the corresponding omega-hydroxyalkanoic carboxylic acid protected at the hydroxyl group. Appropriate reaction conditions include acid chloride reaction and carbodiimide coupling. Following deprotection of the protected hydroxyl group, it can be converted to a halide group through use of such reagents as N-bromo or -chloro succinimide. The halo substituted R side chain can be coupled to the corresponding nitrogen heterocycle by an amine substitution reaction. The 2- carbon substituted compounds can be prepared by formation of an imine (Schiff Base) group between the 2 carbon and the nitrogen of the heterocycle and reaction of the appropriate organo lithium compound with the imine group. The organo lithium compound can be formed from the halo substituted R side chain or alkyl halide and a reagent such as n-butyl lithium. The imine can be prepared from the glucopyranose by oxidative condensation. These methods are known in the art. See for example J. March "Advanced Organic Chemistry", 4^th Ed. John Wiley & Sons, New York, NY (1992), the disclosure of which is incoφorated herein by reference.

The cyclo mannitol (pyrrolidine) compounds can be synthesized by rearrangement of the corresponding iminoglucitol compound using a catalytic amount of an organic acid such as p-toluene sulfonic acid.

V. Routes and Doses for Administration

Any of the therapeutic agents described above can be formulated as pharmaceutical compositions. A pharmaceutical composition of the invention includes a therapeutic agent in combination with a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels. An oral dosage form may be formulated such that the therapeutic agent of the invention is released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein.

Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non- aqueous vehicles (which may include edible oils), or preservatives. A therapeutic agent of the invention can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the pharmaceutical compositions containing therapeutic agents of the invention may be in powder form, obtained by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile saline, before use. Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.

Pharmaceutical compositions of the invention may also contain other ingredients such as flavorings, colorings, anti-microbial agents, or preservatives. It will be appreciated that the amount of a therapeutic agent required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage. The dosage administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired. A daily dosage of active ingredient can be expected to be about 0.001 to about 1000 milligrams per kilogram of body weight, with the preferred dose being about 0.001 to about 100 mg/kg, preferably administered several times a day.

The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms. Additives may also be included in the formulation to enhance the physical appearance, improve stability, and aid in disintegration after administration. For example, liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours or days. Sustained release products can also be formulated for implantation or transdermal/transmucosal delivery. Such formulations typically will include a polymer that biodegrades or bioerodes thereby releasing a portion of the active ingredient. The formulations may have the form of microcapsules, liposomes, solid monolithic implants, gels, viscous fluids, discs, or adherent films.

Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

Film-coated tablets are compressed tablets, which are covered with as thin layer of film or water-soluble material. A number of polymeric substances with film-forming properties may be used. Film coating imparts the same general characteristics as sugar coating with the added advantage of a greatly reduced time period required for the coating operation.

Enteric-coated tablets are compressed tablets coated with substances that resist solution in gastric fluid but disintegrate in the intestine. Enteric coatings can be used for tablets containing drug substances which are inactivated or destroyed in the stomach, for those which irritate the mucosa, or as a means of delayed release of the medication. Multiple compressed tablets are compressed tablets made by more than one compression cycle.

Layered tablets are prepared by compressing additional tablet granulation on a previously compressed granulation. The operation may be repeated to produce multilayered tablets of two or three layers. Special tablet presses are required to make layered tablets.

Press-coated tablets, which are also referred to as dry-coated, are prepared by feeding previously compressed tablets into a special tableting machine and compressing another granulation layer around the preformed tablets. They have all the advantages of compressed tablets, i.e., slotting, monogramming, speed of disintegration, etc., while retaining the attributes of sugar-coated tablets in masking the taste of the drug substance in the core tablets. Press-coated tablets can also be used to separate incompatible drug substances; in addition, they can provide a means to give an enteric coating to the core tablets. Both types of multiple-compressed tablets have been widely used in the design of prolonged-action dosage forms.

Compressed tablets can be formulated to release the drug substance in a manner to provide medication over a period of time. There are a number of types which include delayed-action tablets in which the release of the drug substance is prevented for an interval of time after administration of until certain physiological conditions exist; repeat-action tablets which periodically release a complete dose of the drug substance to the gastrointestinal fluids; and the extended-release tablets which continuously release increments of the contained drug substance to the gastrointestinal fluids. The non-aqueous carrier, or excipient, can be any substance that is biocompatible and liquid or soft enough at the mammal's body temperature to release the active ingredient into the animal's bloodstream at a desired rate. The carrier is usually hydrophobic and commonly organic, e.g., an oil or fat of vegetable, animal, mineral or synthetic origin or derivation. Preferably, but not necessarily, the carrier includes at least one chemical moiety of the kind that typifies "fatty" compounds, e.g., fatty acids, alcohols, esters, etc., i.e., a hydrocarbon chain, an ester linkage, or both. "Fatty" acids in this context include acetic, propionic and butyric acids through straight- or branched-chain organic acids containing up to 30 or more carbon atoms. Preferably, the carrier is immiscible in water and/or soluble in the substances commonly known as fat solvents. The carrier can correspond to a reaction product of such a "fatty" compound or compounds with a hydroxy compound, e.g., a mono-hydric, di- hydric, trihydric or other polyhydric alcohol, e.g., glycerol, propanediol, lauryl alcohol, polyethylene or -propylene glycol, etc. These compounds include the fat-soluble vitamins, e.g., tocopherols and their esters, e.g., acetates sometimes produced to stabilize tocopherols. Sometimes, for economic reasons, the carrier may preferably comprise a natural, unmodified vegetable oil such as sesame oil, soybean oil, peanut oil, palm oil, or an unmodified fat. Alternatively the vegetable oil or fat may be modified by hydrogenation or other chemical means which is compatible with the present invention. The appropriate use of hydrophobic substances prepared by synthetic means is also envisioned.

