NL2021840B1 - Pharmacological Chaperones For Enzyme Treatment Therapy - Google Patents
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- 0 *[C@](C[C@@]([C@]([C@]1COCc2ccccc2)OCc2ccccc2)OCc2ccccc2)[C@@]1O Chemical compound *[C@](C[C@@]([C@]([C@]1COCc2ccccc2)OCc2ccccc2)OCc2ccccc2)[C@@]1O 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
- A61K31/39—Heterocyclic compounds having sulfur as a ring hetero atom having oxygen in the same ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D291/00—Heterocyclic compounds containing rings having nitrogen, oxygen and sulfur atoms as the only ring hetero atoms
- C07D291/08—Heterocyclic compounds containing rings having nitrogen, oxygen and sulfur atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D327/00—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms
- C07D327/02—Heterocyclic compounds containing rings having oxygen and sulfur atoms as the only ring hetero atoms one oxygen atom and one sulfur atom
- C07D327/04—Five-membered rings
Abstract
The present invention relates to therapeutic methods for the management of a patient’s lysosomal storage disease by enzyme replacement therapy. The methods involve the use of combinations of, and kits containing: a) an exogenous lysosomal hydrolase, and b) a reversible cyclophellitol-derived glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase.
Description
Pharmacological Chaperones For Enzyme Treatment Therapy
FIELD OF THE INVENTION
The present invention relates generally to the field of therapeutics for lysosomal storage diseases and glycosidase deficiency related diseases. More specifically, the invention relates to various combinations of enzyme replacement therapy or gene therapy, for the treatment of lysosomal storage diseases. It further relates to cyclophellitol cyclosulfamidates as pharmacological chaperones of glycosidases, compositions comprising these compounds, and their therapeutic uses, as well as pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.
BACKGROUND OF THE INVENTION
Lysosomal storage disorders (LSDs) are diseases typically involving a compromised enzyme, in particular a lysosomal hydrolase. Typically, the activity of a single lysosomal hydrolytic enzyme is compromised, either reduced, or lacking altogether, usually due to the inheritance of an autosomal recessive mutation. As a consequence, the nominal substrates of the compromised enzyme accumulate in the lysosomes, producing a severe disruption of the cellular architecture, and resulting in various manifestations of the disease. Lysosomal storage disorders include sphingolipidoses and other complex carbohydrate metabolism disorders, such as those include diseases known as Gaucher, Niemann-Pick, Farber, GM1gangliosidosis, GM2-gangliosidosis, Tay-Sachs, Krabbe, Fabry, Schindler, Pompe, Fucosidosis, Mannosidosis, and Wolman's disease, as well as disorders that show aberrant accumulation of α-synuclein (a-syn) or such as Parkinson's disease (PD) or Lewy-body dementia. A successful approach to treating many of the acute LSDs is Enzyme Replacement Therapy (ERT). This therapy involves intravenous administration of an exogenously-produced natural or recombinant, replacement enzyme to a patient. In particular, a recombinant human agalactosidase, such as Fabrazyme® from Genzyme, see also US patent 7,011,831, has been found to be particularly useful for treating a deficiency in α-galactosidase A (α-gal A) which generates an LSD known, such as Fabry disease. This ERT usually involves bi-weekly infusions of Fabrazyme®at a dose of 1.0 and 0.2 mg/kg body weight.
Fabry disease is characterized by the toxic accumulation of globotriaosylceramide (Gb3) in lysosomes and globotriaosylsphingosine (Lyso-Gb3) in a patient's plasma and tissue.An unsolved problem with ERTs for treating LSDs has remained in the development of immunity by patients to the enzymes of the ERTs upon repeated treatment. Antibodies developed by patients against ERT enzymes not only reduce the enzymes' efficacy but also put patients at risk of anaphylactic shock. This is particularly pronounced in hemizygous male patients, which produce no α-Gal A at all.
As set out above, the elicited antibody response to the therapeutic protein also usually results in a decreased effect of the ERT during further treatment of Fabry patients.
Another problem with ERTs for treating LSDs remains the lack of stability of the ERT enzymes in the human body. As a result, only a small amount of the injected enzymes reaches in active form the target lysosome of the patients.
Another problem with conventional ERT treatments for treating LSDs is the stressful high frequency of required infusions, typically involving intravenous injections on a weekly or bi-weekly basis, with comparatively high amounts of enzymes.
An alternative therapy approach for lysosomal storage disease has focussed on the use of small molecules for treatment, whereby a class of molecules was found to inhibit upstream generation of lysosomal hydrolase substrates to relieve the input burden to the defective enzyme, in the so-called substratereduction therapy (SRT). An example of this class of molecules are deoxynojirimycin (DNJ) and derivatives thereof, that inhibit glucosylceramide synthase (GCS). Certain uses of GCS inhibitors DNJ type either alone (WO 00/62780) or in combination with a glycolipid degrading enzyme (WO 00/62779) have been described. Another example of the substrate deprivation class of molecules are the amino ceramide-like small molecules which have been developed for glucosylceramide synthase inhibition. Glucosylceramide synthase catalyzes the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. Glucosylceramide itself is the precursor of hundreds of different glycosphingolipids. Also, amino ceramide-like compounds have been developed for use in Fabry and Gaucher's disease, see for instance US-B-5,916,911 and US6,051,598. However, these are directed towards the defective enzyme, and hence will disturb the molecular pathways whereby patients are effectively deprived of the products of normal metabolism.
Generally, ERT methods are known, as set out for instance in AU2017268649, which discloses a method to predict response to pharmacological chaperone treatment of diseases; US2011152319, which discloses Methods for Treatment of Fabry Disease; US2010113517, which discloses a method for the treatment of Fabry disease using pharmacological chaperones; JP2018027945, which discloses high concentration aglucosidase compositions for the treatment of Pompe disease; US2016051528 which discloses a method for the Treatment of Pompe Disease Using 1-Deoxynojirimycin Derivatives; US2011136151, which discloses assays for diagnosing and evaluating treatment options for Pompe disease; CN103373955, which discloses multivalent azasugar derivatives and synthetic method thereof; JP2009137933 disclosing a method for treating Gaucher's disease; W02004037373 (A2): Chemical chaperones and their effect upon the cellular activity of beta-glucosidase; US5242942 (A): Anticonvulsant fructopyranose cyclic sulfites and sulfates and WO2016162588 (Al): Compositions for treating diseases related to lysosomal storage disorders.
However, none of the disclosed methods results in an in-situ reversible composition that maintains the activity of the enzyme sufficiently high.
An approach to stabilize the enzyme is disclosed in EP2874648 which suggests an agalactosidase A and 1-deoxygalactonojirimycin (also known as migalastat or galacto-DNj) co-formulation for the ERT treatment of Fabry disease. Herein, the 1deoxygalactonojirimycin is disclosed as to act acts as a pharmacological chaperone for a specific mutant «-galactosidase A, by selectively binding to the enzyme, thereby increasing its , and to increase amount of active enzyme in a patients lysosomes. However, this composition is limited to specific conformations and mutations of this particular enzyme.
Accordingly, there has remained a need to improve ERTs for treating LSDs to overcome significant limitations associated with these treatment modalities when used alone or with existing combination regimens. A further need to improve ERTs has involved reducing the frequency and/or size of doses of ERTs.
A multigene therapy is under phase 2 clinical trials for Fabry disease (AVR-RD-01), for the endogenous expression of a modified lysosomal a-A/-acetyl-galactosaminidase (aNAGAL) with increased α-galactosidase activity (a-NAGALEL). This is however still in the early stages. It is considered that also modified lysosomal α-NAGAL with increased agalactosidase activity (a-NAGALEL) may require stabilisation.
Accordingly, there emerges a need to stabilize endogenously expressed modified lysosomal enzymes for treating LSDs. The present invention meets this need by providing improved approaches utilizing a combination of enzyme replacement therapy and a chaperone therapy for treating LSDs. In this chaperone therapy; the compounds according to the invention aim to pharmacological chaperone, i.e. stabilize a therapeutic recombinant enzyme.
SUMMARY OF THE INVENTION
This invention provides various combinations of enzyme replacement therapy or (multi)gene therapies for the treatment of lysosomal storage diseases and glycosidase deficiency related diseases. Several general approaches are provided. Each general approach involves enzyme replacement therapy (ERT) with an enzyme chaperone in a manner which optimizes the clinical benefit, i.e., treatment, while minimizing disadvantages associated with ERT.
