HEMITARTARATE SALT AND PHARMACEUTICAL COMPOSITION UNDERSTANDING THE SAME

专利摘要:
hermitatarate salt, uses thereof and pharmaceutical compositions that comprise it. the present invention relates to the hemitheartate salt of a compound represented by means of the following structural formula: (hemitartarate of formula i), which can be used in pharmaceutical applications. the particular individual crystalline forms of the hemitartrate of formula (i) are characterized by means of a variety of physical properties and measurements. , as well as, crystalline hermitardate production methods of formula (i), and are used to inhibit glycosylceramide synthase or decrease glycosphingolipid concentrations in individuals for the treatment of a number of diseases. pharmaceutical compositions are also described.
公开号:BR112012012947B1
申请号:R112012012947-3
申请日:2010-11-24
公开日:2021-04-06
发明作者:Hanlan Liu；Chris Willis；Renu Bhardwaj；Diane P. Copeland；Abizer Harianawala；Jeffrey Skell；John Marshall；Jianmei Kochling；Gerard Palace；Judith Peterschmitt；Craig Siegel；Seng Cheng
申请人:Genzyme Corporation；
IPC主号:

专利说明:

[0001] [0001] This patent application claims the benefit of U.S. Provisional Patent Application 61 / 264,748, filed on November 27, 2009, the teachings in their entirety which are incorporated into the present invention. BACKGROUND
[0002] [0002] Glycosphingolipids (GSLs) are a class of naturally occurring compounds that have a multitude of biological functions, including the ability to promote cell growth, cell differentiation, adhesion between cells or between cells and matrix proteins, the attachment of microorganisms and viruses to cells, and metastasis of tumor cells. GSLs are derived from glycosylceramide (GlcCer), which is produced from ceramide and glUDP-glucose by means of the enzyme UDP-glucose: N-acylsphingosine glycosyltransferase (GlcCer synthase). The ceramide structure is shown below:
[0003] [0003] The accumulation of GSLs has been associated with a number of diseases, including Tay-Sachs, Gaucher, and Fabry disease (see, for example, U.S. Patent No. 6,051,598). GSLs have also been linked to certain types of cancer. For example, it has been found that certain GSLs occur only in tumors or in high concentrations at abnormal levels in the tumors; exert stimulatory actions or marked inhibitory actions on the growth of tumors, when added to tumor cells in culture media, and the inhibition of the body's normal immune defense system when shed by tumors in the surrounding extracellular fluid. The composition of GSLs in the tumor changes as tumors become increasingly malignant and antibodies to certain GSLs inhibit tumor growth.
[0004] [0004] Compounds that inhibit GlcCer synthase can decrease GSL concentrations and have been reported to be useful for treating an individual with one of the diseases mentioned above. A number of potent GlcCer inhibitors, referred to in the present invention as "amino ceramide type compounds", are described in US Patent Nos. 6,051,598, 5,952,370, 5,945,442, 5,916,911 and 6,030,995. The compound of Formula (I), shown below, is an inhibitor of GlcCer synthase currently in clinical trials for the treatment of Gaucher disease:
[0005] [0005] There is a need for salt forms of the present drug candidate which are crystalline and otherwise have physical properties that are amenable to manufacture on a large scale. There is also a need for pharmaceutical formulations in which this candidate drug is stable and effective when administered to the patient, as well as with regard to improved treatment methods using this compound. SUMMARY OF THE INVENTION
[0006] [0006] It has been found that the hemitartrate salt of the compound of Formula (I) (hereinafter "Hemitartarate of Formula (I)") can be crystallized under well-defined conditions in order to provide certain non-hygroscopic crystalline forms. Formula (I) hemitartarate has several advantageous properties when compared to other Formula (I) salts. As described in Example 1, many Formula (I) salts, including citrate, malate, fumaric, methylsulfonic, and acetic salts, do not could be obtained in solid form. Although the hydrochloric salt and 1: 1 of the formula (I) tartrate salt were obtained in solid form, neither was crystalline and both were too hydroscopic for the formulation. Formula (I) hemitartarate is easier to formulate and synthesize than the free base and the other salts. Formula (I) hemitartarate is also crystalline, non-hygroscopic, soluble in water and flows better than the corresponding free base (hereinafter referred to as "Free Base Formula (I)") and other salts. In this way, these favorable properties make Formula (I) Hemitartarate liable to be manufactured on a large scale as a candidate drug.