Typically, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts, and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field. In addition to the active or therapeutic ingredient, tablets contain a number of inert materials. The latter are known as additives or "adds." They may be classified according to the part they play in the finished tablet. The first group contains those, which help to impart satisfactory compression characteristics to the formulation. These include (1) diluents, (2) binders, and (3) lubricants. The second group of added substances helps to give additional desirable physical characteristics to the finished tablet. Included in this group are (1) disintegrators, (2) colors, and in the case of chewable tablets, (3) flavors, and (4) sweetening agents. Frequently the single dose of the active ingredient is small and an inert substance is added increase the bulk in order to make the tablet a practical size for compression. Diluents used for this purpose include dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, dry starch, and powdered sugar.

Most tablet formulators tend to use consistently only one or two diluents selected from the above group in their tablet formulations. Usually these have been selected on the basis of experience and cost factors. However, the compatibility of the diluent with the drug must be considered. When drug substances have low water solubility, it is recommended that water-soluble diluents be used to avoid possible bioavailability problems.

Agents used to impart cohesive qualities to the powdered material are referred to as binders or granulators. They impart a cohesiveness to the tablet formulation which insures the tablet remaining intact after compression, as well as improving the free-flowing qualities by the formulation of granules of desired hardness and size. Materials commonly used as binders include starch, gelatin, and sugars as sucrose, glucose, dextrose, molasses, and lactose. Natural and synthetic gums which have been used include acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Beegum, and larch arabogalactan. Other agents which may be considered binders under certain circumstances are polyethylene glycol, ethylcellulose, waxes, water and alcohol.

The quality of binder used has considerable influence on the characteristics of the compressed tablets. The use of too much binder or too strong a binder will make a hard tablet which will not disintegrate easily. Alcohol and water are not binders in the true sense of the word; but because of their solvent action on some ingredients such as lactose and starch, they change the powdered material to granules and the residual moisture retained enables the materials to adhere together when compressed.

Lubricants have a number of functions in tablet manufacture. They improve the rate of flow of the tablet granulation, prevent adhesion of the tablet material to the surface of the dies and punches, reduce interparticle friction, and facilitate the ejection of the tablets from the die cavity. Commonly used lubricants include talc, magnesium stearate, calcium stearate, stearic acid, and hydrogenated vegetable oils. Most lubricants with the exception of talc are used in concentrations less than 1%. Lubricants are in most cases hydrophobic materials. Poor selection or excessive amounts can result in "waterproofing" the tablets, result in poor tablet disintegration and dissolution of the drug substance.

A disintegrator is a substance, or a mixture of substances, added to a tablet to facilitate its breakup or disintegration after administration. The active ingredient must be released from the tablet matrix as efficiently as possible to allow for its rapid dissolution. Materials serving as disintegrates have been chemically classified as starches, clays, celluloses, aligns, or gums.

The most popular disintegrators are corn and potato starch which have been well-dried and powdered. Starch has a great affinity for water and swells when moistened, thus facilitating the rupture of the tablet matrix. However, others have suggested that its disintegrating action in tablets is due to capillary action rather than swelling; the spherical shape of the starch grains increases the porosity of the tablet, thus promoting capillary action.

In addition to the starches a large variety of materials have been used and are reported to be effective as disintegrators. This group includes Veegum HV, methylcellulose, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, and carboxymethylcellulose. Sodium lauryl sulfate in combination with starch also has been demonstrated to be an effective disintegrant.

Colors in compressed tablets serve functions other than making the dosage from more esthetic in appearance. Any of the approved certified water- soluble FD&C dyes, mixtures of the same, or their corresponding lakes may be used to color tablets.

In addition to the sweetness which may be afforded by the diluent of the chewable tablet, e.g., mannitol or lactose, artificial sweetening agents may be included. Among the most promising are two derivatives of glycyrrhizin, the glycoside obtained from licorice. Compressed tablets may be characterized or described by a number of specifications. These include the diameter size, shape, thickness, weight, hardness, and disintegration time.

The following experiments further illustrate aspects of the invention. They are not meant as limitations of the invention, which has been fully described in the foregoing text. The detailed explanations of the Figures are provided following the examples section. These Figures are referred to in the example section.

The examples that follow illustrate preferred embodiments of the present invention and are not limiting of the specification and claims in any way.

Biological Protocols

Fibroblast Culture

Primary skin fibroblast cultures were established from a patient homozygous for the N370S (c.1226 A G) mutation. Type 2 (infantile) Gaucher disease fibroblasts (GM0877) and normal fibroblast cultures (GM05659, GM00498) were obtained from the Coriell Cell Repositories. Fibroblasts were cultured in minimum essential medium with Earle's salts and non-essential amino acids (Gibco) supplemented with 16.5% fetal bovine serum (Irvine Scientific) and 1% glutamine pen-strep (Irvine Scientific) at 37°C in a 5% CO₂ atmosphere in air. Monolayers were passaged upon reaching confluency with trypsin-EDTA (Irvine Scientific). All cells used in this study were between the fourth and eighteenth passage. Total cell protein was determined using Coomassie Plus (Pierce) or Micro BCA (Pierce) assay reagent.