An embodiment of the invention provides for a composition for treating a lysosomal storage disease and/or glycosidase and/or β-glucosidase deficiency related diseases, particularly Fabry, Gaucher or Pompe disease, comprising:
a) an exogenously produced, natural or recombinant lysosomal hydrolase, or an endogenous produced natural mutant lysosomal hydrolase by (multi)gene therapy; and
b) one or more reversible cyclophellitol-derived glycosidase inhibitors capable of competitively blocking the active site of the lysosomal hydrolase.
Preferably, the composition concerns treatments where the lysosomal hydrolase is an «-galactosidase, an «-glucosidase, or a β-glucosidase.
More preferably, the reversible glycosidase inhibitor is a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group, more preferably a reversible glycosidase inhibitor comprising a cyclic sulfamidate functional group.
In a preferred embodiment, the reversible glycosidase inhibitor has the structure according to formula I:
O
I I o-s=o
wherein each of R1 to Rs individually equals H, a lower alkyl, a lower alkenyl, and/or a lower alkynyl group.
Preferably, the α-galactosidase is a recombinant human α-galactosidase, preferably Fabrazyme®, and preferably, the reversible glycosidase inhibitor comprises an a-gai-cyclic sulfamidate, or a form suitable for administration and for forming the inhibitor in situ. A particularly preferred a-gal-cyclic sulfamidate is an a-gal 1,6 cyclophellitol cyclosulfamidate of the formula I, wherein each of R1 to R5 individually equals H, a lower alkyl, a lower alkenyl, a lower alkynyl group and/or an optionally substituted (hetero)aryl group.
The present invention also relates to the composition according to the invention for administering to a patient at substantially the same time the lysosomal hydrolase and the reversible glycosidase inhibitor.
Another embodiment of the invention provides a composition useful for treating a lysosomal storage disease, particularly Fabry disease, comprising: a) the exogenously produced, natural or recombinant lysosomal hydrolase; b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase.
Another embodiment of the invention provides a kit useful for treating a lysosomal storage disease, particularly Fabry disease, comprising: a) the exogenously produced, natural or recombinant lysosomal hydrolase; (b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase.
Another embodiment of the invention provides the compounds of structure according to formula II:
o ρΛ...,·λ -A u γ γ
O-R:.S (II), wherein each of R1 to Rs individually equals H, a lower alkyl, a lower alkenyl, a lower alkynyl group and/or an optionally substituted (hetero)aryl group, preferably wherein all rests R1 to R5 are H; and more preferably wherein the compounds are in a chair conformation.
Another embodiment of the invention provides for a composition, wherein the reversible glycosidase inhibitor has the structure according to formula II, wherein each of R1 to R4 individually represents H, a lower substituted alkyl such as (CHz)nX wherein X is -CH3, OH, -NH2, -I, -Br, -Cl or -CF3 and n ranges of from 0 to 5; a lower alkyl, a lower alkenyl, and a lower alkynyl group, and wherein R5 represents -OH, -CH2OH, -CH2O-alkyl, preferably a Ci to C5 alkyl, or a -CH2O-glycoside. Preferably, each of R1 to R4 in (II) individually represents H, a lower substituted alkyl such as -(CH2)nX wherein X is -CH3, -OH, -NH2, -I, -Br, -Cl or -CF3, and n ranges of from 0 to 5; and wherein R5 represents OH, CH2OH, CH2O-alkyl, or CH2Oglycoside. More preferably, R1 to R5 represent H. Yet more preferably, the hexyl ring is predominantly in a 4Ci conformation.
Another preferred embodiment of the invention provides a composition according to formula I or II, wherein R1 equals a substituted alkyl such as (CH2)nY wherein Y represents CH3, OH, NH2,1, Br, Cl or CF3 and n ranges from 0 to 5; wherein R1 is H, or a carboxy group (CO)Z wherein Z represents (CH2)nCH3, OH, NH2, or CF3 and n ranges from 0 to 5.
Another preferred embodiment of the invention provides a composition according to formula I or II herein-below, wherein R1 equals a substituted alkyl such as (CH2)nX, wherein X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5; and wherein R2 to R4 represent H or a carboxy group (CO)X, wherein X represents (CH2)nCH3, OH, NH2, or CFsand n = 0-5, and/or an optionally substituted (hetero)aryl group.
Preferably, the α-galactosidase is a recombinant human α-galactosidase, preferably Fabrazyme®. Preferably, the reversible glycosidase inhibitor is an α-gal-cyclic sulfamidate.
A particularly preferred embodiment of the invention provides for a composition, wherein the α-gal-cyclic sulfamidate is an a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III:
HO'
HO’
'OH
OH wherein R1 is H or lower substituted alkyl (CHz)nX, wherein X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5; a lower alkyl, preferably a Ci to Cs-alkyl; or an optionally substituted carboxyl group (CO)X wherein X is (CHzjnCHs, OH, NH2, or CF3,and n = 0-5. More preferably, R1represents H.
The present invention preferably also relates to a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III wherein R1 is H or lower, optionally substituted alkyl, such as (CH2)nX where X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5; wherein R1 is H or lower alkyl such as (CO)X where X is (CH2)nCH3, OH, NH2, or CF3 and n = 0-5; preferably H.
Another embodiment of the invention provides for a composition as set out herein above, for administering to a patient at substantially the same time with the lysosomal hydrolase and the reversible glycosidase inhibitor, and/or wherein the components are administered such that they form a reversible complex in situ.
Advantageously, a composition as set out herein above, may also be employed for administering to a patient having been treated with gene therapy and endogenously expressing an α-galactosidase formed by a recombinant mutated a-NAGALEL.
Another embodiment of the invention provides for a kit useful for treating a lysosomal storage disease, particularly Fabry disease, comprising: a) the lysosomal hydrolase; and b) the reversible glycosidase as set out above, pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof; wherein the components are administered such that they form a reversible complex in situ.
Preferred compounds include a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III wherein R1 is H or lower alkyl, preferably H.
Another preferred embodiment of the present invention relates to a method to prepare the compounds according to formula I, in line with the reaction scheme disclosed in Figure 2. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
Another preferred embodiment relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry or Pompe disease, comprising:
a) subjecting the patient to a gene therapy to endogenously express a natural or recombinant lysosomal hydrolase; and
b) administering to the patient a reversible glycosidase inhibitor according to the invention capable of competitively blocking the active site of the lysosomal hydrolase.
It is to be understood that both the foregoing Summary of the Invention and the following Description of Preferred Embodiments are exemplary and explanatory only and are not restrictive of the invention(s) herein.
The present invention also relates to novel 1,6 cyclophellitol cyclosulfamidates,, cyclosulfamidates or cyclosulfamides of the formula (I):
wherein X represents -N(R1)- or oxygen, preferably wherein one of X represents -N(R1)-, the other X represents oxygen, more preferably wherein the X at the exposition representsN(R1)- and the X at the exposition represents oxygen, wherein R1 each individually represent H, a lower substituted alkyl such as (CHz)nX wherein X is CHs, OH, NHz, I, Br, Cl or CF3, and n ranges of from 0 to 5; an optionally substituted (hetero)aryl group; or a carboxy group (CO)Z wherein Z represents (CHzjnCHs, -OH, or -NHz; wherein each of R2 to R4 individually represents H, a lower substituted alkyl such as (CHz)nY wherein Y represents CH3, ΌΗ, -NHz, -I, -Br, -Cl or -CF3, and n ranges of from 0 to 5 and/or an optionally substituted (hetero)aryl group; or a carboxy group (CO)X, wherein X represents (CHz)nCH3, OH, NHz, or CF3 and n = 0-5, and/or an optionally substituted (hetero)aryl group;
wherein R5 represents -OH, -CH2OH, -CHzO-alkyl, or -CHzO-glycoside; and/or pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof. More specifically, the present invention relates to the 1,6 cyclophellitol cyclosulfamidate of the formula I, wherein R1 is H.
Another embodiment of the invention provides for 1,6 cyclophellitol cyclosulfamidates according to formula II, wherein each of R1 to R4 individually represents H, a lower substituted alkyl such as (CHzjnX wherein X is -CH3, -OH, -NH2, -I, -Br, -Cl or -CF3 and n ranges of from 0 to 5; a lower alkyl, a lower alkenyl, and a lower alkynyl group, and wherein R5 represents -OH, -CH2OH, -CH2O-alkyl, preferably a Ci to Cs alkyl, or a -CH2Oglycoside. Preferably, each of R1 to R4 in (II) individually represents H, a lower substituted alkyl such as -(CH2)nX wherein X is -CH3, -OH, -NH2, -I, -Br, -Cl or -CF3, and n ranges of from 0 to 5; and wherein Rs represents OH, CH2OH, CH2O-alkyl, orCH2O-glycoside. More preferably, R1 to Rs represent H. Yet more preferably, the hexyl ring is predominantly in a 4Ci conformation.