[0007] [0007] It has also been found that stable granules for Formula (I) Hemitartarate capsule formulations can be prepared using defined proportions of a water-insoluble filling agent, a water-soluble filling agent and Formula (I Hemitartarate). ). Based on this discovery, stable pharmaceutical formulations of Formula (I) Hemitartarate are described.
[0008] [0008] It has also been found that the compound of Formula (I) or pharmaceutically acceptable salts thereof (including Formula (I) Hemitartarate) are metabolized by the liver, mainly by cytochrome P450 enzymes. Based on this discovery, methods of treatment with the Formula (I) compound or pharmaceutically acceptable salts thereof (including Formula (I) Hemitartarate), which reduce the potential for drug / drug interactions are described.
[0009] [0009] It has also been found that the Gaucher mice that were given recombinant glycocerebrosidase and then Formula (I) Hemitartarate showed lower levels of GL1 in visceral organs and a reduced number of Gaucher cells in the liver in comparison with treatment with glycocerebrosidase alone or Formula (I) Hemitartarate alone. Based on this finding, combination therapies with the compound of Formula (I) or pharmaceutically acceptable salts thereof (including Formula (I) Hemitartarate) are also described.
[0010] [00010] One embodiment of the present patent application is the hemitartrate salt of the compound represented by Formula (I). As noted above, the hemitartrate salt of the compound represented by Formula (I) is referred to in the present invention as "Formula (I) hemitartrate." The compound represented by Formula (I) is referred to in the present invention as "Formula (I) free base".
[0011] [00011] Another embodiment of the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and Hemitartarate of Formula (I).
[0012] [00012] Another embodiment provides a method of glycosylceramide synthase which inhibits or decreases the concentrations of glycosphingolipids in an individual in need, by administering to the individual an effective amount of Formula (I) Hemitartarate.
[0013] [00013] Another modality provides for the use of Formula (I) Hemitartarate for the manufacture of a drug to inhibit glycosylceramide synthase or decrease the concentrations of glycosphingolipids in an individual in need of them.
[0014] [00014] Another modality provides the use of Formula (I) Hemitartarate to inhibit glycosylceramide synthase or decrease the concentrations of glycosphingolipids in an individual in need of them.
[0015] [00015] Another modality is a method of treating an individual with Gaucher disease. The method comprises administering to the individual an effective amount of a first therapeutic agent in combination with an effective amount of a second therapeutic agent. The first therapeutic agent is represented by means of Formula (I) or a pharmaceutically acceptable salt thereof, and the second therapeutic agent is effective for the treatment of Gaucher disease.
[0016] [00016] Another modality is a method of treating an individual with Fabry's disease. The method comprises administering to the individual an effective amount of a first therapeutic agent in combination with an effective amount of a second therapeutic agent. The first therapeutic agent is represented by means of Formula (I) or a pharmaceutically acceptable salt thereof, and the second therapeutic agent is effective for the treatment of Fabry's disease.
[0017] [00017] Another embodiment provides the pharmaceutical composition comprising: the hemitartrate salt of a compound represented by Formula (I); at least one water-soluble filler, at least one water-insoluble filler, at least one binder, and at least one lubricant.