Intact Cell Lysosomal β-glucosidase Assay

The intact cell assay was modified from a previously reported procedure (21). Cells were plated into 24-well assay plates in media. The media were replaced with media containing potential chemical chaperones dissolved in DMSO after cell attachment. The total DMSO content in the media was less than 1% and had no effect on cell viability or β-glucosidase activity according to control experiments. Untreated and chaperone-treated media was replaced every 3-4 days when incubations longer than 4 days were utilized. Potential chaperones were evaluated at all concentrations in quadruplicate. The enzyme activity assay was performed after removing media supplemented with potential chaperones. The monolayers were washed with Dulbecco's phosphate buffered saline solution (PBS, Irvine Scientific, pH 7.2). Eighty μL of PBS and 80 μL 0.2M acetate buffer (pH 4.0) were added to each well. The reaction was started by the addition of 5 mM 4-methylumbelliferyl β-D glucoside (100 μl, Sigma) to each well, followed by incubation at 37°C for 1 h. The cells appeared intact when examined by light microscopy after treatment as described. The reaction was stopped by lysing the cells with 2 mL of 0.2M glycine buffer (pH 10.8). Liberated 4-methylumbelliferone was measured (excitation 365 nm, emission 445 nm) using an Aviv fluorimeter. The fluorescence was also evaluated on a SpectraMax Gemini plate reader (Molecular Devices) in 24-well format using identical excitation and emission wavelengths. The fluorescence of untreated cells was compared with those treated with potential chaperones. Conduritol B epoxide (CBE, Toronto Research Chemicals) was routinely added to control wells to evaluate the extent of non-specific β-glucosidase activity (22).

Lysed Cell β-glucosidase Assay

Intact cells were harvested and the pellet lysed in PBS by sonication. Prior to assay, the cell lysate was diluted with an equal volume of pH 5.0 acetate buffer. All lysed cell β-Glu assays were performed in the presence of 0.1% triton X-100 (Fisher) and 0.2% taurodeoxycholic acid (TDC, Calbiochem). Substrate (20 mM 4-methyllumbelliferyl β-D glucoside) was added to initiate the reaction, and the samples were incubated at 37°C for 30 min. The reaction was terminated by the addition of glycine buffer (pH 10.8) and the fluorescence measured as described above. IC₅₀ values were determined by preincubating acidified lysate with small molecules for 15 min in the presence of TDC and triton X-100 at 37°C. Small molecule concentrations ranged from 10.0 nM - 1.0 mM. Substrate (20 mM 4-methyllumbelliferyl β-D glucoside) was added, and the samples were incubated for an additional 30 min. The reaction was quenched with glycine buffer and the fluorescence recorded. In vitro stabilization of β-glucosidase

An assessment of the ability of chemical chaperones to stabilize β-Glu against denaturation was performed using a commercially available highly purified human placental β-Glu preparation, Ceredase® (alglucerase injection, Genzyme), remaining in vials after patient administration. Ceredase samples containing albumin were used without purification. Enzyme aliquots (20 μl, pH 7.4) were incubated with 0 μM, 50 μM, or 100 μM chemical chaperone on ice for 1 h. The samples were heated at 48°C as a function of time in an attempt to heat inactivate (denature) β-Glu, and then the samples were diluted into 3 volumes of 0.1 M acetate-phosphate buffer (pH 5.0). The enzyme was immediately incubated with 20 μL of substrate (20 mM 4-methyllumbelliferyl β- D glucoside) in the presence of 0.1 % triton X-100 and 0.2% TDC for 10 min at 37°C before quenching with glycine buffer. Liberated 4-methylumbelliferone was measured (excitation 365 nm, emission 445 nm) using an Aviv fluorimeter. Enzyme activity was reported relative to unheated enzyme.

Preparation of Molecules

N-f«-Butyl)deoxynojirimycin, Ν-(7-oxadecyl)deoxynojirimycin, N-(n- nonyl)deoxynojirimycin, and N- -dodecyl)deoxynojirimycin were purchased from Toronto Research Chemicals. Νonanoic acid, nonyl aldehyde, decyl alcohol, 1-nonanesulfonic acid, dodecylamine, morpholine, 1- (hydroxyethyl)piperazine, and diethylamine were purchased from Aldrich. Trans-3-hydroxy-L-proline was purchased from Fluka. Following published procedures, 1 -deoxynojirimycin (DΝJ) was prepared from 2,3,4,6-tetra-O- benzyl-α-glucopyranose (23). 2,5-Anhydro-imino-D-glucitol was prepared from 5-keto-D-fructose according to Reitz's method (24, 25). Ν-Νonyl compounds (2, 3, 4, 5, and diethyl nonylamine) were prepared via reductive amination of nonyl aldehyde with 2,5-anhydro-imino-D-glucitol, morpholine, 1- (hydroxyethyl)piperazine, trans-3-Hydroxy-L-proline, and diethylamine, respectively. Likewise, reductive amination of octyl aldehyde and DΝJ gave N- ( -octyl) deoxynojirimycin (ΝO-DΝJ). Compound 6 was generated by the treatment of N-nonyl-2,5-anhydro-imino-D-glucitol 2 and 2-methoxypropene in the presence of p-toluene-sulfonic acid (catalytic amount) (26, 27). Structural characterization was determined by 1H-, ¹³C-NMR, and high resolution mass spectrometry (see supplemental information).

Similarly, the C3, C5 adamantyl amido nojirimycin compounds having hydroxyl groups or acetyloxy groups at carbon positions 3-5 and 7 and the C8 amido substituted mannitol compound were prepared (Figure 5, compounds 7- 12).

Example 1 Alkylated deoxynoiirimycin analogues increase lysosomal β-glucosidase activity.