The present invention also relates to 1,6 cyclophellitol cyclosulfamidates according to formula III, wherein R1 is H, a lower alkyl, a lower substituted alkyl (CH2)nX, wherein X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5; or an optionally substituted carboxyl group (CO)X wherein X is (CH2)nCH3, OH, NH2, or CF3, and n = 0-5.
The present invention also relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry, Gaucher or Pompe disease, comprising administering to the patient:
a) an exogenously produced, natural or recombinant lysosomal hydrolase; and
b) a reversible cyclophellitol-derived glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase as set out herein above, more preferably wherein the lysosomal hydrolase is an α-galactosidase or α-glucosidase, such as wherein the α-galactosidase is a recombinant human α-galactosidase, preferably Fabrazyme®. Preferably, the reversible glycosidase inhibitor is a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group.
Another preferred embodiment of the present invention relates to a method as se tout above, herein the lysosomal hydrolase is a recombinant α-glucosidase, preferably Myozyme®, and wherein the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase is an α-glucose-configured cyclosulfamidate.
Preferably the lysosomal hydrolase and the reversible glycosidase inhibitor are administered together, at substantially the same time, to the patient, and/or wherein the components are administered, to the patient, such that they form a reversible complex in situ.
The present invention also relates to the use of a composition and/or a compound according to the invention, and pharmaceutically acceptable salts, solvates, chelates, noncovalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof capable of competitively blocking the active site of a lysosomal hydrolase, for treating a lysosomal storage disease and/or glycosidase deficiency related diseases, particularly Fabry, Gaucher or Pompe disease.
The present invention also relates to a pharmaceutical composition comprising a coformulation of between about 0.5 and about 20 μΜ α-galactosidase A; and between about 50 and about 20,000 μΜ a compound according to the invention, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is formulated for parenteral administration to a subject.
The present invention also relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry or Pompe disease, comprising:
a) subjecting the patient to a gene therapy to endogenously express a natural or recombinant lysosomal hydrolase; and
b) administering to the patient a reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 demonstrates the reaction coordinates of α-galactosidases and inhibitors. A. Reaction itinerary of retaining α-galactosidase, showing conformations of the Michaelis complex, transition state, and covalent intermediates. B. Glucose configured cyclosulfates 1 and 2 inhibits a- and β-glucosidases respectively. New galactose configured cyclosulfates 3 and 4, and galactose configured cyclosulfates biosiosters 5-9 mimic the Michaelis complex 4Ci conformation. a-Ga/-cyclophellitol 10 and a-Ga/-cyclophellitol aziridine 11 inhibit irreversibly a-gal A by mimicking the oxocarbenium ion transition state 4H3.
Figure 2 discloses a preferred reaction scheme for obtaining compounds according to the invention: Reagents and conditions: a) (i) BCI3, DCM, -78 °C, 3.5 h; (ii) BzCI, Pyr, rt, 18 h, 79%; b) RuCI3-3H2O, NalO4, EtOAc, ACN, 0 °C, 2 h, 15: 0% and 16: 78%; c) OsO4, NMO, H2O, acetone, rt, 3 days, 17: 44% and 18: 34%; d) (i) SOCI2, Et3N, imidazole, DCM, 0 °C; (ii) RuCI3, NalO4, CCI4, ACN, 0°C, 3 h, 19: 56%, 20: 69%, 21:82%, 30: 62% over 3 steps and 39: 59% over 3 steps; e) NH3, MeOH, rt, 3 h, 34%; f) H2, Pd(OH)2, MeOH, rt, 18 h, 4: 90%, 5: 92%, 6: 90%, 7: 57%, 8: 28% and 9: 94%; g) m-cpba, DCM, rt, 18 h, 23: 76%, a-epoxide: 19%; h) NaN3, LiCIO4, DMF, 80100 °C, 18 h, 24: 40%, 25: 38% and 42: 69%; i) MsCI, Et3N, DCM, rt, 4 h, 26: 92% and 35: quant; j) H2O, DMF, 140 °C, 3 days, 67%; k) PtO2, H2, THF, rt, 4 h, 28: 80%,33: 99%, 43: 88%; I) Boc2O, Et3N, DCM, rt, 18 h, 29: 99%, 34: 93%; m) TFA, DCM, rt, 8 h, 31: 99% and 40: 71%; n) 1iodooctane, K2CO3, TBAI, DMF, 18 h, 32: 62% and 41: 66%; 0) DMF, 140 °C, 3 days, 47% over 2 steps; p) 1 M NaOH, EtOH, 70 °C, 2 h, 86%; q) SO2(NH2)2, Pyr, reflux, 18 h, 61%.
Figure 3. Crystal structures of a-go/-cyclosulfate 3 and a-ga/-cyclosulfamidate 7 in Fabrazyme. (A) a-ga/-cyclosulfate 3 reacts with Aspl70 nucleophile and adopts a 1S3 covalent intermediate conformation in complex with Fabrazyme®. (B) Unreacted 7 in complex with Fabrazyme® adopts a 4Ci Michaelis complex conformation in the active site.
Figure 4 discloses the effect of comparative compounds α-cyclosulfate 3 and Gal-DNj, and α-cyclosulfamidate 7 on thermal stability and cell culture medium stability of Fabrazyme. A. Heat-induced melting profiles of lysosomal Fabrazyme® recorded by thermal shift. Temperature melting profiles of Fabrazyme® were recorded at optimum pH 4.5 in the presence of α-cyclosulfamidate 7 and Gal-DNJ. The protein (0.5 mg/mL) was heated from 25 to 95° at rc/min in the presence of Sypro Orange. Data were shown as normalized curves. B. Schematic representation of stabilization effect assay. Fabrazyme® was incubated with inhibitor for 15 min in DMEM/F-12 (Ham) medium and subsequently incubated with ConA sepharose beads for 1 h at 4 C and washed (x3) to remove bound inhibitor. a-Gal activity was finally determined by 4-MU-a-Gal assays. C. Percentage of Fabrazyme® residual activity after 15 min incubation in DMEM/F-12 (Ham) medium in the presence of inhibitors 3 and 7 (at 0, 100, 200, 500 μΜ) and Gal-DNJ (0, 1, 10 and 50 μΜ), followed by final ConA purification. Percentages are calculated considering the 100% activity of Fabrazyme® observed at 0 min incubation time (n=2, error bars indicate mean ± standard deviation).
Figure 5 demonstrates the effect of α-cyclosulfamidate 7 and Gal-DNJ in cultured fibroblasts from Fabry disease. A. FD fibroblasts of WT (cl04, classic Fabry (R301X and D136Y) and variant Fabry (A143T and R112H) were incubated with α-cyclosulfamidate 7, Gal-DNJ, Fabrazyme® or the combination of enzyme and chaperone for 24 h. Then, the medium was collected, cells were harvested and α-Gal A activity was measured in the cell homogenates by 4-MU-a-Gal assay. In all cell lines co-administration of α-cyclosulfamidate 7 or Gal-DNJ with Fabrazyme® increased intracellular α-Gal A activity when compared to cells treated Fabrazyme alone. B. α-Gal A activity in cell culture medium samples was measured after ConA purification. a-Gal A activity is at least two times higher in all the cell lines treated with α-cyclosulfamidate 7 (200 μΜ) or Gal-DNJ (at 20 μΜ). Reported activities are mean ± standard deviation from two biological replicates, each with two technical replicates.
Figure 6 discloses Gb3 and Lyso-Gb3 quantification after α-cyclosulfamidate 7 and Gal-DNJ co-treatment with Fabrazyme® in cultured fibroblasts. Gb3 (A) and lysoGb3 (B) levels measured by LC-MS/ MS, in Fabry fibroblasts from WT (cl04), 2 different classic Fabry patient cell lines (R301X and D136Y) and 2 different variant Fabry patient cell lines (A143T and R112H), with 24 h treatment of Fabrazyme® (400 ng/ml of culture medium) with or without α-cyclosulfamidate 7 (200 μΜ) and Gal-DNJ (20μΜ). Reported values are mean ± standard deviation from at least three biological replicates.
DESCRIPTION OF PREFERRED EMBODIMENTS
The therapeutic methods of the invention described herein provide treatment options for the management of various lysosomal storage diseases and glycosidase deficiency related diseases. More specifically, the therapeutic methods of the invention involve various combinations of enzyme replacement therapy for the treatment of lysosomal storage diseases and glycosidase deficiency related diseases. The therapeutic methods involve the use of combinations of:
a) an exogenously produced, natural or recombinant lysosomal hydrolase, preferably an agalactosidase, more preferably a human α-galactosidase, more preferably a recombinant human α-galactosidase, particularly Fabrazyme® (registered trademark of Genzyme Therapeutic Products Ltd. Partnership);
b) a reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase, preferably a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group and derived from a cyllophelitol.
