Patent Application
20120035120
This invention relates to methods of treating and amliorating diseases of cancer, hyperinsulinemia, hypoglycemia and atypical parkinson's disease, tauopathies, and other neurological diseases, and/or a ganglioside deficiency state or disorder by administering a suitable glycolipid, ganglioside or ganglioside analog.
Besides the administration of antibodies or tyrosine kinase inhibitors for malignancies, the typical treatment for many cancers is the administration of radiation therapy and chemotherapeutic agents. However, side effects are a limiting factor in radiation treatments. Combination chemotherapy has some success in reaching partial or complete responses. Unfortunately, the remissions obtained through chemotherapy are often not durable.
Congenital hyperinsulinism (CHI, OMIM 256450) is a genetic disorder of pancreatic β-cell function characterized by failure to suppress insulin secretion in the presence of hypoglycemia, resulting in brain damage or death if inadequately treated.
Neuroendocrine tumors including such cancers as insulinoma, hepatomas, mesotheliaoma and fibrosarcoma may also cause hyperinsulinemia accompanied by hypoglycemia.
Gangliosides are sialic acid containing glycosphingolipids. Gangliosides are normal components of plasma membranes which are particularly abundant in the nervous system. In humans, gangliosides are most abundant in the gray matter of the brain, particularly in nerve endings. As a result, the addition of one or more glycolipids for cancer (e.g., during immunotherapy, such as antibodies or tyrosine kinase inhibitors), patients suffering from hyperinsulinemia, hypoglycemia or hypersinsulinemia with hypoglycemia or neurological disorders (e.g., Huntington's Disease, multiple systems atrophy, atypical Parkinson's disease, tauopathies) may reduce the mortality and/or morbidities associated with each disease. Attempts have been made to use gangliosides in the treatment of disorders of the nervous system. This has led to the development of synthetic gangliosides as well as natural ganglioside containing compositions for use in the treatment of disorders of the nervous system (see, U.S. Pat. Nos. 4,476,119; 4,593,091; 4,639,437; 4,707,469; 4,713,374; 4,716,223; 4,849,413; 4,940,694; 5,045,532; 5,135,921; 5,183,807; 5,190,925; 5,210,185; 5,218,094; 5,229,373; 5,260,464; 5,264,424; 5,350,841; 5,424,294; 5,484,775; 5,519,007; 5,521,164; 5,523,294; 5,677,285; 5,792,858; 5,795,869; and 5,849,717).
There exists a need in the art for ganglioside compounds and methods of treatment which use the compounds to treat diseases such as cancer, hyperinsulinemia, atypical
Parkinson's disease, multiple systems atrophy, Huntington's disease, tauopathies or ganglioside deficient states or disorders, including neurological conditions. Ideally, such compounds act in a manner similar to, or better than, the natural gangliosides for the prophylaxis, treatment and cure of such states or disorders.
In various aspects, this invention provides compounds, compositions, and methods for treating congenital and non-congenital diseases. Exemplary diseases treatable according to the invention include those characterized by ganglioside deficiency. Exemplary diseases treatable according to the invention include cancer (e.g., in combination with one or more of antibodies, antibody fragments and antibody conjugates, tyrosine kinase inhibitors), atypical Parkinson's disease, Parkinson's Disease, other neurological diseases and conditions (e.g., multiple systems atrophy, Huntington's disease, GM3 synthase deficiency, GM2 synthase deficiency); a tauopathy (e.g., a neurological disorder or condition associated with an increased aggregation of tau protein), hyperinsulinemia, hypoglycemia or hyperinsulinemia with hypoglycermia.
In a first aspect, the invention provides novel synthetic compositions of formula (1)-(5):
including pharmaceutically acceptable salts, isomers, hydrates, solvates, and prodrugs thereof in which Z can be O, S, C(R10)2 and NR2, X can be H, D, —OR11, —NR11R12, —S11, and —CHR11R12, and R10, R11 and R12 can be independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, —C(═B)R13, —C(═B)—Z—R13, —SO2R13, and —SO3 functional moieties. Further, a novel ganglioside of the present invention can have B, B′, and Z independently selected from O, NR14 or S, and Y can be selected from H, D, —OR15, —SR15, —NR15R16, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl moieties. Further still, a novel ganglioside of the invention can have R1, R2, R3, R4, R5, R6, R7, R8, R9, R13, R14, independently selected from H, D, —OR17, —NR17R18, —SR19, and —CHR19R20, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl moieties. Further R15, R16, R17, R18, and R19 are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl moieties, —C(═B,)R20, —C(═B′)R20, —SO2R20, and —SO3 functional moieties. Further, R20 is selected from H, D, —OR21, —SR21, —NR21R22, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl moieties. Further, R21 and R22 are independently selected from H, D, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl moieties. Further, double bonds can be E, Z or a mixture of E and Z.
In some embodiments in which X is NHR that contains an amide (i.e., R is hydrogen), a substituted or unsubstituted fatty acid amide of from 1 to 40 carbon atoms in length or from 2 to 40 carbon atoms in length or C(O)CHCl2; saccharide is a moiety of a naturally occurring mammalian ganglioside or glycosphingolipid or sulfatide; Y is substituted or unsubstituted alkyl, alkenyl, or alkynyl from 12 to 40 carbon atoms in length; and R′″ is hydrogen or hydroxy. In some embodiments, the Y member is unsubstituted. In some embodiments, the Y member consists of an unsubstituted linear or branched alkyl chain or an unsubstituted linear or branched alkenyl chain. In some embodiments, the Y member is alkenyl having from 2 to 4 double bonds in a branched or linear carbon chain. In some embodiments, the R fatty acid is unsubstituted. In other embodiments, the R fatty acid member is from 14 to 24 carbon atoms in length. In some exemplary embodiments, the R fatty acid is unsubstituted and the Y member consists of an unsubstituted linear or branched alkyl chain or an unsubstituted linear or branched alkenyl chain. In a further set of such embodiments, R is a long chain saturated or polyunsaturated fatty acid; in some further embodiments the long chain polyunsaturated fatty acid is DHA, EPA, or GLA. In some embodiments of the above, R is an α-hydroxy fatty acid (e.g., α-hydroxy palmitic acid) joined at the amide bond to the sphingoid base. In some embodiments, R is a α-hydroxy fatty acid (e.g., α-hydroxy palmitic acid) joined at the amide bond to a 4,8-sphingadiene base of from 16, 18 or 20 carbons in length.
In other embodiments of any of the above, the saccharide moiety is selected from galactosyl glycosphingolipids, digalactosyl glycosphingolipids, sulfated glycosphingolipids, glucosyl glycosphingolipids, lactosyl glycosphingolipids, the lacto series glycosphingolipids, the neo-lacto series of glycosphingolipids, globoside glycosphingolipids, or the ganglioside glycosphingolipids (Chen, “Handbook of Carbohydrate Engineering”, Chapter 1, pages 1-48, CRC Press, Yarema, ed., (2005)). In preferred embodiments, the saccharide moiety is a glycosphingolipid selected from GA2, GA1, GM1b, GD1c, GD1α, GM3, GM2, GM4, GM1a, GD1a, GT1a, GT1α, GD3, GD2, GD1b, GT1b, GQ1b, GQ1bα, GT3, GT2, GT1c, GQ1c, GP1c, sulfatide, globoside, sialyl paraglobosides and GPlcα. In a particularly preferred embodiment, the compound is the ganglioside GM1 or another compound of the above formula having the saccharide moeiety of GM1. In other embodiments, the compound is the ganglioside GM3 or GD3 or another compound of the above formula having the saccharide moeiety of GM3 or GD3. In still other embodiments, the compound or its saccharide moiety corresponds to that of GT1b, sulfatide, or, more preferably, Gb3.
In some exemplary embodiments, the present invention also provides a novel ganglioside compound in which the saccharide component can be
and such saccharide moieties may or may not be deacetylated. These structures are optionally incorporated into the compounds of formulae 1-5, above.
The invention further provides pharmaceutical compositions including at least one compound of the invention and a pharmaceutically acceptable carrier.
In some exemplary embodiments of any of the above, the compound is of formula (6)-(10):
wherein n is an integer from 1 to 40, NHR is NH2 or an amide of a saturated, unsaturated, or polyunsaturated (i.e., having at least two double bonds in the fatty acid chain) fatty acid having from 0 to 40 carbons in the chain. In some embodiments, n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17; More particularly, in some embodiments, n can be 7, 10, 11, 13 or 14. In some embodiments, the fatty acid is unsubstituted or alpha-hydroxylated. In other embodiments the long chain fatty acid is substituted. In other embodiments still, the R fatty acid member has from 14 to 28 carbon atoms in the fatty acid chain. In some embodiments, the long chain fatty acid is an unsubstituted or an alpha-hydroxylated C16, C18, C20, C22, or C24 fatty acid. In some embodiments, the fatty acid is an unsubstituted omega-3 or omega-6 or omega 9 fatty acid which may be optionally substituted with an alpha-hydroxyl group. In some further embodiments of such, the fatty acid is a C16, C18, or C18 fatty acid. In some embodiments, the compound comprises a 4,8-sphingadiene backbone. In some further embodiments of any of the above, the long chain fatty acid is a polyunsaturated fatty acid. In some embodiments of the above, the long chain fatty acid is an α-hydroxy fatty acid (e.g., a substituted or unsubstituted α-hydroxy palmitic acid or stearic acid) joined at the amide bond to a sphingoid base. In other embodiments of any of the above, R′ is the saccharide moiety of a glycosphingolipid selected from GA2, GA1, GM1b, GD1c, GD1α, GM3, GM2, GM4, GM1a, GD1a, GT1a, GT1α, GD3, GD2, GD1b, GT1b, GQ1b, GQ1bα, GT3, GT2, GT1c, GQ1c, GP1c, Gb3, Gb4, sailyl-paragloboside, globoside, sulfatide and GP1cα. Accordingly, the saccharide moiety can be a mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nano-, or deca-saccharide. In some embodiments, the saccharide moiety can be the moiety of GM3, GM2, GM1a, GD1a, GT1a, or GT1α. In exemplary embodiments, the compound of formula (1)-(10) has the stereoisomerism of a corresponding naturally occurring glycolipids. In some embodiments, wherein the glycolipid has one or more double bond, the double-bond may be independently cis or trans, or a mixture thereof. In some embodiments, the double bonds are in the trans configuration.