Since DNJ analogues are known to bind to the active site of wt lysosomal β-Glu, these compounds served as a starting point for chemical chaperone screening. DNJ analogues of varying alkyl chain length and sterically hindered alkyl substitutions were cultured with fibroblasts for 5 days, employing a homozygous N370S cell line derived from a Gaucher patient. An intact cell assay was performed at pH 4 (see Methods) using conduritol B epoxide (CBE) as a control to specifically measure lysosomal β-Glu activity (22). CBE covalently inhibits lysosomal β-Glu, but not non-lysosomal β-Glu. As expected, CBE insensitive activity was negligible (typically less than 5%) at pH 4. Several of the DNJ and mannitol analogs increased intracellular N370S β-Glu activity at lower concentrations and inhibited the enzyme at high concentrations (Fig. 1). N-f«-nonyl)deoxynojirimycin (ΝΝ-DΝJ) displayed the activity profile expected of a chemical chaperone. At low concentrations (< 30 μM), β-Glu activity was elevated when compared to untreated cells. Up to a 1.65-fold increase in Ν370S β-Glu activity was observed after adding 5 μM NN- DNJ to the culture media for five days. Dose-dependent inhibition occurred at concentrations exceeding 60 μM, with less than 20% activity remaining at a culture concentration of 100 μM. While NN-DNJ did not cause cell death over a 4-5 day incubation period, notable cell death occurred at high concentrations (> 60 μM) during longer incubation periods. N-(n-dodecyl)deoxynojirimycin (ΝD- DΝJ) was highly inhibitory at all concentrations evaluated, even at nanomolar concentrations (data not shown). At ΝD-DΝJ concentrations higher than 30 μM, complete cell death occurred within 24 h, potentially due to membrane disruption. The known β-Glu inhibitor N-(«-butyl)deoxynojirimycin (ΝB-DΝJ) showed no activity, even at concentrations as high as 100 μM. This is consistent with Priestman and coworkers' demonstration that β-Glu was not inhibited by low doses of ΝB-DΝJ in mice receiving β-Glu infusions (32). The β-Glu IC₅₀ value of ΝB-DΝJ as determined in wt fibroblast lysates was =300 μM, hence the small molecule concentration in tissue culture may simply have been too low to observe chaperone activity. Both N-(7-oxadecyl)deoxynojirimycin and N-(n- octyl)deoxynojirimycin (ΝO-DΝJ) increased β-Glu activity over a wide range of concentrations, resulting in no inhibition through 100 μM. Decreasing compound lipophilicity with oxygen-substituted alkyl chains (N-(7- oxadecyl)deoxynojirimycin) has been shown to decrease toxicity (29). ΝO-DΝJ was well tolerated by the cells after 9 days of exposure at concentrations as high as 100 μM.

To be confident that the β-Glu activity assessed in intact cells was not due to changes in fibroblast membrane permeability, experiments were also performed with membrane-lysed cell preparations as described above in the methods protocol. Ν370S cells that had been cultured with NN-DNJ were sonicated, and an activity assay was performed on the lysates. These data were compared to those obtained in intact cells (Table 1). β-Glu activity assays have been typically performed on lysates at pH 5.0 in the presence of triton X-100 and taurodeoxycholate or deoxycholate, and therefore these conditions were utilized (33). It is known that the activity of the N370S mutation is extremely sensitive to assay conditions; however, these were not optimized further for these studies. At longer incubation periods, NN-DNJ-induced activity increases were greater in detergent-stimulated lysates than in intact cells. However, these differences were small at shorter incubation periods (Table 1). These results demonstrate that substrate permeability is entirely adequate in intact cells, and further demonstrate that the activity increase in the presence of NN-DNJ is not due to changes in fibroblast membrane permeability. It is clear that the length and steric bulk of the alkyl chain has a significant effect on the ability of the putative chaperones to increase intracellular β-Glu activity. The amphiphilic character of alkylated DNJ mimics glucosylceramide, and increasing the alkyl chain length and steric bulk increases enzyme affinity by exploiting hydrophobic recognition elements in the active site. The β-Glu IC₅₀ of NN-DNJ is 100-fold lower than NB-DNJ in lysates, which contributes to the observed difference in chaperone activity. The alkyl chain length also affects cell permeability and partitioning in cell culture experiments. Potential chemical chaperones with longer alkyl chains should be able to insert into the membrane, and increase local concentration near membrane-associated β-Glu.

Example 2 The N-/fø-nonyl)deoxynoiirimvcin mediated increase in Ν370S β-glucosidase activity increases with culture time and persists upon removal of NN-DNJ from fibroblast cultures.