The present invention preferably relates to a composition wherein the reversible glycosidase inhibitor has the structure according to formula I wherein X represents -NfR1)or oxygen, preferably wherein one of X represents -MR1)-, the other X represents oxygen, more preferably wherein the X at the exposition represents-NfR1)- and the X at the C1position represents oxygen, wherein R1 each individually represent H, a lower substituted alkyl such as (CHz)nX wherein X is CH3, OH, NH2,1, Br, Cl or CF3, and n ranges of from 0 to 5; an optionally substituted (hetero)aryl group; or a carboxy group (CO)Z wherein Z represents (CH2)nCH3, -OH, or -NH2; wherein each of R2 to R4 individually represents H, a lower substituted alkyl such as (CH2)nY wherein Y represents -CH3, -OH, -NH2, -I, -Br, -Cl or -CF3, and n ranges of from 0 to 5 and/or an optionally substituted (hetero)aryl group; or a carboxy group (CO)X, wherein X represents (CHzJnCHj, OH, NH2, or CF3 and n = 0-5, and/or an optionally substituted (hetero)aryl group; wherein R5 represents -OH, -CH2OH, -CMO-alkyl, or -CH2O-glycoside; and/or pharmaceutically acceptable salts, solvates, chelates, noncovalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.
Preferably, the present invention relates also to the use of kits comprising a) the exogenously produced, natural or recombinant lysosomal hydrolase or endogenously produced by (multi)gene therapy, natural or recombinant lysosomal hydrolase; b) the reversible glycosidase inhibitor capable of reversibly blocking the active site of the lysosomal hydrolase.
More preferably, the present invention furthermore preferably relates to a composition more preferably an α-gal-cyclic sulfamidate, still more preferably a a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III wherein R1 is H or lower, optionally substituted, alkyl, including but not limited to (CMjnX where X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5; a carboxyl such as (CO)X wherein X is (CMjnCHs, OH, NH2, or CFsand n = 0-5; preferably H.
It is believed that co-administration of a) the exogenously produced, natural or recombinant lysosomal hydrolase or endogenously produced by (multi)gene therapy, natural or recombinant lysosomal hydrolase with b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase can be effective in making it possible to treat a larger number of patient mutations with reduced α-Gal A activity with the lysosomal hydrolase than the pharmacological chaperone alone which would just stabilize specific mutations. Secondly, the stabilization of the lysosomal hydrolase by the reversible glycosidase inhibitor and preferably also 1-deoxygalactonojirimycin may reduce the required enzyme dosages or extend IV injections intervals, and therefore reduce side effects and treatment costs.
Herein, the term effective amount, in relation to delivery of an enzyme to a subject in a combination therapy of the invention, preferably means an amount sufficient to improve the clinical course of a lysosomal storage disease.
According to the application, a subject or patient is a human or non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. Although the animal subject is preferably a human, the compounds and compositions of the application have application in veterinary medicine as well, e.g., for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.
The term cyclophellitol relates to a compound with the IUPAC nomenclature name (l/?,2/?,3R,4S,5R,65)-2-(hydroxymethyl)-7-oxabicyclo[4.1.0]heptane-3,4,5-triol. The term cyclophellitol-derived implies compounds with a cyclohexane ring and various substitutions which may be derived chemically from the cyclophellitol, or synthesized differently, whereby the stereochemistry of the substituents at the carbon atoms of the cyclohexane ring may be varied in line with the required or desired conformation.
The term enzyme replacement therapy or ERT refers to refers to the introduction of a non-native, purified enzyme into an individual having a deficiency in such enzyme. The administered enzyme can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual otherwise requiring or benefiting from administration of a purified enzyme, e.g., suffering from protein insufficiency. The introduced enzyme can be a purified, recombinant enzyme produced in vitro, or enzyme purified from isolated tissue or fluid, such as, e.g., placenta or animal milk, or from plants.
The term adjuvant or adjuvant therapy refers to any additional substance, treatment, or procedure used for increasing the efficacy, safety, or otherwise facilitating or enhancing the performance of a primary substance, treatment, or procedure.
The term co-formulation refers to a composition comprising an enzyme, such as an enzyme used for ERT, for example, an α-Gal A enzyme (e.g., a human recombinant α-Gal A enzyme that is formulated together with a chaperone for the enzyme.
In certain embodiments the chaperone is a compound according to structure (I), or a pharmaceutically acceptable salt, ester or pro-drug thereof. In certain embodiments, treating a subject with the co-formulation comprises administering the co-formulation to the subject such that the α-Gal A enzyme and chaperone are administered concurrently at the same time as part of the co-formulation.
As used herein, the term glycoside relates to a material containing a saccharide structure which may be represented by the formula (IV)
-(OG)Z(IV) wherein O represents an oxygen atom and (G) is a saccharide structure (backbone) of the glycoside. The value z represents the number of monosaccharide units in the glycoside. The oxygen atom is attached to the saccharide in an ether linkage or substituted by a sulfur (S) atom linkage. Where the aldehyde or ketone structure of the saccharide is involved in the glycoside formation the product may be termed an acetal or ketal respectively. The favoured reaction is the acetal or ketal formation to give the glycoside with the oxygen being attached to the carbon in the one position. It is less likely that the hydrophobic moiety will be attached through one of the remaining hydroxyl groups present on the starting saccharide. The glycosides which are suggested for use in the present invention include those selected from the group consisting of fructoside, glucoside, mannoside, galactoside, taloside, aldoside, altroside, idoside, arabinoside, xyloside, lyxoside, iduronide, glucuronide and riboside and mixtures thereof. Preferably the glycoside is a fructoside and most preferably a galactoside or a glucoside. All of the aforementioned glycosides may be obtained from sugars (saccharides) with the preferential fructose and glucose starting materials being obtained from corn syrup. Complex glycosides, those containing one or more different saccharide units, may also be used as starting materials.
The glycoside has the ability to utilize the monomeric saccharide unit to promote chain growth of the glycoside. Thus z in the above formula may vary between 1 and 10, preferably from about 1.2 to about 5, and most preferably from about 1.3 to about 3.5. The value z, may also be referred to as the degree of polymerization (D.P.) of the glycoside. This number is an average degree of polymerization. In obtaining a glycoside, polymerization of the monosaccharide units occurs to some extent usually through a 1,6 linkage. The saccharide portion of the glycoside molecule enhances water solubility and thus for detergent purposes it is advantageous to have the D.P. somewhat greater than 1. It is also desirable for detergents that the hydrophobic moiety have sufficient length to give a proper HLB e.g. C4 and above.
As used herein, the term active site refers to the region of a protein that has some specific biological activity. For example, it can be a site that binds a substrate or other binding partner and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. Active sites in this application can encompass catalytic sites of enzymes, antigen biding sites of antibodies, ligand binding domains of receptors, binding domains of regulators, or receptor binding domains of secreted proteins. The active sites can also encompass transactivation, protein-protein interaction, or DNA binding domains of transcription factors and regulators.
As used herein, the term chaperone refers to a compound that specifically interacts reversibly with an active site of a protein and enhances formation of a stable molecular conformation. As used herein, chaperone does not include endogenous general chaperones present in the ER of cells, or general, non-specific chemical chaperones such as water
The term combination therapy refers to any therapy wherein the results are enhanced as compared to the effect of each therapy when it is performed individually. The individual therapies in a combination therapy can be administered concurrently or consecutively.
The term modified lysosomal enzyme may also comprise endogenously expressed modified naturally occurring enzymes, such as a-/V-acetyl-galactosaminidase (aNAGAL) with increased α-galactosidase activity (a-NAGALEL).AIso herein, the term lysosomal storage disease or glycosidase deficiency related enzymes preferably means any disease that can be treated in accordance with the invention with an exogenously produced natural or recombinant lysosomal hydrolase. Lysosomal storage diseases include the following diseases: Fabry, Gaucher, Pompe, Schindler, Krabbe, mucolipidosis I, NiemannPick, Farber, GMl-gangliosidosis, GM2-gangliosidosis (Sandhoff), Tay-Sachs, Krabbe, Schindler, sialic acid storage, fucosidosis, mannosidosis, aspartylglucosaminuria, Wolman, and neuronal ceroid lipofuscinoses. Glycosidase deficiency related diseases include pathological conditions driven by the deficiency or malfunction of glycosidases, including Parkinson's Disease or Alzheimer related to glucocerebrosidase (GBA) or a- galactosidase malfunction or mutations.
