WO2011146804A2

WO2011146804A2 - Gm3 ganglioside replacement therapy

Info

Publication number: WO2011146804A2
Application number: PCT/US2011/037286
Authority: WO
Inventors: Jay S. Schneider; Matthew Michael Kremer; Kevin A. Strauss
Original assignee: Lazarus Therapeutics, Inc.
Priority date: 2010-05-20
Filing date: 2011-05-20
Publication date: 2011-11-24
Also published as: WO2011146804A3

Abstract

A method of treating individuals lacking the enzyme GM3 synthase, The method includes the use of purified GM3 ganglioside, derived from natural sources, as a replacement therapy. The GM3 can be administered by multiple routes including injection and intranasal delivery. The GM3 source can be dairy, synthetic or tissue.

Description

GM3 GANGLIOSIDE REPLACEMENT THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application seeks priority to U.S. provisional application 61/346,723 filed May 20, 2010, herein incorporated by reference in its entirety. The application is related to PCT Patent Application Nos. PCT/US2010/047520, PCT/US2010/047522, PCT/US2010/047524, PCT/US2010/047527, PCT/US2010/047528, filed September 1, 2010, based on US Provisional applications 61/238,712, 61/238,775, 61/238,726, 61/238,748, and 61/238,735 respectively, each of which is incorporated by reference in its entirety.

FIELD

[0002] Described herein are methods of using purified GM3 ganglioside as replacement therapy in individuals lacking the enzyme GM3 synthase.

BACKGROUND

[0003] Gangliosides are glycosphingolipids produced mostly in ganglions especially at nerve endings and are involved in cell-signaling function. Ganglioside GM3 is the simplest ganglioside with the inclusion of two sugars (i.e., glucose, galactose) and a single sialic acid. Ganglioside GM3 is a common precursor of the major ganglio-series ganglio-sides, and is distributed in almost all mammalian tissues. GM3 is synthesized by the transfer of sialic acid from CMP-sialic acid to non-reduced terminal galactose residue of lactosylceramide through the alpha 2,3 glycosyl bond, and the reaction is catalyzed by GM3 synthase (CMP-NeuAc:lactosylceramide alpha 2,3-sialyltransferase: EC 2.4.99.9). The enzyme is active at the branch point of the extension of the sugar chain on glycosphingolipids, and the regulatory expression of SAT-I activity is considered to affect the biosynthesis not only for ganglio-series but also for lacto/neolacto-,globo- and/or isogloboseries glycosphingolipids.

[0004] There are a number of human disorders characterized by disruption of ganglioside biosynthesis. For example, a nonsense mutation in S1AT9 on chromosome 2pl2-pl l .2 spanning 5.1 cM, results in the premature termination of the Gm3 synthase enzyme. See for example, Simpson et al, Nature Genetics, 2004; 36: 1225-1229. This study identifies an autosomal recessive infantile-onset symptomatic epilepsy syndrome associated with development stagnation and blindness. Biochemical analysis of plasma glycosphingolipds confirmed that affected individuals lack GM3 synthase activity. Also see Proia, Nature Genetics, 2004; 36: 1147-1148.

[0005] GM3 synthase is an enzyme that catalyzes the initial step in the biosynthesis of complex gangliosides from lactosylceramide. Affected individuals lack GM3 synthase activity and a complete lack of GM3 ganglioside and its downstream biosynthetic derivatives, primarily the a- and b- series gangliosides, including GM2, GMla, GDI a, GTla, GD3, GD2, GDlb, GTlb, and GQlb. There is also an accumulation of lactosylceramide and alternative pathway gangliosides.

[0006] This neonatal developmental disorder has its onset in the first months of life, usually beginning with poor feeding and generalized seizures resistant to anticonvulsants. There is a complete absence of responsiveness to most social stimuli and infants seem to take little interest in their surroundings. The individual often seem highly irritable, is unable to sit alone, or to communicate verbally. They eventually become unable to take oral nourishment and require tubal feedings. An infant may seem limp and floppy.