In yet other embodiments, the compounds according to the invention, include the compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) comprise an omega-3 fatty acid (e.g., DHA (docahexaenoic acid), EPA (eiocosapentaenoic acid)) or an omega-6 fatty acid (e.g., GLA (gamma linolenic acid)) as the polyunsaturated fatty acid. In a further embodiment of such compounds, n is 7, 10, 11, 12, 13 or 14. In a still further embodiment, the saccharide moiety can be the oligosaccharide moiety of GM3, GM2, GMla, GD1a, GT1a, sialyl-paragloboside or GT1α.
In another embodiment of the compounds of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10), the polyunsaturated fatty acid has non-conjugated double bonds. In a further embodiment of such, the polyunsaturated fatty acid is arachidonic acid. In yet other embodiments, the polyunsaturated fatty acid has 2, 3, 4, 5, or 6 non-conjugated double bonds;
or at least two, three, four, or five non-conjugated double bonds. In still further embodiments, n is 7, 10, 11, 12, 13 or 14.
In another embodiment of compounds of formulae (1)-(10), the saccharide moiety, is selected from the glycans of the glycolipid families of Gala (Ga) (e.g., galactosyl-ceramides, sulfatides, sulfated digalactosides), Gluco, Globo (Gb), Isoglobo (iGb), Lacto (Lc), Neolacto (nLc), Ganglio (Gg), Isoganglio (iGg), Lactoganglio (LcGg) and Gangliosides (for example the a-, b- and c-series). In a further set of such embodiments, the fatty acid is palmitic, stearic or oleic acids.
In another embodiment of compounds of formula (1)-(10), the saccharide moiety, is selected from the group consisting of
In another aspect, the invention provides pharmaceutical compositions for treating a disorder selected from cancer, malignancy, hyperinsulinemia, hypoglycemia and hyperinsulinemia with hypoglycemia, a neurological disorder (e.g., Huntington's Disease, GM3 synthase deficiency, GM2 synthase deficiency, Parkinson's Disease, multiple systems atrophy, atypical Parkinson's Disease, a tauopathy) comprising a compound of the invention and a pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical compositions are in unit dose format and each unit dose provides a therapeutically effective amount of one or more compounds for use according to the invention. The compositions, are formulated for oral, intraperitoneal, intranasal, topical, transcutaneous, subcutaneous, or intravenous administration.
In some embodiments, the above compositions can comprise a plurality of glycolipid analogs wherein each has a saccharide moiety of a different glycolipid. In some such embodiments, the administered glycosphingolipid consist of at least two or three such glycosphingolipids. In still other embodiments, the administered glycosphingolipid consist of at least 80%, 90%, 95%, or 98% of a single glycosphingolipid having a saccharide moiety of a naturally occurring mammalian glycosphingolipid.
In yet another aspect, the invention provides methods for treating a disease by administering a therapeutically effective amount of a glycolipid. This method optionally includes the administration of a therapeutically effective amound of at least a second therapeutic agent to a subject in need thereof. Exemplary glycolipids include, for example, those of the present invention and a those disclosed and/or claimed in any of U.S. patent applications Ser. Nos. 10/485,195; 10/485,892; 10/487,841; 60/452,796; 11/666,577; 60/626,678; and 10/547,566 which are each incorporated herein by reference particularly with respect to such compound subject matter
Exemplary indications for this treatment include malignancies that are resistant or unresponsive to one or more antibodies or tyrosine kinase inhibitors, hematologic and solid tumors, congenital and non-congenital hyperinsulinemia, hypoglycemia, hyperinsulinemia with hypoglycemia, Parkinson's disease, Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, Parkinson's Disease, multiple systems atrophy, atypical Parkinson's Disease, and a tauopathy.
In another embodiment, the present invention provides a method of treating or inhibiting development of a post-prandial hypoglycemia, fasting hypoglycemia, neonatal hypoglycemia, hypoglycemia secondary to dialysis. The method includes the step of administering to the subject a therapeutically effective amount of a glycolipid. The post-prandial hypoglycemia treated or inhibited by methods and compositions of the present invention is, in another embodiment, associated with a Nissen fundoplication or gastric-bypass surgery
In another embodiment, administration of a therapeutically effective amount of a glycolipid to a subject suppresses insulin secretion by the subject.
Methods of the invention can be used for treating different cancers, both solid tumors and soft-tissue tumors alike. Non-limiting examples of cancers amendable to the treatment of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer (NSCLC), non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, sarcoma, renal cell carcinoma, carcinoid carcinoma, head and neck cancer, glioblastoma, melanoma, ovarian cancer, gastric cancer, mesothelioma, and multiple myeloma. In certain aspects, the cancers are metastatic. In other aspects, the cancers are non-metastatic.
In another embodiment, a compound of the invention utilized in methods and compositions of the present invention exhibits an improvement in a desirable biological property relative to the natural glycolipid. In another embodiment, the biological property is improved biological half-life. In another embodiment, the biological property is improved affinity for the insulin receptor. In another embodiment, the biological property is improved potency for antagonism of the insulin receptor. In another embodiment, the biological property is any other desirable biological property known in the art. Each possibility represents a separate embodiment of the present invention.
The present invention provides novel glycolipid compositions and methods of using these compositions to treat a congenital or non-congenital disorder, such as a disease characterized by a ganglioside deficiency. Exemplary disorders include cancer, atypical Parkinson's disease; Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, hyperinsulinemia, hypoglycemia, hyperinsulinemia with hypoglycemis, a tauopathy, or another neurological disorder or condition associated with an increased aggregation of tau protein. The method includes administering to a subject in need thereof of a composition of the invention in a therapeutically effective amount.
Exemplary compounds of the invention have improved structures over lyso-GM1, GM1(stearate) and GM3 (stearate) when injected or given orally. These structures exhibit improved biodistribution, (e.g., brain delivery), improved pharmacokinetics, and improved oral bioavailability.
In some embodiments, the compound of Formula (1) to (10) has a saccharide moiety selected from those set forth in the following table of gangliosides:
Additionally the invention provides for the use of other glycolipids in treating a disorder selected from cancer, atypical Parkinson's disease; Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, a tauopathy, or another neurological disorder or condition associated with an increased aggregation of tau protein including, for instance, the glycolipids of disclosed herein. These compounds to be administered to treat a disorder (e.g., cancer, atypical Parkinson's disease; Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, a tauopathy, or another neurological disorder or condition associated with an increased aggregation of tau protein) include compounds according to the formula:
wherein Z, X, Y and R1 have their identities disclosed herein including in those references incorporated herein by reference (e.g., U.S. Patent Application Publication No. 2007-0275908; U.S. Patent Application Publication No. US 2005/0239741; U.S. Patent Application Publication No. US 2005/0245735; and U.S. Patent Application Publication No. US 2005/0032742).
These compounds also include the neutral glycosphingolipids and neutral glycosyl sphingosines disclosed U.S. Patent Application Publication No. US 2005/0245735 which is incorporated by reference in its entirety.
These compounds also include sialylated oligosaccharide glycolipids disclosed in U.S. Patent Application Publication No. US 2005/0032742 which is incorporated herein by reference. In some embodiments, the oligosaccharide is a glycosylated ganglioside, ceramide, or sphingosine or an analogue of same.
Other compounds of use according to the invention include those disclosed in commonly owned U.S. Patent Application Publication Nos. US 2005/0245735; US 2005-0032742; US 2005-0239741; US 2007-0275908; and US 2008-0125392, each of which is incorporated by reference therein in its entirety for all purposes.
In exemplary embodiments, a compound of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) associates with the insulin receptor inhibiting cellular signalling. In a more preferred embodiment, the glycolipid inhibits the upregulation and transport of Glut-4 or Glut-2 to the cell surface. In another embodiment, the glycolipid reduces glucose uptake by the cell. In another exemplary embodiment, the saccharide of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) is a sugar of GM3, sialylparagloboside. Further, administration of compounds from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) based on the glycan structures of sialylparagloboside, GD1a or GM3 to a subject with hypoinsulinemia, hypoglycemia or hypoinsulinemia with hypoglycemia will reduce the hypoglycemic state, decrease insulin secretion, or reduce the morbidities and mortalities associated with the disease state of the subject.
In some embodiments, the compound selected from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) is administered to a subject or patient to replace a missing or low level glycolipid in the subject. For example, type II diabetes is associated with reduced levels of sulfatide (Buschard, Diabetes, 55: 2826-2834 (2006)). Supplementation of sulfatide (e.g., ceramide fatty acid of C16:0) inhibited glucose stimulation of insulin secretion by activation of the KATP channels, in vitro. In addition, the exogenous sulfatide increased the expression of adiponectin while reducing inflammatory responses by decreasing the levels of TNF-alpha, IL-6 and IL-8 in human adippose tissue, in vitro (Bruun, Mol Cell Endocrinol, 263:142-148 (2007)). In a preferred embodiment, a compound selected from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) will reduce insulin secretion from congenital or noncongenital diseases. In a more preferred embodiment, the congenital disease is Congenital Hyperinsulinemia (e.g., Familial Hyperinsulinemia). In an even more preferred embodiment, a compound from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) contains the saccharide moiety of sulfatide or GM4.
In another aspect, the invention provides methods of treating cancer, atypical Parkinson's disease; Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, a tauopathy, or another neurological disorder or condition associated with an increased aggregation of tau protein a subject by administering to the subject one or more of the compounds selected from formula (1) to (10) in which the compound has a saccharide moiety of a deficient ganglioside or of a ganglioside which is downstream of a deficient ganglioside in the subject. In some embodiments, the ganglioside deficiency state may be mediated, exacerbated or caused by the condition or disease to be treated or by a reduced level of activity or absence of any of the enzymes involved in mammalian ganglioside synthesis or anabolism (e.g., ceramide glycosyl transferases, galactosyltransferases, sialyltransferases, and GalNAc transferases).