[0018] [00018] Another embodiment of the present invention is a method of treating an individual with Fabry's disease. The method comprises the steps of: a) administering to the individual an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof; b) testing the individual to determine whether the individual is a slow, intermediate or extensive / ultra-fast P450 metabolizer; c) if the individual is an intermediate or extensive / ultrafast P450 metabolizer, determining an effective adjusted value of the compound, and d) administering to the individual an effective adjusted amount of the compound of Formula (I), if the individual is an intermediate or extended / ultrafast P450 metabolizer and administering to the individual an effective amount of the compound of Formula (I), if the individual is a slow P450 metabolizer.
[0019] [00019] Another embodiment of the present invention is a method of treating an individual with Gaucher disease. The method comprises the steps of: a) administering to the individual an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof; b) testing the individual to determine whether the individual is a slow, intermediate or extensive / ultra-fast P450 metabolizer; c) if the individual is an intermediate or extensive / ultrafast P450 metabolizer, determining an effective adjusted value of the compound, and d) administering to the individual an effective adjusted amount of the compound of Formula (I), if the individual is an intermediate or extended / ultrafast P450 metabolizer, and administering to the individual an effective amount of the compound of Formula (I), if the individual is a slow P450 metabolizer.
[0020] [00020] Another embodiment of the present invention is a method of treating an individual with Fabry's disease. The method comprises the steps of: a) administering to the individual an effective amount of a compound represented by means of the following structural formula:
[0021] [00021] Another embodiment of the present invention is a method of treating an individual with Gaucher disease. The method comprises the steps of: a) administering to the individual an effective amount of a compound represented by means of the following structural formula:
[0022] [00022] Figure 1 shows the experimental XRPD standard (room temperature) for the Formula (I) Hemitartarate.
[0023] [00023] Figure 2 is a graph of the effectiveness of the enzyme and substrate reduction therapies in reducing the levels of glycosylceramide in the liver of Gaucher mice. GL1 liver levels were measured in 3-month-old untreated Gaucher mice (A) and after 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glycocerebrosidase were analyzed 10 weeks later, without additional treatment (C) or after therapy with Formula (I) Hemitartarate (D) at 150 mg of feed / kg. The levels of GL1 in the liver of mice that were administered with Formula (I) Hemitartarate alone throughout the study period (E) and untreated, in similar age controls (f) are also shown. The data are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.
[0024] [00024] Figure 3 is a graph of the effectiveness of the enzyme and substrate reduction therapies in reducing the levels of glycosylceramide in the spleen of Gaucher mice. Spleen GL1 levels were measured in 3-month-old untreated Gaucher mice (A) and after 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glycocerebrosidase were analyzed 10 weeks later, without additional treatment (C) or after therapy with Formula (I) Hemitartarate (D). GL1 levels in the spleen of mice administered with Formula (I) Hemitartarate alone throughout the study period (E) and untreated, in similar age control (F) are also shown. The data are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.
[0025] [00025] Figure 4 is a graph of the effectiveness of the enzyme and substrate reduction therapies in reducing the levels of glycosylceramide in the lungs of Gaucher mice. GL1 levels in the lungs were measured in 3-month-old untreated Gaucher mice (A) and after 2 weeks of treatment with recombinant glucocerebrosidase (B). Mice treated with recombinant glycocerebrosidase were analyzed 10 weeks later, without additional treatment (C) or after therapy with Formula (I) Hemitartarate (D). GL1 levels in the lungs of mice administered Formula Hermitate (I) alone throughout the study period (E) and untreated, in similar age control (f) are also shown. The data are expressed as mean ± standard error of the mean (SEM) (n = 5). Statistical significance was determined using the unpaired t test.
[0026] [00026] Figure 5 is a graph showing the quantification of the degree of CD68 staining in the liver. The extent of CD68-positive staining on the liver sections was quantified using the MetaMorph software. Levels are shown in the liver of untreated 3-month-old Gaucher mice (A) or after treatment with glucocerebrosidase (B). Mice treated with the enzyme and then analyzed 10 weeks later, without additional therapeutic intervention (C) or after therapy with Formula (I) Hemitartarate (D) are also illustrated. The extent of staining in the liver of Gaucher mice administered Formula Hermitate (I) alone (E) and in untreated mice, adjusted to the control age (f) are also shown. The data were collected from an analysis of ten 400x images per section of each of the mice. Statistical significance was determined using the unpaired t test.