The increase of N370S β-Glu activity observed with low concentrations of NN-DNJ (up to =30 μM) increased with the duration of the putative chaperone incubation period (Fig. 2A). However, inhibition of enzyme activity persisted at concentrations exceeding 60 μM. Interestingly, cells that had been "pulsed" with NN-DNJ for 4 days and chased with untreated media also showed a significant activity enhancement for at least 6 days after removal of putative chemical chaperone from the fibroblast media (Fig 2B). Although the shape of the activity curve shifted with time, all pulse concentrations resulted in elevation of enzyme activity during the chase periods, including those that initially were inhibitory. Importantly, no inhibition was observed even at high pulse concentrations, suggesting that we have not yet optimized the utilization of these compounds. Fan and co workers report similar elevation of mutant α- galactosidase A activity in Fabry lymphoblasts 5 days after removal of the pharmacological chaperone 1-deoxy-galactonojirimycin (DGJ) (11). They believe that mutant α-galactosidase A synthesized in the presence of DGJ is stable for at least 5 days. While this may also be the case for N370S β-Glu, an alternative possibility is that NN-DNJ and related alkylated iminocyclitols are incorporated into the cell membrane as discussed above, leading to sustained activity enhancement. Example 3 NN-DNJ increases the activity of wt and N370S β-glucosidase, but not L444P β-glucosidase. Both wt and Gaucher disease type 2 cells homozygous for the c.1448 T_C (L444P) mutation were also incubated with NN-DNJ for 4 days (Fig. 3). The enzyme activity of the cells containing the N370S mutation exhibited the greatest enhancement, while the activity of the L444P mutant cells was only inhibited over the concentration range assayed. The wt cells consistently showed a 1.2-fold maximum increase in activity under these conditions. Interestingly, Asano and coworkers also observed α-Gal A activity elevation in wt lymphoblasts upon incubation with DGJ, the chaperone that corrects the activity deficit in one variant associated with Fabry disease (12). Both wt and N370S β-Glu activity can be increased by culturing with putative chaperones, whereas the L444P mutant is not amenable to this approach. Since not all Gaucher-associated β-Glu variants are defective in folding and trafficking (some mutations destroy the active site or prevent the activator protein, saposin C, from binding for example), NN-DNJ chaperoning should be ineffective in some cases. The inability of NN-DNJ to enhance the cellular activity of the L444P variant is reassuring in that it would be surprising if all mutant enzymes were amenable to this approach.

Example 4 Probing the requirements for N370S β-glucosidase chaperoning with simple amphipathic molecules and alkylated nitrogen heterocycles. β-Glu activity, especially in the case of the N370S mutant, is stimulated by detergents, bile salts, phosphatidylserine, and the activator protein Saposin C (19). The mechanisms by which these molecules enhance enzyme activity are not known, hence it was decided to test different types of amphipathic molecules. A series of charged and neutral amphipathic molecules were evaluated over a concentration range of 5-200 μM. We compared both wt and N370S cell lines because both proteins could potentially be stabilized by these molecules. Nonanoic acid, nonanal, 1-nonanesulfonic acid, decan-1-ol, dodecylamine, and diethyl-nonylamine were incubated with fibroblasts for 4 days. Dodecylamine was toxic to both wt and N370S fibroblasts at concentrations greater than 10 μM. A similar toxicity effect was observed when cells were treated with dodecyl-DNJ. All of the other compounds exhibit activity within 5% of untreated cells at low concentrations (<50 μM). At very high concentrations (200 μM), up to 20% inhibition was observed. In comparison, NN-DNJ chaperoned wt cells show a 1.2-fold increase in activity and N370S cells show a 1.5-fold increase in activity under the same conditions. The activity of β-glucosidase is not increased by positively charged, negatively charged, or neutral simple amphipathic molecules. A series of alkylated nitrogen heterocycles were prepared to study the core structure requirements for N370S activity enhancement (Table 2). While all of the molecules contained the same nonyl alkyl chain and a nitrogen that would be protonated under the cellular conditions utilized, a range of activities was observed (see supplemental information for activity profile for each compound). The iminocyclitol 2, a known transition state mimetic, only slightly increased β-Glu activity, whereas related 5-membered ring N-heterocycles 5 and 6 were inactive (34). Surprisingly, the morpholine and piperazine based molecules 3 and 4 showed some activity despite their inability to form numerous hydrogen bonds in the active site thought to be important for the binding of 1 to β-Glu. However, the latter compounds may be able to form an ion pair with the putative active site carboxylate because of their structures. The piperazine and morpholine compounds had measurable IC₅₀ values (high μM range) while 5 and 6 had IC₅₀ values in the mM range. This may explain why the former compounds are active and the latter are not. NN-DNJ possessed the lowest IC₅₀ in wt and N370S lysates, and was the most promising compound at concentrations below 10 μM. When NN-DNJ was incubated in conjunction with the morpholine 3, the activity profile was identical to the expected profile for the tighter binding NN-DNJ (data not shown), suggesting that they competed for the same site in β-Glu. The concept that the best inhibitors are the best chaperones demonstrated by the Fabry disease study also seems to be recapitulated by β-Glu chaperones (12). Example 5 NN-DNJ protects β-glucosidase from thermal denaturation. Ceredase® (alglucerase injection) was pre-incubated with 0, 50 μM, or 100 μM NN-DNJ before being subjected to heat inactivation (48 °C). Untreated β-Glu lost the most activity under these conditions, with only 20% activity remaining after 1 h of heating, β-glucosidase activity was retained by the inclusion of NN-DNJ, and the effect was improved with increasing NN-DNJ concentration (Fig. 4). Enzyme incubated with 100 μM NN-DNJ retained twice as much activity as untreated enzyme at all time points evaluated, suggesting that NN-DNJ binding stabilized wt β^Glu against thermal denaturation in vitro. These results imply that small molecule binding may also stabilize wt and N370S β-Glu within cells allowing proper folding and trafficking, leading to increased enzyme activity (Fig 1). These data are also consistent with observations that NB-DNJ-treated mice receiving enzyme infusions exhibited an increased β-Glu serum half-life (32).