In certain aspects, the present invention provides a method for treating hyperinsulinemia, by administering effective amounts of compounds from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) and combining this treatment with the administration of drugs that reduce insulin or insulin like growth factor (e.g., IGF-1 and IGF-2) secretion, drugs and methods for increasing glucose levels, diet, diuretic, chemotherapy, radiation and surgery.
In an exemplary embodiment, the treatment uses a compound of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) in combination with one or more of additional treatment methods including sulfonylureas (e.g., diazoxide), somatostatin analogs (e.g., octreotide®), calcium channel blockers (e.g., nifedipine), glucagon, IGF-1, insulin, glucocorticoids, growth hormone, glucagon like peptides (e.g., GLP-1, exendin-4, exendin peptide derivatives, exendin peptide antagonists and extended formulations thereof), glucose (e.g., IV, oral or gastrostomy), pancreatectomies, and tumor resection.
In some embodiments the glycolipid is capable of inhibiting cell proliferation, intiating apoptosis or is capable of killing the malignant cells independent of any additional antineoplastic therapies including antibodies. For example, GM3 can be used to treat certain CNS malignancies including glioma and bladder cancer (PCT Patent Application Serial No. WO9852577; Fujimoto, J Neuro-Oncology, 71: 99-106 (2005); Watanabe, Cancer Res, 62: 3850-3854 (2002), 9-O-acetyl-GD1b can treat CNS malignancies and breast cancer (Abad-Rodriguez, J Neurochemistry, 74: 2547-2556 (2006), sialyl-paragloboside can inhibit leukemias and Burkitt lymphoma (Schaade, Med Microbiol Immunol, 188: 23-29 (1999) and GD1b, GT1b and GQ1b suppress the growth of melanoma (Kanda, J Invest Dermatol, 117: 284-293 (2001)).
In certain aspects, the present invention provides a method for treating cancer, by administering effective amounts of compounds from Formula (1) to (10) combined with one or more antibodies directed against the tumor or tumor enviroment and combining this treatment with the administration of radiation treatment or chemotherapeutic agents to a patient susceptible to, or diagnosed with cancer. A variety of chemotherapeutic agents may be used in the combined treatment methods of the invention.
In one embodiment, the present invention can be used for increasing the duration of survival of a human patient susceptible to or diagnosed with a cancer, increasing progression free survival of a human patient susceptible to or diagnosed with a cancer, increasing response rate in a group of human patients susceptible to or diagnosed with a cancer, increases the duration of response in a subject with cancer, or to treat the signs and symptoms, ameliorate, or slow the progression of the malignancy when administered with an antibody directed towards an antigen or tyrosine kinase inhibitor. In an even more preferred embodiment, the response of the malignancy to the combination of agents is synergistic.
As will be understood by those of ordinary skill in the art, the appropriate doses of therapeutic and chemotherapeutic agents will be generally around those already employed in clinical therapies wherein the therapeutics are administered alone or in combination with other therapeutics. Variation in dosage will likely occur depending on the condition being treated. The physician administering treatment will be able to determine the appropriate dose for the individual subject.
In accordance with the invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise.
The terms “cancer”, “cancerous”, “malignancy” and “malignant: refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, Hodgkin's disease, non-Hodgkins lymphoma, anaplastic large T-cell lymphoma, acute myelogenous leukemia (AML), chronic lympocytic leukemia (CLL), cutaneous T-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, peripheral T-cell lympoma, brain, as well as head and neck cancer, and associated metastases. Examples of head and neck cancers include, but are not limited to, adenoid cystic carcinomas localized, for example to the tongue, parotid gland, nose, palate, skin, neck, submandibular gland, glottis, sinus, epiglottis, buccal space, nerves, larynx, mouth, pharynx, or cheek.
“Treatment” is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
Atypical Parkisonian diseases or syndromes refers to a group of disorders whose clinical features overlap those of idiopathic Parkinson's disease. The four major Parkinsonian syndromes embrace three important neurodegenerations, multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration, and a lacunar cerebrovascular disorder, vascular parkinsonism (see, Gilman S., Clin. Geriatr. Med 22:827-42 (2006). In embodiments, the conditions to be treated according to the invention can be any one of these four major syndromes.
The term “congenital disease” as used herein refers to a disease or disorder that is inherited genetically. The term is intended to include familial hyperinsulinemia (OMIM no. 256450, 601820, 606762, 602485, 609968, 609975, 610021) (e.g., persistent hyperinsulinemic hypoglycemia of infancy, ABCC8-related hyperinsulinism, GCK-related hyperinsulinism, HADHSC-related hyperinsulinism, GLUD1-related hyperinsulinism, KCNJ11-related hyperinsulinism, insulin receptor-related hyperinsulinism, UCP2-related hyperinsulinism, exercise induced hyperinsulinism, diffuse hyperinsulinism), Beckwith-Wiedemann Syndrome, Congenital Disorders of Glycosylation (CDG)(e.g., CDG-Ia-n, CDG-IIa-h), type II diabetes, polycystic kidney disease, hypopituitarism, hyperinsulinism, glycogen storage disease (Type I and III), organic acidurias (e.g., maple syrup urine disease, 3-hydroxymethylglutaryl-CoA lysase deficiency, glutaric acidemia type I and II, 3-methylglutaconic aciduria type 1, mevalonic aciduria, alpha-methylacetoacetic aciduria, short-chain acyl-CoA dehydrogenase deficiency, 3-methylcrotonyl-CoA carboxylase I deficiency, long-chain acyl-CoA dehydrogenase deficiency, carnitine-acylcarnitine translocase deficiency, carnitine palmitoyltransferase II deficiency, medium chain acyl-CoA dehydrogenase deficiency), phosphoenolpyruvate carboxykinase deficiency, disorders of fatty acid oxidation (e.g., 3-hydroxyacyl-CoA dehydrogenase deficiency, very long chain acyl-CoA dehydrogenase deficiency, trifunctional protein deficiency), familial leucine sensitive hypoglycemia, insulin secreting tumors, insulin like growth factor (e.g., IGF-1, IGF-2) secreting tumors, Addison's disease, reactive hypoglycemia and idiopathic postprandial syndrome, Doege-Potter syndrome, hyperinsulin hyperammonia syndrome, multiple endocrine neoplasia, neurofibromatosis type 1, adrenal insufficiency (e.g., adrenal hyperplasias including ACTH-independent macronodular adrenal hyperplasia), Simpson-Golabi-Behmel syndrome type 1, idiopathic ketotic hypoglycemia, and reactive hypoglycemia and idiopathic postprandial syndrome. The term is also intended to include GM3 synthase deficiency (OMIM no. 609056; Amish Infantile Epilepsy Syndrome) in which the sialyltransferase (ST3Gal5; SIAT9) that prepares GM3 in the ganglioside biosynthetic pathway has decreased or absent biosynthetic activity. The term also is intended to include GM2 synthase deficiency [GM3 (Hematoside) Sphingolipodystrophy] in which the GalNAc-transferase (B4Galnt1) that prepares GA2, GM2 and GD2 in the glycolipid biosynthetic pathway has decreased or absent biosynthetic activity. The term is also intended to include Huntington's Disease (OMIM no. 143100) which has a glycolipid deficient phenotype that results in a reduction in the amount of higher glycolipids (e.g., GM2, GM1, GD2, GD1a, GT1a, GT1b, GQ1b)(Neurobiol Dis, 27:265-277 (2007)).
The term “noncongenital disease” as used herein refers to a disease or disorder that is not inherited genetically. The term is intended to include type II diabetes, acquired adrenal insufficiency, premature birth, sepsis, acquired hypopituitarism, gastric dumping syndrome, insulin secreting tumors, insulin like growth factor (e.g., IGF-1, IGF-2) secreting tumors, immunopathologic hypoglycemia, bowel bypass surgery or resection, reactive hypoglycemia and idiopathic postprandial syndrome, acquired hypoglycemia resulting from dialysis, hypoglycemia resulting from organ (e.g., kidney, liver, heart, lung) transplantation, medication or toxin induced hypoglycemia, asphyxia, hypocalcemia, opoid withdrawal and Nissen fundoplication.
The compounds for use according to the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds (e.g., D, 13C, 18O) for use according to the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
The term “substituted” as used herein means that a hydrogen atom has been replaced with another monovalent group (e.g., halo, haloalkyl, hydroxy, thiol, alkoxy, thiohaloalkyl, amino, and the like), or that two hydrogen atoms of the same atom have been replaced by a divalent group.
The terms “halo” or “halogen” as used herein refer to Cl, Br, F or I. The term “haloalkyl” and the like, refer to an alkyl group, as defined herein, wherein at least one hydrogen atom of the alkyl group is replaced by a Cl, Br, F or I. A mixture of different halo atoms may be used if more than one hydrogen atom is replaced. For example, a haloalkyl includes chloromethyl (—CH2Cl) and trifluoromethyl (—CF3) and the like.
The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which is saturated, and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by —CH2CH2CH2CH2—. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having six or fewer carbon atoms.
The terms “alkyl,” “alkenyl” and “alkynyl” are meant to include both substituted and unsubstituted forms of the indicated radical. The terms also include, but are not limited to, forms of the radicals having 3 or fewer or 6 or fewer carbon atoms. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl, alkenyl, alkynyl radicals can be one or more of a variety of groups selected from, but not limited to: —ORa, ═O, ═NRa, ═N—ORa, —NRaRb, —SRa, —halogen, —OC(O)Ra, —C(O)Ra, —CO2Ra, —CONRaRb, —OC(O)NRaRb, —NRbC(O)Ra, —NRc—C(O)NRbRa, —NRbC(O)2Ra, —NRe—C(NRaRbRc)═NRd, —NRd—C(NRaRb)═NRc, —S(O)Ra, S(O)2Ra, —S(O)2NRaRb, —NRbSO2Ra, —CN and —NO2 in a number ranging from zero to (2m′+1), wherein m′ is the total number of carbon atoms in such radical. Ra, Rb, Rc, Rd and Re each preferably independently refer to hydrogen or unsubstituted alkyl. When a compound of the invention includes more than one of any Ra, Rb, Rc, Rd or Re group, each of those groups are independently selected as well. For example, if there are two or more Ra groups in a formula, each of those are independently selected. When Ra and Rb are attached to the same nitrogen atom, they can optionally be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NRaRb is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include, but not be limited to, groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., —CF3 and —CH2CF3) and acyl (e.g., —C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like). Preferred substituents are lower alkyl, lower alkoxy, hydroxy, and halo. The term “lower” indicates C1 to C6 carbons in the chain. In some embodiments, the total number of substituents for the alkyl, alkenyl, or alkynyl radical are independently in a number which is 0, 1, 2, 3 or 4. In a further embodiment, these substituents are independently selected from the group consisting of lower alkyl, lower alkoxy, hydroxy, and halo.