[0027] [00027] Figure 6 is a graph showing the effectiveness of Formula (I) Hemitartarate in young D409V / null mice. Formula (I) hemitartrate was administered to 10-week-old D409V / null mice daily via an oral probe at a dose of 75 or 150 mg / kg for 10 weeks. Glycosylceramide levels in the liver, vasculatured lung, and spleen were assessed at the end of the study using HP-TLC. Data are presented as a percentage of GL-1 in untreated mice of similar control age. The dotted lines indicate the levels of glycosylceramide observed in normal wild-type mice. * P <0.05; ** p <0.01 in relation to the control without treatment (two-tailed, unpaired t test). The data are represented as means + standard error of the mean (SEM) n = 5 for 75 mg / kg, n = 6 for 150 mg / kg).
[0028] [00028] Figure 7 shows the effect of Formula (I) Hemitartarate therapy on the accumulation of GL-3 in the liver, heart, kidney, spleen, brain, and blood of the Fabry mouse.
[0029] [00029] Figure 8 shows a graph of the effect of Formula (I) Hemitartarate therapy on the appearance and progression of peripheral neuropathy in Fabry mice.
[0030] [00030] Figure 9 shows graphs of measurements of some of the markers of renal function in Fabry mice treated with Formula (I) Hemitartarate.
[0031] [00031] Figure 10 shows a schedule for the ERT and SRT studies of populations of mice that received different drug therapies: A) Fabrazima bimonthly, no Hemitartarate of Formula (I); B) Bi-monthly Fabrazima Hemitartarate and Formula (I) Hermitatarate in foodstuffs; C) Fabrazima administered at the beginning of the study and at month four of the study and Formula (I) Hemitartarate in foodstuffs; D) no Fabrazima, Hemitartarate of Formula (I) in foodstuffs; and E) no drug therapy.
[0032] [00032] Figure 11 shows the graphs of blood levels of GL-3, in ng / ml of blood in six populations (n = ) Of mice (AE Fabry-Rag; and F of the wild type); the mouse populations received the following therapies: A) Fabrazima bimonthly, no Hemitartarate of Formula (I); B) Bi-monthly Fabrazima and Hemitartarate of Formula (I) in foodstuffs; C) Fabrazima administered at the beginning of the study and at month four of the study and Formula (I) Hemitartarate in foodstuffs; D) no Fabrazima, Hemitartarate of Formula (I) in foodstuffs; E) no drug therapy; and F) no drug therapy.
[0033] [00033] Figure 12 shows graphs of GL-3 levels in the liver and kidney of mouse Fabry-Rag; the mouse populations ( N =) received the following therapies: A) Fabrazima bimonthly, no Hemitartarate of formula (I); B) Bi-monthly Fabrazima and Hemitartarate of formula (I) in foodstuffs; C) Fabrazima administered at the beginning of the study and at month four of the study and Formula (I) Hemitartarate in foodstuffs; D) no Fabrazima, Hemitartarate of Formula (I) in foodstuffs; and E) no drug therapy
[0034] [00034] Figure 13 shows graphs of GL-3 levels in the urine of Fabry-Rag mice; the mouse populations (N = ) received the following therapies: A) Fabrazima bimonthly, no Hemitartarate of formula (I); B) Bi-monthly Fabrazima and Hemitartarate of Formula (I) in foodstuffs; C) Fabrazima administered at the beginning of the study and at month four of the study and Formula (I) Hemitartarate in foodstuffs; D) no Fabrazima, Hemitartarate of Formula (I) in foodstuffs; and E) no drug therapy.