Example 6 Study of Sterically Bulky DNJ as Chaperones for β-Glu

Molecule Preparation Morpholine and l-(hydroxyethyl)piperazine were purchased from

Aldrich. Following literature procedure, 1 -deoxynojirimycin (DNJ) was prepared from 2,3,4,6-tetra-O-benzyl-α-glucopyranose(ref 35). 2,5-Anhydro- imino-D-glucitol (A2) was prepared from 5-keto-D-fructose according to Reitz's method.(ref s 36, 37). N-alkylated compounds (A5, Bl, Dl, and GI) were prepared via reductive amination of N-octyl aldehyde or N-nonyl aldehyde with 2,5-anhydro-imino-D-glucitol (A2), morpholine, l-(hydroxyethyl)piperazine, and isofagamine respectively. Likewise, reductive amination of octylaldehyde and heptaldehyde with DNJ gave N-octyl deoxynojirimycin (NO-DNJ) (FI) and N-heptyl deoxynojirimycin (NH-DNJ) (F2) respectively. F3 and F5 were prepared via N-alkylation of DNJ with different lengths of alkyl bromides with terminal methyl esters. The esters were deprotected and coupled to 1- adamantylamine using standard conditions. Subsequent acetylation of F3 and F5 gave F6 and F7 respectively. The structural characterization is determined by Η-, ¹³C-NMR, and mass spectrometry. The results are presented below. NMR spectra (1H at 300 or 400 MHz, ^,3C at 75 or 100 MHz) were recorded in CDC1₃ or CD₃OD as solvents, and chemical shifts were expressed in parts per million related to internal TMS.

N-Octyl-2,5-anhydro-imino-D-glucitol (A5) 1H (300Mz, CD₃OD) δ 0.91 (t, 3H, J= 7.2 Hz), 1.26-1.36 (br s, 10H), 1.51 (m, 2H), 2.65-2.76 (m, 3H), 3.04 (td, 1H, J = 5.7Hz, 5.1 Hz), 3.57-3.74 (m, 4H), 3.94-4.01 (m, 2H); ^I3C NMR (75Mz, CD₃OD) δ 78.2, 77.7, 71.8, 67.3, 62.3, 61.6, 55.5, 33.0, 30.7, 30.4, 28.5, 28.4, 23.7, 14.5; HRMS for C14H29NO4 [M+H⁺]: calcd 276.2169, found 276.2164.

N-Nonylmorpholine (Bl) 1H (300Mz, CDCI₃) δ 0.83 (t, 3H, J = 7.5 Hz), 1.22 (s, 12H), 1.43 (m, 2H), 2.26 (t, 2H, J- 8.0 Hz), 2.37 (t, 4H, J= 4.5 Hz), 3.65 (t, 4H, J = 4.5 Hz); ¹³C NMR (75Mz, CDCI₃) δ 66.8, 59.1, 53.7, 31.9, 29.6, 29.5, 29.3, 27.5, 26.6, 22.7, 14.1; HRMS for C13H27NO [M+H⁺]: calcd 214.2165, found 214.2163.

l-(Hydroxyethyl)-4-nonylpiperazine (Dl) 1H (400Mz, CDC1₃) δ 0.87 (t, 3H, J = 6.8 Hz), 1.26 (s, 12H), 1.48 (m, 2H), 2.33 (t, 2H, J = 8.0 Hz), 2.40-2.60 (m, 10H), 3.63 (m, 2H), 4.00 (s, 1H); ¹³C NMR (lOOMz, CDC1₃) δ 59.3, 58.6, 57.6, 53.0, 52.7, 31.7, 29.45, 29.41, 29.1, 27.4, 26.6, 22.5, 13.9; HRMS for CιsH32NO [M+H⁺]: calcd 257.2587, found 257.2581.

N-Octyl deoxynojirimycin (NO-DNJ) (FI) 1H (400Mz, CD₃OD) δ 0.89 (t, 3H, J= 6.4 Hz), 1.30 (s, 10H), 1.48 (m, 2H), 2.09-2.19 (m, 2H), 2.55 (m, 1H), 2.78 (m, 1H), 2.98 (dd, 1H, J= 11.2, 4.8 Hz), 3.11 (t, 1H, J= 9.6 Hz), 3.31 (t, 1H, J= 9.6 Hz), 3.45 (m, IH), 3.83 (m, 2H) ; ¹³C NMR (lOOMz, CD₃OD) δ 80.5, 72.0, 70.7, 67.3, 59.3, 57.6, 53.8, 32.9,

30.6, 30.4, 28.6, 25.1, 23.7, 14.4; HRMS for C14H29NO4 [M+H⁺]: calcd 276.2169, found 276.2168.

N-Heptyl deoxynojirimycin (NH-DNJ) (F2) 1H (400Mz, CD₃OD) δ 0.89 (t,

3H, J = 7.2 Hz), 1.20-1.38 (m, 8H), 1.48 (m, 2H), 2.09-2.19 (m, 2H), 2.56 (m, IH), 2.77 (m, IH), 2.98 (dd, IH, J= 11.2, 4.8 Hz), 3.11 (t, 1H, J = 9.6 Hz), 3.33 (t, IH, J = 9.6 Hz), 3.42-3.49 (m, IH), 3.83 (m, 2H) ; ^,3C NMR (lOOMz, CD₃OD) δ 80.5, 72.0, 70.7, 67.3, 59.3,

57.6, 53.8, 33.0, 30.3, 28.5, 25.1, 23.6, 14.4; HRMS for C13H27NO4 [M+H⁺]: calcd 262.2013, found 262.2011.