In some embodiments, according to the invention, the compound of the invention has a saccharide moiety with the structure:
The term “sphingoid,” as used herein, includes sphingosines, phytosphingosines, sphinganines, ceramides, and the like. Both naturally occurring and synthetically produced compounds are included. The naturally occurring sphingoid base has an alkyl chain length of from 14 to 22 carbons long, but is preferably 18 carbons long. Synthetic sphingoid bases can be longer or shorter (e.g., having from 2 to 40 carbons in the alkyl chain). A ceramide is an N-acetylated sphingoid base. The fatty acids of ceramides vary in chain lengths (14 to 40 carbons in the chain) and the presence or absence of a hydroxyl group at the α- or ω-carbon atom.
The term “glycosphingolipid” is a carbohydrate-containing derivative of a sphingoid or ceramide. The carbohydrate residue is attached by a glycosidic linkage to O-1 of the sphingoid or ceramide.
As used herein, the term “heteroatom” is meant to include oxygen (O), nitrogen (N), and sulfur (S).
Ganglioside analog refers to gangliosides in which the saccharyl moiety, the base (e.g., sphingoid-like backbone), or the fatty acid-derived hydrocarbon is of a structure other than that found in naturally occurring ganglioside. Unless indicated otherwise, the term ganglioside is meant to include ganglioside analogs as well as naturally occurring gangliosides.
The term “sialic acid” (abbreviated “Sia”) refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. Another sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al., J. Biol. Chem. 261: 11550-11557 (1986); Kanamori et al., J. Biol. Chem. 265: 21811-21819 (1990). Also included are 9-substituted sialic acids such as a 9-O—C1-C6 acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetylNeu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992); Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New York (1992). The synthesis and use of sialic acid compounds in a sialylation procedure is described in, for example, International Patent Application Publication No. WO 92/16640, published Oct. 1, 1992.
In some embodiments, the compound of formula (1)-(10) is a glycolipid compound of a formula:
in which each n is independentlyas set forth above. In some embodiments of the above, each n is 1.
The terms “deficiency” and “deficiency state” refer to a large relative reduction in the levels of the referenced ganglioside or enzyme or enzyme activity in tissues or samples from a subject. Such deficiency states may generally exist, for instance, when the levels of a subject glycolipid are zero or undetectable or less than two-thirds, one-half, one-fourth, one-tenth, one-twentieth, or one-one hundredth of the levels for the comparable well-matched control group with a similar malignancy.
The term “fatty acid” refers to an aliphatic monocarboxylic acid which may be substituted or unsubstituted and saturated or unsaturated and have from 1 to 40 carbon atoms.
In some embodiments, the fatty acid is from 2 to 40 carbon atoms in length, including the carboxyl moiety carbon. In further embodiments, the fatty acid is 14 to 24 carbon atoms in length and is saturated or unsaturated. In other embodiments, the fatty acid is unsubstituted and from 14 to 24 carbons in length and is saturated or unsaturated. If unsaturated, the double bond may be in the cis or trans conformation, or be a mixture thereof. In some embodiments, the fatty acid is unsaturated with at least one of the double bonds is in the trans configuration. Unsaturated fatty acids may be polyunsaturated, for instance, having from 1, 2, 3, or 4 double bonds. Exemplary fatty acids are stearic acid and oleic acid as well as the omega-3, omega-6, and omega 9 fatty acids of from 14 to 24 carbons in length. In some embodiments, the fatty acid is optionally substituted with an α-hydroxy or an α-alkoxy group or an acylated α-hydroxy group (acetyl α-hydroxy) which acylated compound may function as a prodrug of a compound of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10). The aliphatic moiety may be linear or branched. In preferred embodiments of any of the above, it is linear.
“Commercial scale” refers to gram scale production of a compound of formulae (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) in a single reaction. In preferred embodiments, commercial scale refers to production of greater than about 50, 75, 80, 90, 100, 125, 150, 175, or 200 grams of a compound of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or 10). In some embodiments, the methods of use and manufacture of the pharmaceutical compositions involve the use of such compounds produced on a commercial scale.
The compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) can be made by any method available to one of ordinary skill in the art. The saccharide moiety of the compounds for use according to the invention can be prepared by any means known in the art including those methods described in U S. Pat. Nos. 5,922,577; 6,284,493; and 6,331,418, each of which is incorporated by reference herein in its entirety. Additional particularly suitable methods for making compounds for use according to the invention are described in U.S. Patent Application Publication No. US 2009-0170155; U.S. Patent Application Publication No. US 2009-0170155; and U.S. Patent Application Publication No. US 2009-0170155; which are assigned to the same assignee as the instant application; and in U.S. Pat. Nos. 6,440,703 and 6,030,815; and PCT International Patent Application Publications Nos. WO2004/080960, WO2003/016469, WO2005/118798 and WO2004/080960. Each of these references is incorporated herein by reference particularly with respect to their teachings as to how to synthesize various glycolipids.
The compounds of the invention and of use in the methods of the invention can be prepared using, unless otherwise indicated, conventional methods and protocols in chemistry and enzymology known in the art. For example, compounds for use according to the invention may be prepared by chemical and enzymatic processes as outlined further below.
Additional suitable synthetic approaches are disclosed in WO 2004/080960 A2 which is incorporated by reference herein in its entirety.
If the acceptor is a ceramide, the enzymatic step is optionally preceded by hydrolysis of the fatty acid moiety from the ceramide. Methods of removing a fatty acid moiety from a glycosphingolipid are known to those of skill in the art. Standard carbohydrate and glycosphingolipid chemistry methodology can be employed, such as that described in, for example, Paulson et al., Carbohydrate Res. 137: 39-62 (1985); Beith-Halahmi et al., Carbohydrate Res. 5: 25-30 (1967); Alais and Veyrieries, Carbohydrate Res. 207: 11-31; (1990); Grudler and Schmidt, Carbohydrate Res. 135: 203-218 (1985); Ponpipom et al.; Tetrahedron Lett. 1717-1720 (1978); Murase et al., Carbohydrate Res. 188: 71-80 (1989); Kameyama et al. Carbohydrate Res. 193: cl-c5(1989); Hasegawa et al. J. Carbohydrate Chem. 10: 439-459(1991); Schwarzmann and Sandhoff, Meth. Enzymol. 138: 319-341 (1987); Guadino and Paulson, J. Am. Chem. Soc. 116: 1149-1150 (1994) (including supplemental material, which is also incorporated herein by reference). For example, the fatty acid moiety can be removed by base hydrolysis. Once the glycosylation reactions are completed, the same or a different fatty acid can be attached to the product of the glycosylation reactions.
Methods for coupling a fatty acid are generally known in the art and examples are discussed herein. The N-acyl group (NHR) of a compound selected from formulae (1) to (10) can be derived from a wide variety of polyunsaturated fatty acids (or corresponding activated derivative, e.g., active ester, acid halide, etc.). Acylation can be carried out in the conventional way, for example, by reacting the starting products with an acylating agent, particularly with a reactive functional derivative of the acid, whose residue is to be introduced.
The invention also provides metal or organic base salts of the glycosphingolipid compounds for use according to the present invention having free carboxy functions, and these also form part of the invention. Also forming part of the invention are acid addition salts of glycosphingolipid derivatives, which contain a basic function, such as a free amino function, for example, esters with aminoalcohols.
Another suitable method of synthesizing a glycolipid of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) can use wild type or mutant endoglycoceramidase as taught in WO/2005/118798 which is incorporated herein by reference in its entirety with respect to the methods of synthesis and enzymes used therein.
Further modifications can be made to the glycolipids synthesized using the endoglycoceramide synthase of the present invention. Exemplary methods of further elaborating glycolipids produced using the present invention are set forth in WO 03/017949; PCT/US02/24574; US2004063911 (although each is broadly directed to modification of peptides with glycosyl moieties, the methods disclosed therein are equally applicable to the glycolipids and method of producing them set forth herein). Moreover, the glycolipid compositions of the invention can be subjected to glycoconjugation as disclosed in WO 03/031464 and its progeny (although each is broadly directed to modification of peptides with glycosyl moieties, the methods disclosed therein are equally applicable to the glycolipids and method of producing them set forth herein).
The products produced by the above processes can be used without purification. However, for some applications it is desirable to purify the compounds. Standard, well-known techniques for purification of substrates are generally suitable. For example, thin or thick layer chromatography, column chromatography, ion exchange chromatography, or membrane filtration can be used. Moreover, membrane filtration, preferably utilizing a reverse osmotic membrane, or one or more column chromatographic techniques, can be utilized. For instance, membrane filtration wherein the membranes have molecular weight cutoff of about 3,000 to about 10,000 can be used to remove proteins such as glycosyl transferases. Nanofiltration or reverse osmosis can then be used to remove salts and/or purify the product saccharides (see, e.g., WO 98/15581).
Another exemplary purification strategy makes use of a membrane in conjunction with an organic solvent. Both glycolipids and glycosphingolipids can be purified by this method. Moreover, any of the intermediate enzyme reaction products described herein can be purified according to this method. The method includes concentrating a reaction product in a membrane purification system with the addition of an organic solvent.