[0007] Affected individuals have developmental stagnation and regression, profound motor dysfunction, visual and hearing deficits, and seizures. Interestingly, brain MRI shows no gross structural abnormality in affected children. Contribution to the signs and symptoms in individuals lacking GM3 synthase and thus GM3 may be attributable to either lack of GM3, per se, or lack of downstream biosynthetic derivatives. It is impossible to say at present the lack of which ganglioside is responsible for which sign or symptom of the disease, but the administration of purified GM3 ganglioside will correct the deficiency of GM3 and provide adequate precursor for the conversion to the a- and b- series gangliosides such as GM2, GMla, GDla, GTla, GD3, GD2, GDlb, GTlb, and GQlb since the enzymes need for the subsequent conversion are not lacking in these GM3 synthase deficient individuals.

[0008] There currently is no treatment available for individuals lacking GM3 synthase activity. These individuals have profound neurological problems. For example, individuals may live into adolescence but in a general vegetative state requiring total custodial care. They are usually blind and non-responsive to their environment

SUMMARY

[0009] An embodiment of the invention provides a method of treating individuals having ganglioside deficiency disorders by providing purified ganglioside such as purified GM3 ganglioside

[0010] An embodiment of the invention provides a method of treating individuals lacking the enzyme GM3 synthase by providing purified GM3 ganglioside

[0011] The ganglioside can be GM3. In another aspect, the gangliosides comprise GM3 and at least one other ganglioside for which GM3 serves as a precursor, including but not limited to GM2, GM 1, GMla, GDla, GTla,GTlaa, GD3,GD2, GDlb, GTlb, GQlba, GT3, GT2, GTlc, GPlca. or any combination thereof.

[0012] Additional features may be understood by referring to the following detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 illustrates exemplary pathways for the biosynthesis of gangliosides.

DETAILED DESCRIPTION

[0014] Described herein is a method for treating individuals lacking the enzyme GM3 synthase. The method relates to the use of purified GM3 ganglioside, derived from natural sources as replacement therapy. The GM3 can be administered by multiple routes. In one embodiment the GM3 is administered by injection. In an alternative route the GM3 is administered via a formulation allowing for intranasal delivery of the drug. The method provided herein also includes administering a therapeutically effective amount of one or more gangliosides.

[0015] Provided herein is a composition for treating or preventing cognitive dysfunction in a human patient in need thereof by administering a therapeutically effective amount of one or more gangliosides to the patient. The gangliosides used in the present composition can be selected from the group consisting of GM3, GM2, GM 1, GMla, GDla, GTla,GTlaa, GD3,GD2, GDlb, GTlb, GQlba, GT3, GT2, GTlc, GPlca or any combination thereof.

[0016] In a specific embodiment, the ganglosides can be GM3. In another aspect, the gangliosides comprise GM3 and at least one other ganglioside selected from the group consisting of GM2, GM 1, GMla, GDla, GTla,GTlaa, GD3,GD2, GDlb, GTlb, GQlba, GT3, GT2, GTlc, GPlca or any combination thereof.

[0017] In another embodiment, the method of treating or preventing cognitive dysfunction in a human patient in need thereof can be by administering a therapeutically effective amount of one or more gangliosides to a patient. The gangliosides can be selected from the group consisting of GM3, GM2, GM 1, GMla, GDI a, GTla,GTl a, GD3,GD2, GDlb, GTlb, GQlba, GT3, GT2, GTlc, GPlca, or any combination thereon. In another aspect, the gangliosides comprise GM3 and at least one other ganglioside selected from the group consisting of GM1, GD3, GM2, GDla, GDlb, GT1 or any combination thereof. The gangliosides can be administered as a co-therapy. As a therapeutic measure gangliosides can be administered with other known neuroprotective therapies such as leutpeptin. Furthermore, it is possible to administer gangliosides in conjunction with therapies to reduce accumulation of abnormally high level of GM3 precursors such as lactosylceramide and its alternative derivatives,

[0018] In general, gangliosides are the group of glycosphingolipids that show the greatest structural variation and also the more complex structure and are characteristic of nervous tissues. The main gangliosides of the brain are GM1, GDla, GDlb and GT1. GM3 is present mainly in nervous tissues outside brain tissues. GM1 activates trkB signaling and potentiates neurotrophins, and exogenous administration of GM1 has been shown to reduce nerve cell degeneration. Gangliosides are glycosphingolipids that localize in the outer leaflet of the plasma membrane of vertebrate cells. Gangliosides are highly concentrated in the nervous system and play a critical role in the normal development, growth and function of neurons.