N-Octyl isofagamine (GI) Η (400Mz, CD₃OD) δ 0.89 (t, 3H, J - 7.2 Hz), 1.30-1.36 (m, 10H), 1.74 (m,

IH), 2.70 (t, IH, J = 11.2 Hz), 2.82 (t, IH, J = 12.4 Hz), 3.05 (m, 2H), 3.37(t, IH, J = 9.6 Hz), 3.43-3.51 (m, 2H), 3.63-3.75 (m, 2H), 3.80 (dd, IH, J = 10.8 Hz, 3.2 Hz); ¹³C NMR (lOOMz, CD₃OD) δ 72.9, 70.4, 62.8, 60.8, 58.4, 56.6, 54.7, 43.0, 32.8, 30.1, 27.7, 25.5, 23.6, 14.4; HRMS for C14H29NO3 [M+H⁺]: calcd 260.222, found 260.2217.

(F3-C4-OH)

1H (400Mz, CD₃OD) δ 1.70 (s, 6H), 1.95-2.05 (m, 11H), 2.38 (m, 2H), 2.98-3.21 (m, 3H), 3.39-3.55 (m, 3H), 3.61 (t, IH, J= 9.4 Hz), 3.75 (m, IH), 3.95 (dd, IH, J = 12.4Hz, 2.6 Hz), 4.12 (d, IH, J= 12. 4Hz); HRMS for C2θH34N2θs [M+H⁺]: calcd 383.254, found 383.2549. (F5-C6-OH)

1H (400Mz, CD₃OD) δ 1.31 (m, 2H), 1.54-1.66 (m, 10H), 1.95-2.05 (br s, 9H),

2.10 (t, 2H, J = 7.2 Hz), 2.63-2.71 (m, 2H), 2.92 (m, IH), 3.11 (m, IH), 3.24-

3.30 (m, 2H), 3.47 (t, IH, J= 9.6 Hz), 3.60 (m, IH), 3.85 (dd, IH, J- 12.4 Hz, 2.8 Hz), 3.96 (dd, IH, J = 12.4 Hz, 2.0 Hz); ¹³C NMR (lOOMz, CD₃OD) δ 175.2, 78.9, 69.9, 68.8, 67.3, 56.7,

55.8, 53.7, 52.7, 42.3,37.5, 37.4, 30.8, 27.3, 26.6, 24.2; HRMS for C22H38N2O5

[M+H⁺]: calcd 411.2853, found 411.2853.

(F6-C4-OAc)

1H (400Mz, CDCb) δ 1.62 (br s, 6H), 1.70 (t, 2H, J = 6.8 Hz), 1.93-2.04 (m, 23H), 2.31 (t, IH, J = 10.4 Hz), 2.56-2.78 (m, 3H), 3.15 (dd, IH, J = 11.6 Hz, 5.2 Hz), 4.13 (s, 2H), 4.90-5.11 (m, 4H); HRMS for C28H42N2O9 [M+H⁺]: calcd 551.2963, found 551.2956.

(F7-C6-OAc)

1H (400Mz, CDCb) δ 1.25(m, 2H), 1.42 (m, 2H), 1.60 (m, 2H),1.70 (br s, 6H), 1.95-2.10 (m, 23H), 2.31 (t, 1H, J = 10.4 Hz), 2.50-2.78 (m, 3H), 3.20 (dd, 1H, J = 11.6 Hz, 5.2 Hz), 4.15 (s, 2H), 4.91-5.20 (m, 4H); HRMS for C3oH4₆N₂O9 [M+H⁺]: calcd 579.3276, found 579.3284.

The C5 and C3 amidoadamantyl DNJ's, alkyl DNJ's, alkylamidomannitols, alkyl morpholine, alkyl piperazine and isofagamine compounds of Figure 5 were assayed in the fibroblast culture using an N370S cell line derived from a Gaucher patient as described in Example 1. The assay and analysis were conducted as described in Example 1. The activity increases at designated concentrations are provided in Figure 5. Table 1. A comparison of β-Glu activity in intact and lysed cells.

10 μl NN-DNJ was added to the culture medium of N370S fibroblasts for 4 or 9 days before the intact cells were assayed for activity at pH 4.0. Cell lysates pretreated in the same fashion were assayed at pH 5.0 with 0.1% Triton X-100 and 0.2% TDC. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. ICA = intact cell β-Glu activity, LCA = lysed cell β-Glu activity.

Relative 4 day Relative 4 day Relative 9 day Relative 9 day

ICA increase LCA increase ICA increase LCA increase

1.50±0.1 1.5±0.05 1.7±0.1 2.0±0.05

Table 2. The effect of variable core structure with constant Λ-nonyl substructure on N370S and wt β-Glu activity.

Various core structures N-alkylated with the n-nonyl group were added to the culture medium of fibroblasts for 5 days. β-Glu activity was assayed at pH 4.0 using 4-methyllumbelliferyl β-D glucoside as a substrate. IC₅₀ values were determined in wt and Ν370S lysates at pH 5.0 in the presence of 0.1% Triton X- 100 and 0.2% TDC. Enzyme activity is normalized to untreated cells, assigned a relative activity of 1. ICA = intact cell β-Glu activity.