Suitable solvents include, but are not limited to alcohols (e.g., methanol), halocarbons (e.g., chloroform), and mixtures of hydrocarbons and alcohols (e.g., xylenes/methanol). In a preferred embodiment, the solvent is methanol. The concentration step can concentrate the reaction product to any selected degree.
In yet another embodiment, the invention provides a pharmaceutical formulation comprising a glycolipid as set forth or incorporated by reference herein, e.g., of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10), in admixture with a pharmaceutically accecptable carrier. The pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249: 1527-1533 (1990).
The pharmaceutical compositions can be administered by a number of routes, for instance, the parenteral, subcutaneous, intravenous, intranasal, topical, oral or local routes of administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. Commonly, the pharmaceutical compositions may be administered parenterally, e.g., intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
The compositions containing the glycolipid compounds can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will depend, as discussed further below, on the particular compound, the severity of the disease and the weight and general state of the subject, as well as the route of administration, but generally range from about 0.5 mg to about 2,000 mg of substrate per day for a 70 kg subject, with dosages of from about 5 mg to about 200 mg of the compounds per day being more commonly used.
In prophylactic applications, compositions containing the compound for use according to the invention are administered to a subject susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a “prophylactically effective dose.” In this use, the precise amounts again depend on the subject's state of health and weight, and the route of administration but generally range from about 0.5 mg to about 2,000 mg per 70 kilogram subject, more commonly from about 5 mg to about 200 mg per 70 kg of body weight.
Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the substrates of this invention sufficient to effectively treat the subject.
Labeled substrates can be used to determine the locations at which the substrate becomes concentrated in the body due to interactions between the desired oligosaccharide determinant and the corresponding ligand. For this use, the compounds can be labeled with appropriate radioisotopes, for example, 125I, 14C, or tritium, or with other labels known to those of skill in the art.
The dosage ranges for the administration of the compounds for use according to the invention are those large enough to produce the desired effect. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician monitoring the therapy.
Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to conjugate, complex or adsorb the glycosphingolipid.
In an exemplary embodiment, the invention provides a method of treating Parkinson's disease by providing neuroprotection from damage caused by toxic metabolites of dopamine. The method comprises administering to a subject in need thereof an amount of a glycolipid as set forth herein, which is sufficient to provide the neuroprotection. In an exemplary embodiment, the administration results in an amelioration of Parkinson's symptoms or a cure of the disease.
In another exemplary embodiment, the invention provides a method of binding a protein implicated in a disease state. Exemplary proteins include tau protein, beta-amyloid proteins, α-synuclein and huntingtin. The method comprises administering to a subject in need thereof an amount of a glycolipid as set forth herein, which is sufficient to protein implicated in the disease state. In an exemplary embodiment, the binding yields an amelioration or cure of the disease state by reducing or eliminating protein in the form in which it is implicated in the disease state.
A ganglioside deficiency state can be identified by assaying blood, plasma, CSF or other tissue samples from the subject for the pertinent ganglioside. A deficiency state exists when a particular ganglioside is absent or less than two-thirds that of an otherwise comparable control population. In some embodiments, the deficiency state exists when tissue levels of the pertinent ganglioside are one-half, one-quarter, one-tenth or one-twentieth of a normal, control, or reference level.
Many methods of analyzing biological samples for ganglioside and glycosphingolipid content are known to one of ordinary skill in the art. The use of such methods has been exemplified by Simpson, et al., Nature Genetics 36(11):1225 (2004) using HPLC-based methods developed by Neville, et al., Anal Biochem. 331(2):275-82 (2004) which are each incorporated herein by reference in their entirety, and particularly, with respect to such methods. These methods provide a rapid and sensitive method to both analyze and characterize the full complement of glycosphingolipid structures present in various cells and tissues. The method characterizes the oligosaccharides released from glycosphingolipids following ceramide glycanase digestion.
Additional methods for analyzing gangliosides are taught by Anumula and Dhume, Glycobiology 8:685 (1998), Svennerholm L, et al., Biochim Biophys Acta 617:97-109 (1980) and Wang B. et al., Comp Biochem Physiol A Mol Integr Physiol 119:435-9 (1998); and Wang B., e al., Am J Clin Nutr. 78(5):1024-9 (2003). Such methods can also be used to identify and quantify compounds for use according to the invention in tissue samples.
Glycolipid deficiency states suitable for treatment can also be identified by measuring the levels of the pertinent glycolipid in a sample from the subject and comparing the measured level with levels of the pertinent ganglioside measured or already established for suitable control populations as would be known to one of ordinary skill in the art. Referent levels of glycolipid in plasma for a control population have been reported previously (Simpson, Nature Genetics, 36:1225-1229 (2004)).
It is contemplated that when used to treat various diseases such as cancer, atypical Parkinson's disease; Huntington's disease, multiple systems atrophy, GM3 synthase deficiency, GM2 synthase deficiency, hyperinsulinemia, hypoglycemia, hyperinsulinemia with hypoglycemis, a tauopathy, or another neurological disorder or condition associated with an increased aggregation of tau protein, the compounds of the invention can be combined with other therapeutic agents suitable for the same or similar diseases.
The compounds, antibodies and chemotherapeutic agents of the invention are administered to a human patient, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred.
In one embodiment, the treatment of the present invention involves the combined administration of a compound from Formula (1) to (10), an antibody and one or more chemotherapeutic agents. Preparation and dosing schedules for chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede, or follow administration of the compound, the antibody or may be given simultaneously therewith.
A typical therapeutically effective dosage of a compound of the invention ranges from about 0.1 mg/kg to about 1000 mg/kg, preferably from 0.1 mg/kg to about 100 mg/kg, more preferably from about 0.1 mg/kg to about 30 mg/kg, more preferably from about 0.1 mg/kg to about 10 mg/kg, and more preferably 0.1 mg/kg to about 3 mg/kg. Advantageously, the compounds for use according to the invention , alone or as part of a pharmaceutical composition, maybe administered several times daily, and other dosage regimens may also be useful. A compound of the invention may be administered on a regimen in a single or multidose (e.g., 2 to 4 divided daily doses or applications). The regimen may be tailored over time according to the response of the subject. The dosage regimen may be hourly, daily, weekly, monthly, acute, subacute, subchronic or chronic. Up to 30 g (e.g., 1 to 2, 2 to 5, 5 to 10, 10 to 20, or 20 to 30 g), for instance, may be administered in one dose.
The effectiveness of the compounds for use according to the invention may be determined using screening protocols known in the art. The biological properties of the compounds for use according to the invention can be readily characterized by methods that are well known in the art including, for example, in vitro screening protocols (e.g., cell cultures and in vivo studies for effectiveness). Exemplary methods for identifying or screening suitable compounds for use according to the invention are set forth below.
The invention is further described with reference to the following Examples. The Examples are provided for the purpose of illustration only and the invention not be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
The synthesis of this compound uses the process described by Samain (Samain and Priem, Method for Producing Oligopolysaccharides. WO 01/04341 (2001); Priem, Glycobiology, 12:235 (2002); Cottaz, EP Application No. 06291569.9 (2006)) with E. coli JM107-nanA-(Nst-01, pBBnsy) strain using lactose and sialic acid (Kok, J Chem Soc Perkin Trans, 1:2811-2815 (1996)) acid as exogeneous acceptors.
E. coli JM107-nanA-(Nst-01, pBBnsy) strain was grown at low cell density culture in shake flask fermentation. When the OD540 is about 1, isopropyl 1-thio-β-galactopyranoside (IPTG) and the substrates Neu5Ac and lactose (2 equivalents) were added via a sterile filter and the culture is shaken at 28° C. for 16 hr. After 16 hr, the cells were recovered by centrifugation at 8000 g and 4° C. for 10 min. The supernatant was separated for further TLC analysis while the pellet is resuspended in H2O and boiled for 30 min. The resulting suspension was again centrifuged (8000 g, 4° C. for 10 min). The supernatant was mixed with activated charcoal, filtered through Celite and is washed with distilled water. The adsorbed oligosaccharides were elutetd with 50% aq. EtOH. The oligosaccharide was further purified using a DOWEX exchange ions resin (DOWEX 1×4 50) and silica gel column chromatography. The solution of product eluted from the silica gel chromatography was evaporated to dryness to yield a solid that is examined by NMR and MS to confirm the structure.
The [5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranose], and DMAP are dissolved in pyridine. Acetic anhydride was added dropwise and then the reaction mixtureis stirred for 2 days. The reaction mixture was concentrated and the residue treated with methanol. After 30 min, the reaction mixture was concetrated again, the residue dissolved in ethyl acetate and the organic solution washed with water, 5% citric acid in water, water and brine. The organic solution was dried with sodium sulfate, filtered and concetrated to dryness yielding a solid used directly for the next step.
The solid is cooled to −30° C. and then hydrogen fluoride-pyridine was added. The reaction mixture was stirred for 1 hr allowing the reaction to slowly warm to room temperature and then for 4 hrs at room temperature. The reaction mixture was cooled to −10° C. and is then slowly added to a cold solution of 6 N NaOH. Sodium carbonate is then carefully added until the pH of the solution is about 8.0. The solution was then extracted with CH2Cl2 (three times) and the combined organic layers dried over sodium sulfate. Concentration provided a solid used directly for the next step.
The solid was dissolved in anhydrous methanol and sodium methoxide in methanol is added. The reaction mixture was stirred overnight, treated with Dowex HCR-W2 resin (H+), the resin was removed by filtration and the filtrate concentrated to dryness to yield a solid. The product is charaterized by NMR and MS.
The compound 1-fluoro-[(5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside] and D-erythro-sphingosine are coupled using the procedure as described in Vaughan, J Am Chem Soc, 128:6300-6301 (2006). The reaction was performed in 25 mM NaOAc (pH 5.0) containing 0.2% Triton X-100. A typical reaction mixture contained approximately 10 mM 1-fluoro-[(5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside], 20 mM of the acceptor D-erythro-sphingosine, and 0.5 mg/mL of the appropriate EGC mutant in a total reaction volume. When completed, the product was purified using reversed phase (C-18) chromatography, the eluted product was concentrated to dryness, dissolved in water and freeze-dried to yield a white solid. The product is analyzed by NMR and MS.