[0019] Numerous studies have shown that gangliosides, have strong neurotrophic, neuroprotective and immunosuppressive properties. Due to their role in modulating cell signaling pathways, gangliosides can affect multiple cellular processes that are critical to normal cell functioning, cell survival and response to injury. In the majority of mammalian cells GM3 is the main ganglioside. GM3 is involved in a multitude of processes which including influencing cell death, programmed cell death (apoptosis), embryogenesis, differentiation, and cell adhesion and mobility,. Being the precursor needed for the production of higher cellular gangliosides, GM3 has a major influence on their functioning because their expression depends the amount of GM3 synthesis. GM3 influences stimulation of cell proliferation by fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF). Comparative studies of glycosphingolipid composition of actively proliferating and contact inhibited cells indicate that biosynthesis of GM3 sharply increased cell contact formation GM3 has been shown to inhibit invasiveness of tumor cells by promoting the interaction of integrin with anti-metastatic membrane proteins CD9 and CD 82 and enhanced cell adhesion to the intercellular matrix along a loss of invasiveness. GM3 has been shown to have the ability to inhibit the growth of tumor cells and tumor development. This ability is associated with a fundamental property of this ganglioside to suppress tyrosine phosphorylation of growth factor receptors in membranes of tumor cells. GM3 has also been shown to suppress phosphorylation of VEGF receptor. GM3 is involved in the regulation of expression of tumor necrosis factorjx (TNF_a), a multifunctional cytokine that has a major influence in immunity modulation, inflammation and apoptosis Due to its influence on apoptosis, GM3 plays an important role in development of the nervous system in man Likewise, due to the role of GM3 as precursor for production of higher gangliosides it has a great influence on the development and funtioning of the nervous system in man. Accordingly, this invention involves the use of GM3 ganglioside, other gangliosides including GM1, GD3, GM2, GDla, GDlb and GT1, and GM3 derivatives and analogs as a treatment for individuals lacking the enzyme GM3 synthase.

[0020] GM3 alone or in combination with other gangliosides would be effective. Gangliosides may be administered parenterally by intravenous or subcutaneous administration or via nasal or mucosal administration, e.g., alone or with appropriate absorption enhancers. This may include prolonged action dosage forms. Gangliosides may be administered using controlled release formulations (e.g., liposomes, nanoparticles, microspheres, implants) to prolong drug activity, or they may be coupled to appropriate transporter molecules in order to cross the blood brain barrier following systemic administration.

[0021] Gangliosides could be administered parenterally or via nasal administration (alone or with appropriate absorption enhancers). Suitable formulations for intranasal administration may also include a therapeutic dose of GM3 in a gel formulation with in situ gelling and mucoadhesive properties such that there is an increased permeation rate and prolonged nasal residence time and thereby increased nasal absorption. Such formulations would increase retention time of the GM3 solution in nasal cavity resulting in greater bioavailability and greater transfer of GM3 to the brain via the olfactory pathway. Chitosan-based mucoadhesive formulations could be used to enhance the retention time and bioavailability of GM3. Nasal bioadhesive gels could also provide enhanced bioavailability compared with other delivery routes and be combined in a formulation with other absorption enhancers.

[0022] Such a formulation may include a therapeutically active amount of GM3 together with gelling solutions of tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide) (e.g., Pluronic F127 ("PF127")) that exhibit thermoreversible properties. By modulating the gelation temperature of different PF 127 solutions, liquid bases for nasal use can be formulated that form a gel in the nasal cavity at body temperature with suitable gel strength resulting in enhancement of the residence time in the nasal cavity. The high solubilizing capacity and nontoxic properties of PF127 make it suitable for nasal drug delivery. GM3 formulations for intranasal delivery may therefore include thermoreversible polymer PF 127 and a mucoadhesive polymer (such as C934P), which enhances nasal residence time and absorption of drug across nasal-mucosal membrane.