[0035] [00035] Figure 14 is a graph showing the latency in seconds of heat sensitivity of Fabry-Rag Mice that received the following therapies: Bi-monthly Fabrazima, no Formula (I) Hemitartarate; Bi-monthly Fabrazima and Hemitartarate of Formula (I) in foodstuffs; Fabrazima administered at the beginning of the study and at month four of the study and Formula (I) Hemitartarate in foodstuffs; no Fabrazima, Hemitartarate of Formula (I) in foodstuffs; no drug therapy; wild-type mice; and untreated for three months.
[0036] [00036] Figure 15 is a graph showing the total amount of degradation area of an HPLC trace in various mixtures comprising Formula (I) Hemitartrate, lactose monohydrate capsulation grade and Avicel PH 301 (microcrystalline cellulose) ), after being exposed to 85 ° C for 3 days. The area of degradation of the HPLC trace is a ratio between the total area of the peaks corresponding to degradation in relation to the total area of the peaks corresponding to the Formula (I) Hemitartarate and the degradation products. DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0037] [00037] The present patent application provides the only crystalline forms of the Formula (I) Hemitartarate and the new pharmaceutical compositions of the Formula (I) Hemitartarate comprising the crystalline forms of the Formula (I) Hemitartarate described in the present invention. The present patent application also provides methods of inhibiting glycosylceramide synthase or decreasing concentrations of glycosphingolipids in an individual in need thereof. In addition, the present patent application provides methods for the preparation of specific crystalline forms of Formula (I) Hemitartarate. The present patent application also provides stable pharmaceutical formulations of Formula (I) Hemitartarate, combination therapies with the compound of Formula (I) or pharmaceutically acceptable salts (including Formula (I) Hemitartarate) and methods of treatment with the compound of Formula (I) or the pharmaceutically acceptable salts thereof (including Formula (I) Hemitartrate)) which minimize the risk of drug / drug interactions. Crystalline forms of Formula (I) Hemitartarate
[0038] [00038] In a particular embodiment, at least a percentage, by weight, in particular of the Hemitartarate of Formula (I) is crystalline. Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or a percentage between 70% and 100%.
[0039] [00039] In another particular embodiment, at least a percentage by weight, in particular of the Hemitartarate of Formula (I) is a unique crystalline form of Hemitartarate of Formula (I). Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or a percentage between 70% and 100%.,
[0040] [00040] As used in the present invention, the term "crystalline" refers to a solid having a crystal structure, wherein the individual molecules have a normal highly homogeneous chemical configuration blocked therein. The crystalline Formula (I) Hemitartarate can be crystals of a single crystalline form of Formula (I) Hemitartarate, or a mixture of crystals of different individual crystalline forms. A single crystalline form means the Hemitartarate of Formula (I) as a single crystal or a plurality of crystals in which each crystal has the same crystal shape.
[0041] [00041] When a certain percentage by weight of Formula (I) Hemitartarate is a simple crystalline form, the rest of Formula (I) Hemitartarate being a combination of amorphous Formula (I) Hemitartarate and / or one or more other forms crystalline Hermitardate of Formula (I) excluding the only crystalline form. When crystalline Formula (I) Hermitartarate is defined as a specified percentage of a particular crystalline Formula (I) Hermitartarate, the remainder consists of amorphous and / or crystalline forms other than the one or more particular forms which are specified. Examples of a single crystalline form include a form of Formula (I) Hemitartarate characterized by one or more properties as discussed in the present invention.
[0042] [00042] Since tartaric acid has two groups of carboxylic acid, it can form salts with different molar ratios from the compound represented by Formula (I) to tartrate (the conjugate base of tartaric acid). For example, the salt in which there is a 1-1 molar ratio of tartrate to Formula (I) is Formula (I) tartrate (1 tartrate: 1 Formula (I)), and the salt in which there is a molar ratio of about 1 - 2 of Formula (I) tartrate to Formula (I) Hemitartarate (1 tartrate: 2 Formula (I)).