The [(5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl-β-(1−1′)-D-erythro-sphingosine] was dissolved in anhydrous methanol that contains triethylamine. Stearic anhydride was added as a solid to the reaction mixture and the reaction stirred for 24 hrs. The product was purified using silica gel chromatography eluting the product with CHCl3/CH3OH. Concentration of the product fractions provides a solid that is analyzed by NMR and MS.
The synthesis of this compound used the process described by Samain (Samain and Priem, Method for Producing Oligopolysaccharides. WO 01/04341 (2001); Priem, Glycobiology, 12:235 (2002); Cottaz, EP Application No. 06291569.9 (2006)) with E. coli JM107-nanA-(Nst-01, pBBnsy) strain using lactose.
E. coli JM107-nanA-(Nst-01, pBBnsy) strain is grown at low cell density culture in shake flask fermentation. When the OD540 was about 1, isopropyl 1-thio-β-galactopyranoside (IPTG) and the substrates Neu5Ac and lactose (2 equivalents) were added via a sterile filter and the culture was shaken at 28° C. for 16 hr. After 16 hr, the cells were recovered by centrifugation at 8000 g and 4° C. for 10 min. The supernatant was separated for further TLC analysis while the pellet is resuspended in H2O and boiled for 30 min. The resulting suspension is again centrifuged (8000 g, 4° C. for 10 min). The supernatant was mixed with activated charcoal, filtered through Celite and was washed with distilled water. The adsorbed oligosaccharides were elutetd with 50% aq. EtOH. The oligosaccharide was further purified using a DOWEX exchange ions resin (DOWEX 1×4 50) and silica gel column chromatography. The solution of product eluted from the silica gel chromatography was evaporated to dryness to yield a solid that is examined by NMR and MS to confirm the structure.
The (β-D-galactopyranosyl)-(1→3)-(β-D-2-N-acetamido-galactopyranosyl)]-β-(1→4)-[(5-N-acetyl-α-neuraminyl)]-(2→3)-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, and DMAP were dissolved in pyridine. Acetic anhydride was added dropwise and then the reaction mixtureis stirred for 2 days. The reaction mixture was concentrated and the residue treated with methanol. After 30 min, the reaction mixture was concetrated again, the residue dissolved in ethyl acetate and the organic solution washed with water, 5% citric acid in water, water and brine. The organic solution was dried with sodium sulfate, filtered and concetrated to dryness yielding a solid used directly for the next step.
The solid was cooled to −30° C. and then hydrogen fluoride-pyridine was added. The reaction mixture was stirred for 1 hr allowing the reaction to slowly warm to room temperature and then for 4 hrs at room temperature. The reaction mixture was cooled to −10° C. and was then slowly added to a cold solution of 6 N NaOH. Sodium carbonate was then carefully added until the pH of the solution was about 8.0. The solution was then extracted with CH2Cl2 (three times) and the combined organic layers dried over sodium sulfate. Concentration provides a solid used directly for the next step.
The compound 1-fluoro-[(β-D-galactopyranosyl)-(1→3)-(β-D-2-N-acetamido-galactopyranosyl)]-β-(1→4)-[(5-N-acetyl-α-neuraminyl)]-(2→3)-(β-D-galactopyranosyl)-(1→4)-β-D-glucopyranoside] and (1R,2R)-2-amino-1-(1,3-dioxolan-2 yl)-propane-1,3-diol) is coupled using the procedure described in Example 3 using a mutant EGC enzymes (Vaughan, J Am Chem Soc, 128:6300-6301 (2006)). The reaction is performed in 25 mM NaOAc (pH 5.0) containing 0.2% Triton X-100. A typical reaction mixture contained approximately 10 mM 1-fluoro-[(5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranoside], 20 mM of the acceptor D-erythro-sphingosine, and 0.5 mg/mL of the appropriate EGC mutant in a total reaction volume. When completed, the product is purified using reversed phase (C-18) chromatography, the eluted product is concentrated to dryness, dissolved in water and freeze-dried to yield a white solid. The product is analyzed by NMR and MS.
The [(5-N-acetyl-α-neuraminyl)-(2→3)]-[β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl]-β-(1→1′)-O-[2-N-stearoyl-D-erythro-sphingosine], a water stable Grubb's catalyst (Hong, J Am Chem Soc, 128:3508-3509 (2006) and styrene is stirred in water/methanol. The reaction mixture is then concentrated to dryness and purified by silica gel chromatography using CHCl3/CH3OH. The eluted product fractions is concentrated to afford the product as a solid that was characterized by NMR and MS.
The (5-N-acetyl-α-neuraminyl)-(2→3)-(β-D-galactopyranosyl)-(1→4)-(β-D-glucopyranosyl)-β-(1→1′)-O-[(1R,2R)-2-amino-1-(1,3-dioxolan-2 yl)-propane-1,3-diol)]-β-(1→1′)-O-[(2R,3R,E)-2-amino-4-(dioctylamino)-6-phenylhex-5-ene-1,3-diol] is dissolved in anhydrous methanol that contains triethylamine. Stearic anhydride is added as a solid to the reaction mixture and the reaction stirred for 24 hrs. The product is purified using silica gel chromatography eluting the product with CHCl3/CH3OH. Concentration of the product fractions provides a solid that is analyzed by NMR and MS.
The solid is dissolved in methanol, tartaric acid, and water and stirred at room temperature for 24 hrs. The reaction mixture is neutralized with sodium bicarbonate the product extracted with CH2Cl2 (3 times). The organic layers are combined and dried with NaSO4, filtered and concentrated to dryness. The solid is used directly for the next step.
The product from the previous step (20 mg, 0.01 mmol) and dioctylamine (6 mg, 0.024 mmol) are dissolved in dimethylformamide (DMF) at room temperature. A solution of trans-2-phenylvinylboronic acid (9 mg, 0.045 mmol) dissolved in methanol (5 mL) is then added. The resulting solution is stirred at room temperature for three days. The reaction mixture is then concentrated to dryness on a rotovap and the residue purified by solid phase extraction using a 1 g HAX cartridge. The eluant is then purified using HPLC to afford the product as a solid that is characterized by NMR and MS.
The efficacy of compounds of formula (1)-(10) compounds alone and in combination with a kinase inhibitor is measured by a cell proliferation assay employing the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res. 62:5485-5488). An aliquot of 100 μL of cell culture containing about 104 cells (from the ATCC or DZM such as A549, NCI-H441, NCI-1650, NCI-1975, PC-3, DiFi, HN5, SKBR3, MCF-7, HCT-116, CCL-221, NCI-H125, SK-OV-3, MIA PaCa-2, T-47D) in medium are deposited in each well of a 384-well, opaque-walled plate. Control wells are prepared containing medium and without cells. The compound are added to the experimental wells and incubated for 3-5 days. The plates are equilibrated to room temperature for approximately 30 minutes and a volume of CellTiter-Glo Reagent equal to the volume of cell culture medium present in each well is added. The contents are mixed for 2 minutes on an orbital shaker to induce cell lysis. The plate is incubated at room temperature for 10 minutes to stabilize the luminescence signal and the luminescence recorded and reported in graphs as RLU=relative luminescence units.
Alternatively, cells are seeded at optimal density in a 96 well plate and incubated for 4 days in the presence of test compound. Alamar Blue™ is subsequently added to the assay medium, and cells are incubated for 6 h before reading at 544 nm excitation, 590 nm emission. EC50 values are calculated using a sigmoidal dose response curve fit.
As cancer cell lines, human lung cancer A549 cells (ATCC No. CCL-185), human ovarian cancer SK-OV-3 cells (ATCC No. HTB-77), and human colon cancer HCT 116 cells (ATCC No. CCL-247) are used. For the culture of A549 cells, Nutrient Mixture F-12K medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin are used. For the culture of SK-OV-3 cells and HCT 116 cells, McCoy's 5A medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin are used. The cells are cultured at 37° C. in a 5% carbon dioxide atmosphere.
A549 cells (1000 cells/well), SK-OV-3 cells (2000 cells/well), or HCT 116 cells (1000 cells/well) are seeded in each well of 96-well plates, and cultured overnight. Test compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be tested are applied to the appropriate wells at a variety of concentrations and at various time points beginning at 24, 18, 12, 6, and 0 hrs prior to kinase inhibitor addition at three different concentrations. The cells are further cultured for 72 hours. Fifty iut of labeling mixture of Cell Proliferation Kit II (XTT) are added to each well, and the plates are incubated at 37° C. After 1 to 3 hours, the absorbance at 490 nm (reference wavelength: 655 nm) is measured with a plate reader. Growth ratios of the cells in the wells treated with the test compounds are calculated based on the growth ratio of the cells in the control well treated with solvent (dimethyl sulfoxide (DMSO)) for 72 hours, which was defined as 100%. From a plot of test compound concentrations and the cell growth ratios at the concentrations, the concentration of 50% growth inhibition, the GI50 value, are calculated.
As cancer cell lines, human pancreatic cancer MIA PaCa-2 cells (JCRB No. 0070), human cervical cancer HeLa cells (ATCC No. CCL-2), human breast cancer MDA-MB-468 cells (ATCC No. HTB-132), human prostate cancer DU 145 cells (ATCC No. HTB-81), human skin cancer SK-MEL-28 cells (ATCC No. HTB-72), human head and neck cancer KB cells (JCRB No. 9027), human renal cancer 786-0 cells (ATCC No. CRL-1932), and human liver cancer Hep G2 cells (ATCC No. HB-8065) are used. The cells are cultured at 37° C. in a 5% carbon dioxide atmosphere (except for the human breast cancer MDA-MB-468 cells, which are cultured under the condition at 37° C.) by using the mediums mentioned below, respectively.