Inherited lysosomal storage disorders (LSDs) in humans are caused by the deficiency in different lysosomal enzymes: Gaucher disease (GD, deficiency in glucocerebrosidase, (GBA), Pompe disease (defiency in acid alpha-glucosidase) and Fabry disease (FD, deficiency in alpha-galactosidase A, α-GAL A), Krabbe disease (defieciency in beta-glucosidase), Schindler (deficiency in a-/V-acetylgalactosaminidase (α-NAGAL)), mucopolisacaridosis I (deficiency in α-L-iduronidase) among others. Enzyme replacement therapies (ERT) are in use with variable success depending on the disease.
The deficiency in α-galactosidase A (α-gal A) generates a LSD known as Fabry disease which is characterized by the toxic accumulation of glycosphingolipid globotriaosylceramide (Gb3) in lysosomes and globotriaosylsphingosine (Lyso-Gb3) in plasma.
The term Fabry disease refers to classical Fabry disease, late-onset.
Fabry disease, and hemizygous females having mutations in the gene encoding agalactosidase A (α-gal A). The term Fabry disease, as used herein, further includes any condition in which a subject exhibits lower than normal endogenous α-gal A activity.
Various α-Gal A mutations lead to a wide range of different phenotypes, classified as classic and variant Fabry patients, with diverse enzyme and metabolite levels. The accumulation of glycosphingolipid metabolites is thought to be the basis of progressive renal and cardiac insufficiency, and CNS pathology in Fabry patients.
Although intravenous enzyme replacement therapy (ERT) ameliorates Gb3 and lysoGb3 metabolite levels, its long term clinical efficacy is relatively reduced and patients develop neutralizing antibodies upon the recombinant enzyme that can result in inhibition of the ERT. In addition, Fabrazyme® biweekly intravenous (IV) dosage of Fabrazyme® can result in intermittent efficacy of the treatment, and high rate of infusion is associated with stroke incidents.
Recently, a pharmacological chaperone, 1-deoxygalactonojirimycin (Gal-DNJ, Migalastat®) which binds to some specific mutant forms of α-gal A and restores some lysosomal activity, has been approved as a new treatment for Fabry patients with amenable mutations in United States of America, Europe, Australia and Canada among others.
This alternative to intravenous enzyme replacement therapy (ERT) is, however, limited to certain specific mutations and its approval by the FDA is still under consideration.
In addition, α-Gal A deficiency has recently has been detected in late-stage Parkinson's disease (PD) brains and associated to the aberrant accumulation of a-synuclein (a-syn).
On the other hand, carriers of a GBA defect or mutation do not develop its corresponding lysosomal storage disorder (Gaucher Disease, GD) but show a markedly increased risk for PD and Lewy-body dementia. It is still a matter of debate the interconnection between increased levels of Gb3 isoforms cause by deficiency in α-gal A and/or GBA and the pathological accumulation of α-syn. Importantly, α-Gal A positive modulators or PCs may be important therapeutic and biological tools not only for Fabry disease but also for Parkinson's disease.
Carbohydrates are metabolized by their processing enzymes such glycosidases (GHs, enzymes that hydrolize a glycosidic bond) or glycosyltransferases (GTs, enzymes that generate a glycosidic linkage). Glycosidase inhibition or stimulation with pharmacological chaperones (PCs) is the basis of many therapeutic strategies for the treatment of different diseases such as diabetes type II, viral infections, cancer or lysosomal storage disorders among others.
Most glycosidases hydrolyse their substrates through an overall configurational retaining reaction known as Koshland double displacement mechanism.
However, although different GHs and GTs process their substrate through the same mechanistic reaction, many accomplish this by following different conformational itineraries. This is the case for example of a- and β-retaining glucosidases which hydrolyse their substrates through the same Koshland double displacement mechanism but follow opposite conformational reaction itineraries and for which the Michaelis complex -> TS -> intermediate enzymatic half reactions are 4Ci -> 4H3* -> ^3 in α-glucosidases and ^3 -> 4Η3* -> 4Ci in β-glucosidase.
Transition state analogues mimicking the oxocarbenium ion transition state by their half chair conformations have proved to be an important class of glycosidase inhibitors. This transition state mimicry exerted by for example cyclophellitol (aziridines) has also turned into promiscuous glycosidase inhibition, an advantage for the development of broad spectrum glycosidase activity-based probes.
Applicants have now found a novel class of selective and potent glucosidase irreversible inhibitors which favours 4Ci chair conformations are a- and β-cyclophellitol cyclosulfates 1 and 2, respectively (see Figure IB).
However, these compounds which are mimicking both the anomeric stereochemistry and the initial Michaelis complex conformation of the target enzyme, otand β-glucosidase respectively, have a far higher selectivity than cyclophellitol or cyclophellitol aziridine irreversible inhibitors, and thus α-cyclosulfates 1 exhibits exquisite selective inhibition of gastrointestinal α-glucosidase specially relevant for the treatment of type II diabetes. β-Cyclosulfate 2, which also adopts a 4Ci different from the % conformation of typical β-glucosidase Michaelis complexes, reacts much slower than its congener 1, but still inhibits β-glucosidases in the micromolar range. However, the inhibition was surprisingly found to be permanent, at least under the conditions that one expected in a patient's body.
Surprisingly, it was found that in kinetic and activity in vitro studies that whereas ago/-cyclosulfate 3 irreversibly inhibits α-Gal A, a-go/-cyclosulfamidate 7 according to the invention reversibly binds the enzyme and thereby, as demonstrated by in vitro and in situ cell experiments, a-ga/-cyclosulfamidate 7 promotes the stabilization of the human α-gal A in cell culture and consequently increases the lysosomal uptake of a-Gal A.
Accordingly, a composition comprising a-ga/-cyclosulfamidate 7 appears a novel and entirely superior combination ERT therapy for Fabry disease, as well as for the study of a-gal A deficiency linked to PD. Furthermore, the same principle applies to all lysosomal disorders disclosed herein, whereby the chaperone preferably is chosen to mimic the substrate of the respective enzyme.
Yet further, the present compounds may also be employed to stabilize mutant endogenously produced enzymes, such as modified lysosomal a-/V-acetyl-galactosaminidase (α-NAGAL) with increased α-galactosidase activity (a-NAGALEL) expressed after gene therapy, or by cells introduced to a patient and capable of expressing such enzyme.
According to the present application, compounds according to the invention can be administered as the free base or as a pharmacologically acceptable salt form. It can be administered in a form suitable for parenteral administration, including e.g., in a sterile aqueous solution for intravenous administration. The compounds and compositions of the application can be formulated as pharmaceutical compositions by admixture with a pharmaceutically acceptable carrier or excipient.
In certain embodiments, compounds according to the invent and the enzyme, in particular α-Gal A are formulated together in a single composition, i.e., co-formulated together. Such a composition enhances stability of the enzyme both during storage (i.e., in vitro) and in vivo after administration to a subject, thereby increasing circulating half-life, tissue uptake, and resulting in increased therapeutic efficacy of the enzyme. The co formulation is preferably suitable for intravenous administration.
The present application may feature liquid pharmaceutical co-formulations (e.g., formulations comprising α-Gal A and a compound according to the invention, having improved properties as compared to art-recognized formulations.
In certain embodiments, co-formulations of the application include an enzyme and compounds according to the invention that are suitable for intravenous administration.
In certain embodiments, the co-formulation composition comprises a lysosomal enzyme at a concentration of between about 0.05 and about 100 pM, or between about 0.1 and about 75 pM, or between about 0.2 and about 50 pM, or between about 0.3 and about 40 pM, or between about 0.4 and about 30 pM, or between about 0.5 and about 20 pM, or between about 0.6 and about 15 pM, or between about 0.7 and about 10 pM, or between about 0.8 and about 9 pM, or between about 0.9 and about 8 pM, or between about 1 and about 7 pM, or between about 2 and about 6 pM, or between about 3 and about 5 pM.
In certain embodiments, the co-formulation composition comprises compounds according to the invention at a concentration of between about 10 and about 25,000 pM, or between about 50 and about 20,000 pM, or between about 100 and about 15,000 pM, or between about 150 and about 10,000 pM, or between about 200 and about 5,000 pM, or between about 250 and about 1,500 pM, or between about 300 and about 1,000 pM, or between about 350 and about 550 pM, or between about 400 and about 500 pM.
Concentrations and ranges intermediate to the above recited concentrations are also intended to be part of this application.
In certain embodiments, the co-formulation composition comprises compounds according to the invention at a concentration of between about 0.002 and about 5 mg/mL, or between about 0.005 and about 4.5 mg/mL, or between about 0.02 and about 4 mg/mL, or between about 0.05 and about 3.5 mg/mL, or between about 0.2 and about 3 mg/mL, or between about 0.5, and about 2.5 mg/mL, or between about 1 and about 2 mg/mL.