[0043] [00043] The hemitartrate salt can exist in several stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Enantiomers are pairs of stereoisomers whose mirror images are not overlapping, most commonly, because they contain an asymmetrically substituted carbon atom, which functions as a chiral center. Diastereomers are stereoisomers that are not related as mirror images, most commonly, because they contain two or more asymmetrically substituted carbon atoms.
[0044] [00044] When stereochemistry is named (as, for example, L - (+) - tartaric acid) or represented by means of the structure (as, for example, Formula (I)), the named or represented stereoisomer is at least 60 %, 70%, 80%, 90%, 99% or 99.9% by pure relative weight for the other stereoisomers. When a single enantiomer is named (such as, for example, L - (+) - tartaric acid) or represented by means of the structure (such as, for example, Formula (I)), the enantiomer described or named is at least 80%, 90 %, 99% or 99.9%, by weight, optically pure. The percentage of optical purity, by weight, is the ratio of the weight of the enantiomer to the weight of the enantiomer plus the weight of its optical isomer.
[0045] [00045] The term "racemate" or "racemic mixture" means a compound of equimolar amounts of two enantiomers, in which such mixtures do not exhibit optical activity, that is, they do not rotate the polarized light plane.
[0046] [00046] Tartaric acid has three stereoisomers: L - (+) - tartaric acid or dextrotartaric acid and its enantiomer, levotartaric acid or D - (-) - tartaric acid, and in the achiral form, metatartaric acid. The designation L or D does not indicate the acid's ability to rotate the polarized light plane.
[0047] [00047] Any of the stereoisomers of tartaric acid can be used to prepare the Hemitartarate of Formula (I). For example, the hemitartrate can be formed from just one of its stereoisomers, or a combination of them. The hemitartrate salt is selected from D-hemitartrate, L-hemitartrate, hemimesotartaric acid or D, L-racemic L-hemitartrate. In a specific embodiment, the hemitartrate salt is L-hemitartrate. The term "L-hemitartrate" means that the hemitartrate salt is formed from L-tartaric acid. D, racemic L-hemitartrate means that both D-tartrate and L-tartrate were used in the preparation of Formula (I) Hemitartrate. The amount of racemic D, L-hemitartrate D-tartrate can be greater than, equal to, or less than the amount of Ltartarate present.
[0048] [00048] The term "levorotatory" means that the polarized light is turned to the left when passed through an asymmetric compound. The prefix to designate a levorotatory is "L".
[0049] [00049] The term "dextrorotatory" means that the polarized light is rotated to the right when passed through an asymmetric compound. The prefix to designate dextrorotatory is "D". Preparation of Formula (I) Hemitartarate
[0050] [00050] Formula (I) hemitartarate can be prepared by mixing Formula (I) with L-tartaric acid in a suitable solvent. The precipitation of Hemitartarate of Formula (I) can be assisted by adding a seed crystal. The solvents that can be used are methanol, water, ethanol, acetone, ethyl acetate, or combinations thereof.
[0051] [00051] Particularly solid forms of Formula (I) Hemitartarate can be prepared, for example, by means of slow evaporation, slow cooling, and anti-solvent precipitation. Solvents that can be used in these methods include water, heptane, hexane, toluene, dichloromethane, ethanol, isopropyl alcohol, acetonitrile, ethyl acetate, methanol, acetone, methyl butyl tertiary ether (referred to in the present invention as "TBME"), p -dioxane, and tetrahydrofuran (referred to in the present invention as "TF").
[0052] [00052] Solid forms of Formula (I) Hemitartarate can be prepared by evaporating the solvent from a solution of Formula (I) Hemitartarate in a solvent or by means of a mixture of solvents. Suitable solvent mixtures include methanol, ethanol, acetone, water, ethyl acetate and dichloromethane. Preferred solvent mixtures include ethanol, water, methanol and acetone.
[0053] [00053] Solid Formula (I) Hemitartarate forms can be prepared by slowly cooling a heated solution of Formula (I) Hemitartarate in a solvent. Suitable solvents include ethanol, methanol, water, acetone and ethyl acetate.