[0023] For oral or buccal mucosal administration, a transmucosal product can be formulated with GM3 to be administered via the oral/buccal route using mucoadhesive, quick dissolve tablets or an oral spray formulation. Potential mucoadhesive polymers include hydrophilic polymers containing carboxylic group (which exhibit the favorable mucoadhesive properties) such as poly vinyl-pyrrolidone ("PVP"), methyl cellulose ("MC"), sodium carboxy methylcellulose ("SCMC"), hydroxy propyl cellulose ("HPC"), and other cellulose derivatives. Hydrogels that may also be used include carbopol, polyacrylates and their crosslinked modifications, chitosan and its derivatives, Eudragit-NE30D etc. PEGylating various polymers could also enhance mucoadhesion. Carbopol-934, hydroxypropylmethylcellulose, hydroxyethylcellulose, and sodium carboxymethylcellulose may also be used in various combination ratios, together with a therapeutic amount of GM3 for buccal drug delivery. Transmucosal routes of administration are described in PCT/US2010/047527, herein incorporated by reference in its entirety.

[0024] As other aspects of the ganglioside biosynthetic machinery are intact in patients lacking GM3 synthase activity, treatment of these patients with GM3 ganglioside or combinations of gangliosides should provide sufficient substrate with which a- and b- series gangliosides can be produced in the brain to replace those lost by the GM3 synthase mutation. GM3 replacement therapy, particularly if initiated soon after birth, should result in physiologically relevant levels of gangliosides necessary for brain development and function and should result in improved neurological; function and normalization of behavior. Such replacement therapy would need to be maintained through the lifespan.

[0025] Contemplated Studies [0026] The first product in the biosynthetic pathway of the ganglio-series gangliosides is GM3, which serves as a common precursor for the a-series and b-series gangliosides. The sialyl transferase responsible for GM3 synthesis is the GM3 synthase. The following studies will be performed to assess the extent to which GM3 replacement therapy can ameliorate at least some of the behavioral/neurological dysfunction that results from GM3 synthase deficiency and correct the resulting ganglioside abnormalities in brain. These studies will use mice deficient in GM3 synthase that are known to have behavioral abnormalities and degeneration of the organ of Corti, leading to hearing loss (Yoshikawa et al., 2009).

[0027] Startle responses in GM3 null and wild type animals will be assessed at 3 months of age using standard equipment. Briefly, a plexiglass cylinder rests on a piezoelectric transducer that detects the vibrations caused by the movements of the animals. Each test session starts by placing a subject in the Plexiglass cylinder for 10 minutes to acclimate to a background noise of 65 dB. To assess acoustic startle responses, each subject is exposed to 5 consecutive blocks of acoustic stimuli of increasing intensity (70, 75, 80, 85, 90, 100, 110, and 120 dB),each in 20-ms broadband burst and separated by a 40-s interval. The startle response is measured and recorded every 1 ms fori 00 ms starting at the onset of each startle stimulus. To differentiate acoustic startle from tactile sensory startle, responses to airpuff, a 10-ms burst of compressed air (0.2 mPa) is delivered to the dorsal side of each animal through a pipe located above the subject. Bursts are repeated 5 times per animal, and responses are recorded as above. Motor coordination and balance will be measured by using a standard roto-rod test. Mice are placed on a rotating rod and the latency to fall is measured. Rotation speed is 5, 10, and 15 rpm, and increased 5 rpm every day. The test is performed at 10 and 15rpm, 3 times, for 2 days.

[0028] Analysis of brain gangliosides will be performed to confirm the complete shift of a- and b-series gangliosides to o-series, such as GMlb and GDI a, produced by bypassing GM3 synthesis in GM3 synthase deficient animals and to assess the efficacy of replacement therapy to normalize brain ganglioside levels and distribution. Brain weight will also be recorded.

[0029] Animals will be genotyped by standard methods using PCR analyses of genomic DNA isolated from tail biopsies obtained at approximately 5 days of age. Beginning at approximately 7 days of age, some animals will treated daily with GM3 ganglioside, administered in different doses and by different routes of administration (intraperitoneal injection, oral, or intranasal) and others will receive vehicle administration until behavioral testing is performed at approximately three months of age. The degree to which GM3 replacement ameliorates behavioral alterations in GM3 deficient mice will be recorded. The degree to which GM3 replacement normalizes brain lipid content will be recorded. Additionally, CNS pathology and neurodegeneration has been described in GM3 synthase deficient mice (Yamashita et al., 2005). Histological analyses will be performed in order to assess to the extent to which GM3 replacement therapy can counteract the neurodegenerative processes in the brain.