Design and synthesis of comparative α-D-galactosidase cyclosulfates 3 and 4 was investigated. Based on results with a-glu and β-g/u-cyclosulfates 1 and 2, applicants investigated if an α-galactose configured cyclosulfate 3 would be an effective inhibitor of agalactosidases which follow the same 4Ci -> 4Hs 1Ss conformational trajectories by mimicking the initial Michaelis complex conformation. It was found that 3-ga/-cyclosulfate 4 would be conformationally excluded from both a- and galactosidase reaction itineraries and therefore a poor inhibitor (Figure 1).
Additionally, applicants synthesized the novel compounds according to the invention, namely cyclosulfate bioisosteres α-gal- cyclosulfamidates 5-8, as well as cyclosulfamide 9.
The present invention also preferably relates to a process for the preparation of inhibitor compound. In a preferred embodiment first, perbenzylation of starting cyclohexene 14 and subsequent oxidation with OsCU gave diols 15 and 16 as a non-separable mixture of α/β with a 1:4 ratio (Figure 2A).
In parallel, oxidation with RuCh and sodium periodate by in situ formation of RuCk exclusively afforded β-diol 16.
Oxidation reactions on acetylated cyclohexene (following either OsO4 or RuO4 protocol) yielded a mixture of multiple compounds probably due to acetyl migration.
Interestingly, stereoselective oxidation was achieved on perbenzoylated cyclohexene 17. Thus, oxidation with OSO4 afforded a separable mixture of diols 17 and 18 in a 1:0.6 ratio, whereas RuCh and sodium periodate conditions yielded a 0.5:1 ratio of pure a- and β- diols after column chromatography.
Benzoylated diols 17 and 18 were then treated with thionyl chloride and subsequently oxidized to the cyclic sulfate to afford the protected cyclosulfates 19 and 21. Deprotection of β-analogue 21 with different mild/basic conditions (i.e. NH3 in MeOH, KCN or EtsN/HzO under reflux) resulted in the E2-elimination product 22.
a-ga/-cyclosulfate 3 was obtained after benzoyls removal in methanolic ammonia, βGal-cis-cyclosulfate 4 was alternatively synthesized from the perbenzylated β-cyclosulfate 20 after hydrogenation in the presence of Pearlman's catalyst.
Synthesis of α-D-galactosidase cyclosulfate bioisosteres 5-9 according to the invention
Applicants also explored the alkylation of cyclosulfamidates 5 and 7 with an octyl chain: analogues 6 and 8.
In a preferred embodiment, synthesis of cyclosulfamidates 5 and 6 started from cis-1hydroxy-6-azido cyclohexene 27, which was obtained by first nucleophilic addition of sodium azide to β-cyclophellitol 23 followed by mesylation and subsequent inversion of the stereochemistry of the secondary alcohol with optimal yields (Figure 2B). Azido intermediate 27 was reduced and ensuing Boc protection afforded intermediate 29, which was treated with thionyl chloride under basic conditions to form a mixture of sulfite enantiomers that were further oxidized to the boc-protected cyclosulfamidate 30.
Cyclosulfamidate 30 was deprotected with TFA and intermediate 31 was either alkylated with 1-iodooctane and/or directly deprotected by hydrogenolysis to afford the desired final cyclosulfamidates 5 and 6.
In a preferred embodiment, synthesis of sulfamidate 7 and 8 was performed using a strategy involving intramolecular /V-Boc participation to facilitate oxazolidinone 36 formation with simultaneous hydroxyl inversion, and subsequent deprotection yielded the desired cisl-amino-6-hydroxy cyclohexene 37. Then, cyclic sulfamidate synthesis from boc-protected analogue 38, followed by boc deprotection with TFA and either alkylation with 1-iodooctane and/or directly deprotection by hydrogenation in the presence of Pd(OH)2 catalyst afforded desired final cyclosulfamidates 7 and 8.
In a preferred embodiment, cis-diamino 43 was synthesized by nucleophilic addition of sodium azide to mesylated intermediate 26 and subsequent reduction with Ηζ/ΡϊςΟ with excellent yields. Then, treatment with SOzfNFhh in pyridine under reflux to create the cyclic sulfate and benzyl removal by hydrogenation afforded final cyclosulfamide 9.
α-D-Galactosidase cyclosulfate 3 and bioisosteres 7 and 9 inhibit a-gal A in vitro Inhibition and selectivity in pure a- and β-galactosidases (human recombinant agalactosidase, Fabrazyme® from Genzyme, GH27 and purified bacterial β-galactosidase homologue from Cellvibrio japonicus C/GH35A, GH35) was investigated. As initial screening, we determined their apparent IC50 values by using commercial 4-methylumbelliferyl (4MU) a- or β-galactose as substrate at the optimal pH 4.5 (Table 1).
It was found that a-go/-cyclosulfate 3 effectively inhibited a -galactosidase on a par with a-gal-cyclophellitol 10 (IC50 = 25 vs 11 μΜ respectively in Fabrazyme), although resulting in a much weaker inhibitor than α-gal-cyclophellitol aziridine 11 (IC50 = 40 nM). β-cyclosulfate 4 was inactive against β- or α-galactosidase up to 1 mM.
Interestingly, the replacement of the electrophilic cyclic sulfate by a cyclic sulfamidate moiety (compounds 5 and 7) was just tolerated when the NH group was placed close to the anomeric position, suggesting that H bonding may probably be taking place in the neighboring of the acid/base amino acid. The instalment of a NH moiety on the catalytic amino acid site indeed seems detrimental for activity, whereby sulfamidate 5 and sulfamide 9 inactive or massively weak α-galactosidase inhibitors. Remarkably, whereas introduction of an alkyl chain in the aziridine scaffold results in an increase in α-gal A activity, alkylation of sulfamidates 5 and 7 was detrimental for α-galactosidase inhibition (ICso of 6 and 8 > 1 mM).
To study the selectivity of these compounds apparent ICso values were measured in βgalactosidase C/GH35A. Remarkably, following the same trend as glucose configured cyclosulfates, cyclosulfate 3 present an advantage in specificity when compared to aziridine or cyclophellitol type inhibitors, by virtue of mimicking a reactive 4Ci conformation not shared between a- and β-galactosidases (compound 3 inactive up to 1 mM whereas 10,11 and GalDNJ present apparent IC50 values of 18, 0.57 and 331 μΜ respectively in β-galactosidase C;GH35A). Additionally, aziridine 11 and Gal-DNJ showed off-target inhibition of a-Nacetylgalactosaminidase (α-NAGAL) in plasma samples with apparent ICsos of 137 and 15.2 μΜ respectively, as disclosed in Table 1.
Table 1 below shows the apparent IC50 values for in vitro inhibition of human recombinant α-galactosidase A (Fabrazyme), a-A/-acetylgalactosaminidase ((α-NAGAL)) in human plasma and bacterial β-galactosidase homologue (C/GH35A).
Inactivation rates and inhibition constants (Mnact and /<j) in human recombinant agalactosidase (Fabrazyme®); N.D., not determined; adue to weak inhibition; bdue to fast inhibition; N.I., no inhibition observed; *reversible inhibition observed. Reported values are mean ± standard deviation from 3 technical replicates.
Table 1: Apparent IC50 values for in vitro inhibition of human recombinant α-galactosidase A (Fabrazyme), a-/V-acetylgalactosaminidase (α-NAGAL) in human plasma and bacterial βgalactosidase homologue (C/GH35A).