Cell Medium: Human pancreatic Minimum Essential Medium (MIA PaCa-2) containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human cervical cancer Minimum
Essential Medium containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human breast cancer Leibovitz's L-15 Medium (MDA-MB-468) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin; Human prostate cancer Minimum Essential Medium containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 1 mmol/L Sodium Pyruvate Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human skin cancer Minimum Essential Medium (SK-MEL-28) containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human head and neck Minimum Essential Medium (KB cell 11095-080) containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human renal cancer RPMI 1640 Medium (786-O cell) containing 10% fetal bovine serum, 10 mmol/L HEPES Buffer Solution, 1 mmol/L Sodium Pyruvate Solution, 4.5 g/L D-(+)-Glucose Solution, 100 units/mL penicillin and 100 μg/mL streptomycin; Human liver cancer Minimum Essential Medium (Hep G2 cell) containing 10% fetal bovine serum, 0.1 mmol/L MEM Non-Essential Amino Acids Solution, 1 mmol/L Sodium Pyruvate Solution, 100 units/mL penicillin and 100 μg/mL streptomycin.
In the same manner as that in Example 9, the cells are seeded (500 to 4000 cells/well, respectively) in each well of 96-well plates, and the growth ratios of the cells treated with test compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are applied to the appropriate wells at a variety of concentrations and at various time points beginning at 24, 18, 12, 6, and 0 hrs prior to kinase inhibitor addition at three different concentrations. The measurement of absorbance is performed at 1.5 to 3 hours after the addition of the XTT labeling mixture. From a plot of test compound concentrations and the cell growth ratios at the concentrations, the concentration of 50% growth inhibition, the GI50 value, are calculated.
In an exemplary model system, nude (nu/nu) mice are inoculated with MDA-MB-231 cells (human breast carcinoma) (106 cells in 0.2 mL) s.c. in the right flank of the animals. The tumors are staged to 200 mm3 and then treatment with the test compound (1, 30 or 100 mg/Kg; IV or sc) and kinase inhibitor (IV or sc)(e.g., lapatinib). Tumor volumes are obtained every other day and the animals are sacrificed after 2 weeks of treatment. The tumors are excised, weighed and paraffin embedded. Histological sections of the tumors are analyzed, for example, by H and E, anti-CD31, Ki-67, TUNEL, and CD68 staining
A xenotransplant model of the Raji Burkitt's lymphoma in SCID mice is used. Raji cells after s.c. injection cause locally growing tumors. Treatment is started when the tumors have reached a size of 5 mm in diameter. At days 0, 7, and 15, cohorts of five mice receive i.v. either PBS (control group) or in vitro preactivated human PBLs. Four hrs after each PBL inoculation, the mice are treated via tail vein injection either with no antibody, or the following: compounds of the invention alone; compound (1, 50 or 100 mg/Kg) and kinase inhibitor; or compound (1, 50 or 100 mg/Kg) 3 hrs prior to administration of kinase inhibitor. Tumor size is measured using a caliper every 2nd day. Animals are followed until the s.c. tumors reached a maximal tolerated size of 15 mm in diameter and are then killed by cervical dislocation. The days of sacrifice are recorded and are used for survival time analysis.
The compounds of the invention were dissolved in PBS and administered in Gefitinib (AstraZeneca Pharmaceuticals) was dissolved in vehicle containing 1% Tween 80. AEE788 (Novartis Pharmaceuticals) was dissolved in vehicle containing 90% polyethylene glycol 300 and 10% 1-methyl-2-pyrrolidinone. Five days after the implantation of the NCI-H441 or PC14-PE6 tumor cells into their lungs, mice (8-10 per group) are randomized into treatment groups and treated with: compounds of the invention (once daily, s.c. or oral gavage; 1-100 mg/Kg); an EGFR1 inhibitor (e.g., gefitinib, po, once daily, 50 mg/Kg; AEE788 po, 3×weekly, 50 mg/Kg; lapatinib, p.o., once daily, 50 mg/Kg; erlotinib, p.o., once daily, 50 mg/Kg); or compounds of the invention (once daily, s.c. or oral gavage; 1-100 mg/Kg) in combination with an EGFR1 inhibitor (selected from gefitinib, p.o., once daily, 50 mg/Kg; AEE788 po, 3×weekly, 50 mg/Kg; lapatinib, p.o., once daily, 50 mg/Kg or erlotinib, p.o., once daily, 50 mg/Kg) or vehicle control. All of the mice are killed and autopsied when control animals become moribund and primary lung tumor weight (total tumor-bearing lung weight minus the normal lung weight of 0.17 g), incidence and volume of pleural effusion, and the presence of metastasis are measured.
Glucose transport are determined in 3T3-L1 adipocytes or primary human adipocytes as uptake of 2-deoxy-D[1-3H]glucose by the method of Frost, J Biol Chem, 262:9872-9876 (1987). Cells are grown on 13 mm plastic coverslips in 24-well culture dishes. The cells are treated with the compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) at concentrations of 1, 10, 50, 100 μM) and incubated with the cells for at various time points from 0, 30 min, 1 hr and 2 hrs prior to addition of 2-deoxy-D-[1-3H]glucose. The 2-deoxy-D-[1-3H]glucose is then added to a final concentration of 50 mM (0.5 mCi/mL) and the cells are incubated for 6 min. Glucose uptake is stopped by rinsing the coverslips in three successive solutions of ice-cold buffer. Nonspecific uptake is determined in the presence of 25 mM of cytochalasin B. Coverslips are transferred to scintillation vials and the cells are dissolved in 1% SDS. Radioactivity is measured after adding 5 mL of scintillation fluid.
Human Lymphoid cells (IM9) are grown in culture and the insulin receptor isolated as described (Nojiri, J Biol Chem, 266:4531-4537 (1991)). Compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) at concentrations of 0, 0.1, 1, 10, 30, 100 and 200 μM are added to a solution (50 mM HEPES, pH 7.4, 0.1% (v/v) Triton X-100; 30 μL) containing the purified insulin receptor. Insulin (26.3 IU/mg; 5 μL) and MnCl2 (30 mM, 10 μL) are then added to each tube and incubated at room temperature for 1 hr. Phosphorylation is initiated by adding ATP-[γ-32P] (5 μL; 25 μCi) and the reaction mixture incubated at room temperature for 10 min. The reaction is terminated by the addition of Laemmli's buffer (Laemmli, Nature, 227:680-685 (1970)) containing DTT (75 mg/mL) and heating in boiling water for 3 min. The phosphoproteins are separted by SDS-PAGE, the gel stained with Cibracron blue and an autoradiograph taken. The β-subunit of the insulin receptor is cut from the gel and the amount of radioactivity quantitified using a liquid scintillation counter. The amount of inhibition is then plotted.
Experiments are performed in mice fasted for 14 h. The compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered IV, SC, oral gavage or IP to the mice at doses of 0, 0.1, 1, 10, 30 or 100 mg/Kg. Insulin (5 units per animal) is administered at 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr or 24 hr after compound administration through the inferior vena cava under anesthesia. Two minutes after injection, hind limb muscle and adipose tissue are removed and incubated for 30 min on ice with lysis buffer (PBS, pH 7.4/1% Nonidet P-40/0.5% sodium a deoxycholate/0.1% SDS/1 mM PMS/10 mM sodium orthovanadate/3 g/ml aprotinin) Lysates are immunoprecipitated with an anti-insulin receptor subunit antibody. Immunoprecipitated samples are subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The blots are probed sequentially, first with an antiphospho-tyrosine antibody (PY99) and then with an anti-insulin receptor-subunit antibody. Blots are developed by a chemiluminescence detection system. The mice may be normal mice or mice that contain one or more genes that have been deleted, mutated, knocked-in or a combination thereof.
Glucose and insulin tolerance tests are performed in fasted mice (13-15 h) with i.p. injections of glucose (2 g of glucose per kg of body weight) or insulin (0.75 units of insulin per kg of body weight), respectively. For the glucose tolerance test, compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered by IV, SC, oral gavage or IP at a dose of 0, 0.1, 1, 10 and 100 mg/Kg at timepoints of 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr or 24 hrs prior to glucose injection. Blood glucose values are measured immediately before and 15, 30, 60, and 120 min after glucose injection. For the insulin tolerance test, compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered by IV, SC, oral gavage or IP at a dose of 0.1, 1, 10 and 100 mg/Kg at timepoints of 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr or 24 hrs prior to insulin injection. Blood glucose levels are measured immediately before and 15, 30, and 60 min after insulin injection. To induce glucose intolerance, 6- to 8-week-old mice are placed on a 45% high-fat diet (commercial preparation) for 10 weeks (Steppan, Nature, 409:307-312 (2001)). The mice may be normal mice or mice that contain one or more genes that have been deleted, mutated, knocked-in or a combination thereof.
Animals are fasted overnight. The compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered via IV, SC, oral gavage or IP at a dose of 0, 0.1, 1, 10, 30 or 100 mg/Kg. A 120-minute hyperinsulinemic-hypoglycemic clamp is conducted with a continuous infusion of human insulin at a rate of 15 pmol/Kg/min or higher to create a hyperinsulinemic hypoglycemic state that is started at 0 min, 30 min, 1 hr, 2 hr, 6 hr, 12 hr and 24 hrs after compound administration. Blood samples (20 μL) are collected at 20- to 30-minute intervals for the immediate measurement of plasma glucose concentration. Insulin-stimulated whole-body glucose flux is estimated using a prime-continuous infusion of HPLC-purified [3-3H]glucose (10 μCi bolus, 0.1 μCi/min) throughout the clamps. To estimate insulin-stimulated glucose transport activity in individual tissues, 2-deoxy-D[1-14C]glucose (2-[14C]DG) is administered as a bolus (10 μCi) at 45 minutes before the end of clamps. Blood samples (20 μL) are taken at 77, 80, 85, 90, 100, 110, and 120 minutes after the start of clamps for the determination of plasma [3H]glucose, 3H2O, and 2-[14C]DG concentrations. Additional blood samples (10 μL) are collected before the start and at the end of clamps for measurement of plasma insulin concentrations. All infusions are done using microdialysis pumps. At the end of clamps, animals are anesthetized with sodium pentobarbital injection. Within 5 minutes, four muscles (soleus, gastrocnemius, tibialis anterior, and quadriceps) from both hindlimbs, epididymal adipose tissue, and liver are taken. Each tissue, once exposed, is dissected out within 2 seconds, frozen immediately using liquid N2-cooled aluminum blocks, and stored at −70° C. for later analysis.