[0054] [00054] Solid forms of Formula (I) Hemitartarate can be prepared by rapidly cooling a heated solution of Formula (I) Hemitartarate in a solvent, placing the solution in a cooling bath. Suitable solvents include ethanol, methanol, acetone, water, ethyl acetate or mixtures of these solvents.
[0055] [00055] Solid forms of Formula (I) Hemitartarate can be prepared by adding a solution of Formula (I) Hemitartarate in a solvent, as described above, to an antisolvent at a given temperature. More particularly, the antisolvent is ethyl acetate, acetone, acetonitrile, toluene, TF, TBME, p-dioxane, isopropanol, or heptane. Particular solvent / antisolvent mixtures include methanol / ethyl acetate, methanol / acetone, methanol / hexane, methanol / heptane, methanol / acetonitrile, methanol / toluene, methanol / TF, methanol / TBME, methanol / p-dioxane, ethanol / ethyl acetate, ethanol / hexane, ethanol / heptane, ethanol, acetone, ethanol / acetonitrile, ethanol / toluene, ethanol / TBME, ethanol / TF, water / TF, water / isopropanol, water / acetonitrile, water / acetone, dichloromethane / heptane, dichloromethane / dichloromethane acetate, acetone / dichloromethane / acetonitrile acetate, dichloromethane / toluene, dichloromethane / TF, dichloromethane / TBME, dichloromethane / p-dioxane, and dichloromethane / isopropanol.
[0056] [00056] Preferred solvent / antisolvent mixtures include methanol / ethyl acetate, methanol / acetone, methanol / TBME and water / acetone.
[0057] [00057] As used in the present invention, the term "antisolvent" refers to a solvent, in which the Hemitartarate of Formula (I) has a low solubility and causes the Hemitartarate to precipitate out of the solution as a powder fine or crystals.
[0058] [00058] Additional methods for generating the solid forms of Formula (I) Hemitartarate include precipitation of the solid from ethyl acetate / acetone and, optionally, solid drying formed at room temperature. In another method, the solid can then be recrystallized from acetone, with or without the addition of a seed crystal. Alternatively, the Formula (I) Hemitartarate can be precipitated from ethyl acetate / acetone solvents and recrystallized from ethyl acetate. Alternatively, the Formula (I) Hemitartarate can then be recrystallized from isopropanol. Alternatively, the Formula (I) Hemitartarate can be prepared using only acetone without further recrystallization. Alternatively, Formula (I) Hemitartarate can be precipitated from acetone following a brief reflux, without further recrystallization.
[0059] [00059] Alternatively, the Formula (I) Hemitartarate can then be recrystallized from methanol / acetone, with or without the addition of a seed crystal. Alternatively, the Formula (I) Hemitartarate can then be recrystallized from water / acetone, with or without the addition of a seed crystal. Characterization of crystalline forms of Formula (I) Hemitartarate
[0060] [00060] In a particular embodiment, the crystalline form of Formula (I) Hemitartarate, crystalline form A, is characterized by means of one, two, three, four or five large XRPD peaks at angles 2 θ of 5.1 °, 6.6 °, 10.7 °, 11.0 °, 15.9 ° and 21.7 °. In an even more particular modality, the crystalline shape is characterized by means of the XRPD peaks at angles 2 θ of 5.1 °, 6.6 °, 10.7 °, 11.0 °, 13.3 °, 15.1 °, 15.9 °, 16.5 °, 17.6 °, 18.6 °, 18.7 °, 19.0 °, 20.2 °, 21.7 ° and 23.5 °. It is to be understood that a specified angle 2θ means that the specified value ± 0.2 °.