Compd | In vitro α-gal A (Fabrazyme) apparent IC50 (μΜ) | In vitro a-NAGAL apparent IC50 (μΜ) | In vitro β-gal (QGH35A) apparent IC50 (μΜ) | Kinetic Parameters α-gal A (Fabrazyme) kinact(min· ^and ki (μΜ'1) | Kinetic Parameters α-gal A (Fabrazyme) kinact/ki (min' 1μΜ'1) |
3 | 25 ±2 | >200 | >1000 | Kj = 237 k. , = 0.06 inact | 0.25 |
4 | >1000 | >200 | >1000 | N.I. | N.I. |
5 | >1000 | >200 | 250 ± 1 | N.I. | N.I. |
6 | >1000 | >200 | 55% inh. (ImM) | N.I. | N.I. |
7 | 67 ±5 | >200 | >1000 | K' = 110* | - |
8 | >1000 | >200 | >1000 | N.I. | N.I. |
9 | 423 ± 58 | >200 | 331 ±9 | N.D.a | N.D? |
10 | 11 ±0.8 | >200 | 18 ±0.1 | Kf = 430 ^aCt = 0-24 | 0.55 |
11 | 0.040 ± 0.005 | 137 ±8.70 | 0.57 ± 0.02 | N.D? | 16.4 |
Gal- DNJ | 0.079 ± 0.004 | 15.2 ±8.74 | 331 ±21 | Kt = 0.23 ± 0.09* | - |
The reversibility character of the inhibition of this family of compounds was studied, first by time dependent inhibition studies and afterwards determining the kinetic parameters of the inhibition in human α-galactosidase. Thus, compounds at concentrations above their corresponding apparent IC50 values were incubated for different times (30, 60,120, 240 min) and subsequent fluorescent readout with 4-methylumbelliferyl-a-galactopyranose allowed the quantification of the residual enzyme activity (Figure S3). Interestingly, whilst irreversible inhibition (a decrease in «-galactosidase activity with longer incubations) was clearly observed with cyclosulfate 3, cyclosulfamide 9, epoxide 10 and aziridine 11; cyclosulfamidate 7 showed a constant residual activity with different incubation times, pointing to a noncovalent inhibition of Fabrazyme. This was demonstrated by kinetic studies monitoring the absorbance generated by the hydrolysis of 2,4-dinitrophenyl-a-D-galactopyranoside substrate (2,4-DNP-a-Gal) at pH 4.5 (Table 1). Irreversible cyclosulfate 3, epoxide 10 and aziridine 11 follow pseudo-first order kinetics and kinetics parameters of cyclosulfamide 9 could not be measured due to very slow/weak inhibition. Although α-cyclosulfate 3 presents a similar kmaa/K\ ratio than a-cyclophellitol 10 (Amact/AG = 0.25 vs 0.55 min^mM1, respectively), inhibitor 3 shows a stronger initial binding constant (/<) and a slower inactivation rate constant (kinact) than 10 (3: K\ - 237 μΜ and kinact = 0.06 min 1 vs 10: Kt = 430 μΜ and kinact = 0.24 min J, and only a k\ma!K\ ratio could be measured for a-aziridine 11 due to fast inhibition (kinact/ki = 16.4 min^mivr1). Reversible kinetics with increasing 2,4-DNP-a-Gal concentrations demonstrated that cyclosulfamidate 7 reversibly inhibits α-galactosidase with a K = 110 μΜ. Structural analysis of a-qa/-cyclosulfate 3 and a-qa/-cyclosulfamite 7 in complex to Fabrazyme.
Crystal structures of a-ga/-cyclosulfate 3 and a-qa/-cyclosulfamidate 7 in complex with the recombinant human α-gal A (Fabrazyme) were obtained (Figure 3). It was observed that a-ga/-cyclosulfate 3 (Figure 3A) reacts with Aspl70 nucleophile and adopts a χ$3 covalent intermediate conformation in complex with Fabrazyme®. On the other hand, unreacted (Figure 3B) binds reversibly and adopts a 4Ci Michaelis conformation in the active site.
Also herein, the term lower alkyl preferably means a saturated, branched or straight chain hydrocarbon group of 1 to 6 carbon atoms, preferably one to three carbon atoms, such as a methyl, isopropyl or n-pentyl group.
Substituted lower alkyl preferably means substitute lower alkyl groups, such as (CHjjnX, wherein X is CH3, OH, NH2,1, Br, Cl or CF3 and n = 0-5. Particularly preferred compound are those wherein R1 is H or an optionally substituted lower alkyl group such as (CO)X where X is (CH2nCH3, OH, NH2, or CF3 and n = 0-5. Lower alkyls herein in includes straight or branched chains. Examples of such alkyls include alkyl chains containing from 1 to about 30, preferably from about 1 to about 20, and most preferably from about 2 to about carbon atoms. Specific alkyls which may be utilized herein include methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonal, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.
Any conventional method can be used to monitor the status of lysosomal storage disease of a patient being treated in accordance with this invention and the effectiveness of a combination therapy of the invention on the disease. Clinical monitors of disease status can include but are not limited to organ volume (e.g. liver, spleen), hemoglobin, erythrocyte count, haematocrit, thrombocytopenia, cachexia (wasting), and plasma chitinase levels (e.g. chitotriosidase). Chitotriosidase, an enzyme of the chitinase family, is known to be produced by macrophages in high levels in subjects with lysosomal storage diseases (see: Guo et al., 1995, J. Inherit. Metab. Dis. 18, 717-722; den Tandt et al., 1996, J. Inherit. Metab. Dis. 19, 344-350; Dodelson de Kremer et al., 1997, Medicina (Buenos Aires) 57, 677-684; Czartoryska et al., 2000, Clin. Biochem. 33,147 149; Czartoryska et al., 1998, Clin. Biochem. 31, 417-420; Mistry et al., 1997, Baillieres Clin. Haematol. 10, 817-838; Young et al., 1997, J. Inherit. Metab. Dis. 20, 595-602; Hollak et al., 1994, J. Clin. Invest. 93,1288-1292).
Methods and formulations for administering a combination therapy of the invention to a patient include all conventional methods and formulations (see: e.g. Remington's Pharmaceutical Sciences, 1980 and subsequent years, 16th ed. and subsequent editions, A. Oslo editor, Easton Pa.; Controlled Drug Delivery, 1987, 2nd rev., Joseph R. Robinson & Vincent H. L. Lee, eds., Marcel Dekker, ISBN: 0824775880; Encyclopedia of Controlled Drug Delivery, 1999, Edith Mathiowitz, John Wiley & Sons, ISBN: 0471148288; U.S. Pat. No. 6,066,626 and references cited therein).
In the combination therapy of the invention, the lysosomal hydrolase is administered intravenously to a patient to initiate treatment (i.e. to de-bulk the subject). The initial treatment can include administration also of the reversible glycosidase inhibitor. Thereafter, treatment with the lysosomal hydrolase, the reversible glycosidase inhibitor can be administered to the patient over a one to two-hour period on a weekly or bi-weekly basis for one to several weeks or months, or longer.
In certain embodiments, the foregoing combination therapy may also provide an effective reversible inhibition of an enzyme selected from the group consisting of glucocerebrosidase, sphingomyelinase, ceramidase, GMl-ganglioside-8-galactosidase, hexosaminidase A, hexosaminidase B, β-galactocerebrosidase, α-L-iduronidase, iduronate sulfatase, heparan-/V-sulfatase, /V-acetyl-a-glucosaminidase, acetyl CoA:a-glucosaminide acetyl-transferase, α-galactosidase A, a-/V-acetylgalactosaminidase, α-glucosidase, afucosidase, α-mannosidase, aspartylglucosamine amidase, acid lipase, and palmitoyl-protein thioesterase (CLN-1).
Still further, in certain embodiments, the foregoing composition, and combination therapy method may produce a diminution in at least one stored material selected from the group consisting of glucocerebroside, sphingomyelin, ceramide, GMl-ganglioside, GM2ganglioside, globoside, galactosylceramide, glycolipids, glycosphingolipids, globotriaosylceramide, O-linked glycopeptides, glycogen, free sialic acid, fucoglycolipids, fucosyloligosaccharides, mannosyloligosaccharides, aspartylglucosamine, cholesteryl esters, triglycerides, and ceroid lipofuscin pigments.
In certain embodiments, the present specification also relates to a process or method for preparing the compounds (I) to (III) via the reaction pathway as set out below.
The following examples illustrate the present invention further.
EXAMPLES
Example 1: Synthesis of Compound 7 according to the invention
Synthesis of compound 7, herein below, i.e., the a-gal 1,6 cyclophellitol cyclosulfamidate of formula I wherein R is H, via compounds 24 and 38 is disclosed in the flow chart below and starting from compound 14 (see: Y. Harrak Chem. Soc.2011,133, 12079-12084 and L. I. Willems et al, European J. Org. Chem.2014, 2014, 6044-6056) in the flow chart below (and Figure 2):
ΌΒη
DCM, rt, 18 h, 76% m-cpba Bn°
NaN3 LiCIO4 0Bn DMF, 80 C, 18 h OBn
N3
OBn
1) PtOz, H2
TH F, rt, 4 h
2) Boc2O, Et3N DCM, rt, 18 h
24; 40% 25: 38%
O O
O-S;° O-Sh°
BnO^A 'NH HZ’ pd(OH)2 H0^A'NH ΒηΟ'γ'ΌΒη MeOH, rt, 18 h ΗΟ*ηρ 'ΌΗ
OBn OH 7
1)1 M NaOH EtOH, 70 °C, 2 h
2) Boc2O, Et3N DCM, rt, 18 h
OH
.NHBoc 'OBn
OBn
1) SOCI2, Et3N imidazole, DCM, 0 °C
2) RuCI3, NalO4 CCI4, ACN, 0°C, 3 h
Example 2: Thermostability of Fabrazyme® in the presence of compound 7 (as compared to Migalastat)
Fabrazyme® usually presents poor stability in plasma (pH 7.4) and only ~25% of the hydrolytic activity remains after incubation of 1 pg/mL pure enzyme in human plasma for 15 minutes. As result, a very small amount of the injected Fabrazyme® dosage actually reaches the intracellular targeted lysosome. The enormous quantity of required recombinant enzyme is a major drawback of ERT which can be reflected in increased development of neutralizing antibodies in treated patients and elevated costs of the required treatment.