[0030] While this description is made with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope. In addition, many modifications may be made to adapt a particular situation or material to the teachings hereof without departing from the essential scope. Also, in the drawings and the description, there have been disclosed exemplary embodiments and, although specific terms may have been employed, they are unless otherwise stated used in a generic and descriptive sense only and not for purposes of limitation, the scope of the claims therefore not being so limited. Moreover, one skilled in the art will appreciate that certain steps of the methods discussed herein may be sequenced in alternative order or steps may be combined. Therefore, it is intended that the appended claims not be limited to the particular embodiment disclosed herein.

[0031] Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference in their entirety. More generally, documents or references are cited in this text, either in a Reference List before the claims; or in the text itself; and, each of these documents or references ("herein-cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

Claims

1. A composition for treating individuals lacking the enzyme GM3 synthase, comprising a therapeutically effective amount of one or more gangliosides.

2. The composition according to claim 1, wherein said ganglosides comprises GM3.

3. The composition according to claim 1, wherein said gangliosides comprise GM3 and at least one other ganglioside.

4. The composition according to claim 1, wherein said other ganglioside is selected from the group consisting of GM2, GM 1, GMla, GDI a, GTla,GTlaa, GD3,GD2, GDlb, GTlb, GQlba, GT3, GT2, GTlc, GPlca.or a combination thereof.

5. The composition according to claim 1, wherein said ganglioside(s) are administered as a co- therapy.

6. The composition according to claim 1, wherein the individual has a nonsense mutation in S1AT9 on chromosome 2pl2-pl 1.2.

7. The composition according to claim 1, wherein said composition is administered parenterally by intravenous or subcutaneous administration, by nasal administration, or by mucosal administration.

8. The composition according to claim 7, the composition further comprising absorption enhancers.

9. The composition according to claim 7, the composition further comprising prolonged action dosage forms.

10. The composition according to claim 7, the composition further comprising a controlled release formulations.

11. The composition according to claim 10, the composition further comprising liposomes, nanoparticles, microspheres, or implants.

12. The composition according to claim 10, wherein the composition is coupled to transporter molecules.

13. The composition according to claim 7, wherein the composition is administered by intranasal administration, the composition further comprising a chitosan-based mucoadhesive formulations.

14. The composition according to claim 7, wherein the composition is administered by intranasal administration, the composition further comprising a thermoreversible polymer and a mucoadhesive polymer.

15. The composition according to claim 7, wherein the composition is administered by mucousal administration, and wherein said mucousal administration is oral or buccal mucosal administration, said composition further comprising a mucoadhesive polymers.

16. A method for treating individuals lacking the enzyme GM3 synthase, comprising administering a therapeutically effective amount of one or more gangliosides.

17. The method according to claim 16, wherein said ganglosides comprises GM3.

18. The method according to claim 16, wherein said gangliosides comprise GM3 and at least one other ganglioside.

19. The method according to claim 16, wherein said ganglioside(s) are administered as a co- therapy.

20. The method according to claim 16, wherein the individual has a nonsense mutation in S1AT9 on chromosome 2pl2-pl 1.2.

21. The method according to claim 16, wherein administration of said composition is parenteral intranasal, or by mucosal.

22. The method according to claim 21, the composition further comprising absorption enhancers, prolonged action dosage forms, or controlled release formulations.

23. The method according to claim 22, the composition further comprising liposomes, nanoparticles, microspheres, or implants.

24. The method according to claim 22, wherein the composition is coupled to transporter molecules.

25. The method according to claim 16, wherein administration of said composition is intranasal, the composition further comprising a chitosan-based mucoadhesive formulations.

26. The method according to claim 16, wherein administration of said composition is intranasal, the composition further comprising a thermoreversible polymer and a mucoadhesive polymer.

27. The method according to claim 16, wherein administration of said composition is mucousal, and wherein said administration is oral or buccal mucosal administration, said composition further comprising a mucoadhesive polymer(s).