Compound Relative IC₅₀N370S IC₅₀WT

Maximum N370S ICA increase [concentration]

1 NN-DNJ 1.65 [5 μM] 5μM 1 μM

2 1.10 [20 μM] 330 μM 100 μM

3 1.40 [30 μM] 540 μM 175 μM

4 1.50 [5 μM] 450 μM 110 μM

5 No activity >10mM >10mM

6 Slightly inhibitory >1 mM >lmM

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Claims

WHAT IS CLAIMED IS:

1. A method for increasing a beta-glucosidase activity associated with Gaucher disease, the process comprising exposing the beta-glucosidase to an effective activating amount of a deoxynojirimycin compound of formula IA and formula LB

IA IB

which is substituted at the nitrogen or the 2 position carbon by an R group, the R group being -(CH₂)_n-XY, wherein n is an integer from 2 to 10, X is - CH₂-or -CONH- and Y is adamantyl, cyclohexyl or bicyclo[2,l,2]heptane, or a linear alkyl or linear ether group of 6 to 14 carbons provided that only one of the nitrogen and the 2 position carbon is substituted by the R group and the other is substituted by hydrogen; or an effective activating amount of a 2,5-dideoxy-2,5-imino-D-mannitol compound of formula II

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH₂-NHCO-(CH₂)_n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH )_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

2. The method of claim 1 wherein the beta-glucosidase is N370S beta- glucosidase.

3. The method of claim 1 wherein the deoxynojirimycin compound is used and the R group is substituted on the nitrogen.

4. The method of claim 3 wherein n is 5, and Y is adamantyl.

5. A method of activating N370S beta-glucosidase, the method comprising exposing the N370S beta-glucosidase to an effective activating amount of deoxynojirimycin compound of formula IA and formula LB

II which is substituted at the nitrogen by the R group and defined above, or its 2-hydroxymethyl group is converted either to -CH₂-NHCO-(CH2)_n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH2)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2, 5 -imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

6. A method of activating a beta-glucosidase associated with Gaucher disease, the method comprising exposing the beta-glucosidase to an effective activating amount of deoxynojirimycin compound of formula IA and formula LB

IA IB

which is substituted at the nitrogen or the 2 position carbon by an R group, the R group being -(CH₂)_n-XY, wherein n is an integer from 2 to 10, X is - CH₂-or-CONH- and Y is adamantyl, cyclohexyl or bicyclo[2,l,2]heptane, or a linear alkyl or linear ether group of 6 to 14 carbons provided that only one of the nitrogen and the 2 position carbon is substituted by the R group and the other is substituted by hydrogen; or an effective activating amount of a 2,5-dideoxy-2,5-imino-D-mannitol compound of formula II

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH₂-NHCO-(CH2)_n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH₂)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated. A chemical chaperone specific for the beta-glucosidase which is deoxynojirimycin compound of formula IA and formula LB

IA IB

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH₂-NHCO-(CH₂)_π -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH₂)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

8. A method of claim 7 wherein the beta-glucosidase is N370S beta-glucosidase and the chaperone is the deoxynojirimycin compound and Y is adamantyl.

9. The method of claim 7 wherein the chaperone is the 2,5-dideoxy-2,5-imino- D-mannitol compound.

10. A method of stabilizing a beta-glucosidase comprising exposing the beta- glucosidase to an effective stabilizing amount of a deoxynojirimycin compound of formula IA and formula LB

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH2-NHCO-(CH₂)n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH₂)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

11. A method of treating Gaucher disease in a patient in need of such treatment, the process comprising administering to the patient an effective beta- glucosidase activating amount of a deoxynojirimycin compound of formula IA and formula LB

IA IB

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH₂-NHCO-(CH₂)_n -XY wherein X and Y are defined above, or is converted to -CH₂-OCO-(CH₂)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

12. A method of treating Gaucher disease in a patient in need of such treatment, the process comprising administering to the patient a beta-glucosidase together with an effective beta-glucosidase activating amount of a deoxynojirimycin compound of formula IA and formula LB

IA IB

II which is substituted at the nitrogen by the R group as defined above, or its 2- hydroxymethyl group is converted either to -CH2-NHCO-(CH₂)_n -XY wherein X and Y are defined above, or is converted to -CH2-OCO-(CH₂)_n - XY wherein X and Y are defined above, or its 2-hydroxymethyl group is converted to a linear alkyl or linear ether group of 6 to 14 carbons; or derivatives of the deoxynojirimycin compound of formula I and 2,5- dideoxy-2,5-imino-D-mannitol compound of formula II wherein one or more of the hydroxyl groups are acylated.

13. The method of claim 11 wherein the beta-glucosidase and the deoxynojirimycin compound or the 2,5-dideoxy-2,5-imino-D-mannitol compound of are administered together.

14. A method for activating a beta glucosidase mutant comprising exposing the mutant to a chaperoning concentration of a 5 or 6 membered heterocycle compound having one or two nitrogens or nitrogen and oxygen as the heteroatoms wherein the heterocycle mimics a sugar structure and is lipophilic.

15. A method according to claim 14 wherein the heterocycle is a piperidine ring, a piperazine ring, a morpholine ring, a isofagamine ring, or a tetrahydropyran ring wherein each carbon atom of the ring is optionally and individually substituted by an hydroxyl group and the nitrogen atom is substituted by a lipophilic group, the lipophilic group being an alkyl group, an alkyl ester group or an alkyl amide group or a substituted version thereof wherein the substituent is a sterically bulky hydrocarbon group.

16. A pharmaceutical composition suitable for treatment of Gaucher' s disease, comprising a pharmaceutical carrier and an activating amount of a 5 or 6 membered heterocycle compound having one or two nitrogens or nitrogen and oxygen as the heteroatoms, wherein the heterocycle mimics a sugar structure, the heterocycle is a piperidine ring, a piperazine ring, a morpholine ring, an isofagamine ring, or a tetrahydropyran ring, each carbon atom of the ring is optionally and individually substituted by an hydroxyl group and the nitrogen atom is substituted by a lipophilic group, the lipophilic group being an alkyl group, an alkyl ester group or an alkyl amide group or a substituted version thereof wherein the substituent is a sterically bulky hydrocarbon group.

17. A method for treatment of Gaucher' s disease comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 16.