Appropriately configured iminosugars are known to stabilize diverse lysosomal glycosidases with so called pharmacological chaperone (PC) effect.
Ideally, a PC for glycosidases should reversibly bind its enzyme in plasma circulation and in the endoplasmic reticulum at neutral pH, stabilizing it during its transport to the lysosomes and upon dissociation in the acidic compartment aided by the excess of substrate, the enzyme hydrolyzes its natural substrate Gb3.
Thermal shift assays (TSAs) can be used for high-trough put screening of PCs and it has been suggested that the recently approved 2-O-a-D-Galactopyranosyl-ldeoxynojirimycin (Migalastat) is not the ideal PC therapy for Fabry disease, as it stabilizes the enzyme under neutral and acidic conditions. However, the high concentrations of substrate accumulated in the lysosomes aid the displacement of the PC by the natural substrate.
Heat-induced melting profiles of lysosomal Fabrazyme® recorded by thermal shift at pH 4.5, 5.5 and 7.4 showed that Fabrazyme® is more thermostable at pH 5.5.
A comparison whether compound 7, as compared to Migalastat had a stabilization effect on Fabrazyme® was investigated by measuring the heat-induced melting profiles of lysosomal α-gal A at different pHs 4.5, 5.5 and 7.4.
TSAs showed that compound 7 stabilizes Fabrazyme® at pH 7.4 with a ATrnmax of 17.4 °C whereas Migalastat presented a slightly higher thermal stability effect with a ATrnmax of
34.3 °C (Figure 4A, Figure S4).
Thermostability was also measure at acid pH mimicking the lysosomal environment and the stabilization of Fabrazyme® was slightly lower in both cases with ATrnmax of 9.3 and
22.3 °C at pH 5.5 for compound 7 and Migalastat, respectively.
It was then investigated whether compound 7 or Migalastat had stabilization effect on Fabrazyme in Dulbecco's Modified Eagles Medium/Nutrient Mixture F-12 (DMEM/F12, Sigma-Aldrich) medium, supplemented with 10 % fetal calf serum and 1 % penicillin/streptomycin at (pH 7.2), mimicking fibroblasts cell culture conditions. After 15 min incubation of Fabrazyme® (5 mg/mL) in cell culture medium, 80% degradation was observed and this time point was used for further analysis. Thus, compound 7 and Migalastat were incubated with Fabrazyme® (5 mg/mL) at increasing concentrations (starting around the IC50 value) of the corresponding inhibitor in cell medium conditions at pH 7.2 for 15 min and 0 min (for degradation quantification). Samples were then incubated for 1 h at 4 °C with Concanavalin A (ConA) sepharose beads which are known to bind a-Gal A, and washed (3x) to remove the inhibitor bound to a-Gal A. Quantification of agalactosidase activity was then performed via 4MU-a-Gal assays.
In line with TSAs results, compound 7 and Migalastat reversibly bind a-Gal A, preventing its degradation under cell culture conditions at pH 7.2. A comparable value of about 75% stabilized residual α-gal-A activity was observed after 15 min incubation with compound 7 (500 μΜ), or with Migalastat (50 μΜ), whereas just 20% activity remained with no inhibitor.
Example 3
In situ treatment of cultured fibroblasts from patients with Fabry disease.
It was investigated if compound 7 or Migalastat would stabilize α-Gal A activity in situ. In situ studies were carried out in 5 different male cell lines: wild type fibroblast (WT, cl04) which present normal α-Gal A activity, 2 classic Fabry patients fibroblasts (R301X and D136Y mutations) with no α-Gal A activity and 2 variant Fabry patients fibroblasts in latter onset disease (A143T and R112H mutations) with substantially lowered, yet high, enzyme activity that exhibit almost normal plasma Lyso-Gb3 concentration.
Of note, the R112H mutation present a large structural change on the molecular surface but its active site remains intact and the α-Gal A activity and Gb3 and lysoGb3 levels may vary within patients. On the other hand, A143T mutation within the same family also presents diverse activity and metabolites levels.
Fabry disease fibroblasts were incubated with compound 7 (200 μΜ) or Migalastat (at 20 μΜ), and Fabrazyme® (400 ng/ml), or with a combination of all three. After 24 h the cells were harvested, homogenized and the intracellular activity of α-Gal A was measured of the corresponding cell lysates. As expected WT cell line (cl04) presented normal α-Gal A activity whereas untreated classic Fabry patients (R301X and D136Y) and variant mutation samples (A143T and R112H) showed reduced enzymatic activity. None of the cell lines, not classical Fabry fibroblasts R301X and D136Y, showed significant increase in α-Gal A activity when incubated with the inhibitors alone for 24 h, indicating that longer incubation times or higher concentrations may be required to observe this effect (3-5 days for Migalastat at 200 μΜ, see Asano et al, 1995, Eur. J. Biochem. 2000,267, 4179-4186).
Treatment with Fabrazyme® showed a considerable increase in α-Gal A activity in all the studied cell lines. Interestingly, this effect was improved in most cases with the combinatory treatment of Fabrazyme® and PCs after 24 h incubation. In order to investigate whether this increase in α-Gal A activity is due to a stabilization effect on Fabrazyme®, we also measured α-Gal A activity in the medium. Thus, the culture medium was collected before harvesting the cells and α-Gal A activity was measured after ConA purification to remove the bound inhibitor and subsequent 4-MU α-Gal assay. α-Gal A activity was at least two times higher in all the cell lines treated with compound 7 (200 μΜ) or Migalastat (at 20 μΜ), demonstrating that compound 7 and Migalastat prevent Fabrazyme® degradation in cell culture medium.
Gb3 metabolites correction
Normally, Fabry patients present elevated Gb3 levels which can be further metabolized by acid ceramidase into lysoGb3 in lysosomes. These metabolites, which increase with the age of the patient, constitute a distinguishing feature of Fabry disease diagnosis and progression. These metabolites are known to be responsible for the disease manifestations such as neuronophatic pain and renal failure by affecting nociceptive neurons and podocytes.
It was therefore further investigated whether the co-administration of compound 7 and Migalastat with Fabrazyme® would also have a positive effect on the correction of these toxic metabolite levels.
Thus, Gb3 and lysoGb3 levels were measured in the in situ treated cell lysates by LCMS/MS. Normal physiologic Gb3 and lysoGb3 levels are observed in wild type samples (around 1000 pmol/mL and 1 pmol/mL of Gb3 and lysoGb3, respectively). In line with the ERT efficacy, cultured fibroblasts from classic Fabry patients (A143T and D136Y) treated with Fabrazyme® resulted in a reduction of the accumulating Gb3 and is deacylated metabolite lysoGb3.
Interestingly, this reduction was similar when fibroblasts were treated with the combination of α-cyclosulfamidate 7 (200 μΜ) and Fabrazyme®, or Gal-DNJ and Fabrazyme® co-treatment. Variant Fabry cells lines, A143T and R112H, showed instead different patterns. A143T cell line presents normal Gb3 and lysoGb3 basal levels, whereas in R112H fibroblasts these metabolites are increased and they are not corrected by Fabrazyme® itself nor by chaperone-Fabrazyme co treatment.
It has, therefore, been found by applicants that cyclosulfamidates such as compound 7 react reversibly with α-gal A and stabilized the enzyme in vitro and in situ. The in vitro activity and selectivity (ICso values and kinetic parameters) and the thermostability results show that compound 7 and Migalastat are able to stabilize Fabrazyme®.
In vitro incubation of Fabrazyme® in fibroblast culture medium at neutral pH with further purification with ConA beads also showed that compound 7 and Migalastat stabilize Fabrazyme®.
This effect also occurred in in situ cell experiments where increased α-gal A activity was observed in the medium of the cells co-treated with Fabrazyme® and with compound 7 and Migalastat, and this is translated into at least double amount of active Fabrazyme remaining in the cell culture medium, and slightly higher activity in the cell lysates after 24 h incubation.
Regarding toxic accumulation of Gb3 and lysoGb3 metabolites during treatments with Fabrazyme®, we found that the combination of Fabrazyme® and compound 7 treatment in culture fibroblasts has a similar effect when compared to Fabrazyme® alone or Fabrazyme®/Migalastat co-treatment after 24 h incubation.
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