Plasma glucose concentration during clamps are analyzed using 10 μL plasma by a glucose oxidase method on a Beckman glucose analyzer II. Plasma insulin concentration are measured by RIA using kits. For the determination of plasma [3-3H]glucose and 2-[14C]DG concentrations, plasma is deproteinized with ZnSO4 and Ba(OH)2, dried to remove 3H2O, resuspended in water, and counted in scintillation fluid on dual channels for separation of 3H and 14C. The plasma concentration of 3H2O is determined by the difference between 3H counts without and with drying. For the determination of tissue 2-14C]DG-6-phosphate (2-DG-6-P) content, tissue samples are homogenized, and the supernatants are subjected to an ion-exchange column to separate 2-DG-6-P from 2-DG (Ohshima, Am J Physiol, 246:E193-E197 (1984)). The radioactivity of 3H in muscle glycogen is determined by digesting muscle samples in KOH and precipitating glycogen with ethanol (Kim, Diabetes, 45:446-453 (1996)). Skeletal muscle glycogen synthase activity is measured using 14C-UDPG.
The compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) are administered to rats via i.v., s.c., p.o. (oral gavage), or i.p. at doses of 0, 0.1, 1, 10, 30 and 100 mg/Kg. Blood glucose levels and insulin levels are measured by drawing blood from the animal at 0, 30 min, 1 hr, 1.5 hrs, 2 hrs, 4 hrs, 6 hrs, 12 hrs and 24 hrs after administration of the compound. Whole blood is assayed for glucose content using the glucose dehydrogenase based enzymatic assay and quantitated using the Hemocue glucose meter. Insulin levels are assayed in 15 μL of serum using the Rat Insulin RIA kit according to manufacturer's procedure. Intraperitoneal glucose tolerance tests are made on 12- to 20-week-old mice, after 16 hr fast. Animals are injected i.p. with glucose (1 g/Kg). Blood is isolated from the tail vein at times indicated and assayed for glucose content as described above. Insulin content is assayed in isolated islets of equal diameter (˜100 μm). Islets are sonicated in distilled water, then pelleted for 5 min at 1,000×g. Supernatant is removed and diluted 1:6,000 for RIA as described above.
Exendin-(9-39) Significantly Raises Fasting Blood Glucose Levels in SUR-1−/−Mice
Twelve-18 week old male SUR-1−/− and wildtype littermates undergo a baseline evaluation including fasting blood glucose measurements and oral glucose tolerance testing, followed by randomization to treatment with compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) (0, 0.1, 1, 10, 30 or 100 mg/Kg/day; i.v., s.c., p.o. (oral gavage) or i.p.) or vehicle (0.9% NaCl, 1% BSA). Fasting blood glucose levels are determined after an overnight fast on days 3 and 7 after initiation of the study. In addition, oral glucose tolerance and insulin sensitivity are evaluated during treatment.
Pancreatic islets (10 per well), isolated as described in Koster, Proc Natl Acad Sci USA, 99: 16992-16997 (2002), are incubated in glucose-free DMEM supplemented with D-(+)-glucose (1, 7, or 16.7 mM) and either gliblenclamide (1 μM), diazoxide (250 μM) or one or more of the compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10)(0.1, 1, 10, 30 and 100 μM), as indicated. Alteneratively, the islets are first incubated with the compounds of Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) (0.1, 1, 10, 30 and 100 μM) for 0, 30 min, 1 hr, 2 hrs, 4 hrs or 6 hrs prior to glucose, gliblenclamide or diazoxide addition. Islets are incubated for 60 min at 37° C., medium removed and assayed for insulin content with Rat Insulin RIA kit. Pancreatic Islets may be isolated from normal or genetically modified animals or humans.
After an overnight fast, subject receives an intravenous infusion or subcutaneous injection of a compound selected from Formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) (1, 30 or 100 mg/Kg/day). On the second day, the subject is fasted overnight. Blood samples for glucose, insulin, C-peptide, and glucagon are obtained at different intervals after compound administration.
Cancer cell samples are obtained from the affected individual from peripheral blood or bone marrow aspirates. In this examples, either B-cell CLL or MCL cells, depending on the malignancy being studies, were enriched using immunomagnetic sorting (Decker, Blood, 95:999-1006 (2000)). The cells are then cultured in RPMI 1640, supplemented with 30% autologous serum, 50 IU/mL penicillin/streptomycin, glutamine, CpG oligonucleotide DSP30, IL-2 and mercaptoethanol. Cells are plated in 96 well dishes and growth for 48 hrs. Cells are then resuspended in human serum and culture medium and the compound of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be tested is applied to the culture at a variety of concentrations at various time points beginning at 24, 18, 12, 6, and 0 hrs prior to antibody addition (Rituxan). The Rituxan (anti-CD20) is then added and the cells incubated for 24 hrs. Dead and viable cells are discriminated by Annexin V/propidium iodide staining and assessed by flow cytometry.
As cancer cell lines, MCF7, human lung cancer A549 cells (ATCC No. CCL-185), human ovarian cancer SK-OV-3 cells (ATCC No. HTB-77), and human colon cancer HCT 116 cells (ATCC No. CCL-247) are used. For the culture of A549 cells, Nutrient Mixture F-12K medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin are used. For the culture of SK-OV-3 cells and HCT 116 cells, McCoy's 5A medium containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin are used. The cells are cultured at 37° C. in a 5% carbon dioxide atmosphere.
A549 cells (1000 cells/well), SK-OV-3 cells (2000 cells/well), or HCT 116 cells (1000 cells/well) are seeded in each well of 96-well plates, and cultured overnight. Test compounds of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be tested are applied to the appropriate wells at a variety of concentrations and at various time points beginning at 24, 18, 12, 6, and 0 hrs prior to antibody addition at three different concentrations. The cells are further cultured for 72 hours. Fifty μL of labeling mixture of Cell Proliferation Kit II (XTT) are added to each well, and the plates are incubated at 37° C. After 1 to 3 hours, the absorbance at 490 nm (reference wavelength: 655 nm) is measured with a plate reader. Growth ratios of the cells in the wells treated with the test compounds are calculated based on the growth ratio of the cells in the control well treated with solvent (dimethyl sulfoxide (DMSO)) for 72 hours, which was defined as 100%. From a plot of test compound concentrations and the cell growth ratios at the concentrations, the concentration of 50% growth inhibition, the GI50 value, are calculated.
WSU-NHL and Jurkat cells are seeded at 5,000 cells per well in 96-well plates in quadruplicates. Proliferation assays are performed for 96 hours. Tritiated thymidine (3H-TdR) incorporation during the last 16 hours of incubation was used to assess DNA synthesis.
The responses of the compound of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) to be tested is applied to the culture at a variety of concentrations at various time points beginning at 24, 18, 12, 6, and 0 hrs prior to antibody addition (anti-CD70).
To induce apoptosis, mouse cortical cells were cultured and treated with 50 μM hydrogen peroxide for three hours prior to being treated with the ganglioside analogue.
The cells were also treated with the hydrogen peroxide during treatment with the ganglioside analogue and post-treatment for 48 h. Cell death was assayed using the MTT assay.
Approximately 30% of the cells treated with hydrogen peroxide died as a result of the treatment. Treatment with GM1 (approx. 0.1 μM) provided approximately 20% protection of the cells from apoptosis.
To induce non-apoptotic cell death, mouse cortical cells can be cultured and treated with 50 μM hydrogen peroxide and oligomycin (0.01 μM) for three hours prior to being treated with the ganglioside analogue. The cells are also treated with the hydrogen peroxide and oligomycin during treatment with the ganglioside analogue and post-treatment for 48 h. Cell death can be assayed using the MTT assay.
Approximately 30% of the cells treated with hydrogen peroxide can die as a result of the treatment. Treatment of the cells with compounds for use according to the invention protects approximately 20% protection of the cells from death.
C57B1/6 mice 7-8 weeks of age can be treated with MPTP (b.i.d., 20 mg/kg, s.c.). The mice also receive a daily administration of saline, compound of formula (1), (2), (3), (4), (5), (6), (7), (8), (9) or (10) (1, 10 and 30 mg/kg, o.s.) or (0.3. to 3 mg/kg, i.p. or s.c.) for three weeks starting 24 h after the last MPTP injection. The brains are removed and analyzed for striatum and substantia nigra pars compacta dopamine levels. The midbrain is fixed for TH immunohistochemistry and dopamine neuron cell counts.
MPTP alone can cause approximately 76% loss of striatal dopamine. GMla and compounds for use according to the invention increase striatal dopamine levels to approximately the same extent.
Transgenic R6/2 mice, 40 day old, expressing human Huntingtin protein are administered compounds selected from (1) to (10) by intraperitoneal injection (3 or 30 mg/Kg) or oral gavage (30 mg/Kg), daily, for six weeks. Twice weekly, both control mice, receiving saline, and treated mice are assessed for disease progression using the rotorod test by methods known in the art.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the extent not inconsistent with the present disclosure.
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/163,371 filed on Mar. 25, 2009, U.S. Provisional Patent Application No. 61/293,200, filed Jan. 7, 2010, U.S. Provisional Patent Application No. 61/244,735, filed Sep. 22, 2009, U.S. Provisional Patent Application No. 61/220,151, filed Jun. 24, 2009, U.S. Provisional Patent Application No. 61/180,346, filed May 21, 2009 and U.S. Provisional Patent Application No. 61/180,098, filed May 20, 2009, which are incorporated herein by reference in their entirety for all purposes.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/28725 | 3/25/2010 | WO | 00 | 9/26/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61163371 | Mar 2009 | US | |
| 61180098 | May 2009 | US | |
| 61180346 | May 2009 | US | |
| 61220151 | Jun 2009 | US | |
| 61244735 | Sep 2009 | US | |
| 61293200 | Jan 2010 | US |