[0061] [00061] As used in the present invention, the term "main XRPD peak" refers to an XRPD peak with a relative intensity greater than 25%. The relative intensity is calculated as the ratio of the peak intensity of the peak of interest to the peak intensity of the largest peak. Treatment methods using Formula (I) Hemitartarate
[0062] [00062] As used in the present invention, an individual is a mammal, preferably a human patient, but can also be an animal in need of veterinary treatment, such as a pet animal (e.g., dogs, cats, and the like) , a farm animal (for example, cows, sheep, pigs, horses, and the like) or a laboratory animal (for example, rats, mice, guinea pigs, and the like). Individual and patient are used interchangeably.
[0063] [00063] One embodiment of the present patent application is a method of decreasing, for example, inhibiting or reducing the activity of glycosylceramide synthase or decreasing the concentrations of glycosphingolipids in an individual with that need, by administering to the individual an effective amount of Formula (I) of hemitartarate salt, including the crystalline forms thereof, as described above.
[0064] [00064] An individual in need of treatment is an individual with a condition or disease in which the benefits inhibit glycosylceramide synthase or decrease the concentrations of glycosphingolipids in cells, in particular the lysosome or the cell membrane. Glycosylceramide synthase inhibitors have been shown to be useful for the treatment of lysosomal storage diseases, such as Tay-Sachs disease, Fabry Gaucher disease or (see, for example, US Patent Nos. 6,569,889; 6,255,336; 5,916,911; 5,302,609; 6,660,749; 6,610,703; 5,472,969; 5,525,616, the teachings in their entirety which are incorporated by reference in the present invention).
[0065] [00065] Examples of conditions or diseases include polycystic kidney disease and membranous glomerulopathy (see US Provisional Patent Applications 61/130, 401 and 61/102, 541, the teachings in their entirety which are incorporated by reference into the present invention. ), glomerulonephritis and glomerulosclerosis (See Provisional Patent Application 61/137, 214) lupus (See PCT / US2009 / 001773, the teachings in their entirety which are incorporated by reference into the present invention), including type 2 diabetes (see WO 2006/053043, the teachings in their entirety which are incorporated by reference in the present invention); treatment of disorders involving cell growth and division, including cancerous diseases, collagen vascular diseases, atherosclerosis, and renal hypertrophy of diabetic patients (see US Patent Nos. 6,916,802 and 5,849,326, the teachings in all of which are incorporated by reference in the present invention); inhibition of the growth of arterial epithelial cells (see U.S. Patents 6,916,802 and 5,849,326); treatment of patients suffering from infections (see Svensson, M. et al, "Epitelial Glicosphingolipid Expression as a Determinant of Bacterial Adherence and Cytokine Production," Infect. and Immun, 62: 4404 to 4410 (1994), the teachings in their entirety which are incorporated by reference in the present invention); preventing the host, that is, the patient, from generating antibodies against the tumor (see Inokuchi, J. et al, "Antitumor Ativity in Mice of an Inhibitor of Glicosphingolipid Biosyntesis," Cancer Lett, 38: 23-30 (1987 ), the teachings in their entirety which are incorporated by reference in the present invention), and treatment of tumors (see Hakomori, S. "New Directions in Cancer Terapy On Aberrant Expression of Glicosphingolipid: Anti-adhesion and Orto-Signaling Terapy", Cancer Cells 3: 461 to 470 (1991), Inokuchi, J. et al "Inhibition of Experimental Metastasis of Murine Lewis Long Carcinoma by an Inhibitor of Glycosylceramide Syntase and its Possible Mechanism of Action," Cancer Res., 50: 6731 to 6737 (1990) and Ziche, M. et al., "Angiogenesis Can Be Stimulated or Repressed in In Vivo by a Change in GM3: GD3 Ganglioside Ratio," Lab. Invest., 67: 711 to 715 (1992), the teachings in all of which are incorporated by reference in the present invention).
[0066] [00066] Formula (I) hemitartrate can also be used for the preparation of a type of cancer vaccine (see, for example, US Patent Nos. 6,569,889; 6,255,336; 5,916,911; 5,302,609; 6,660,749; 6,610,703; 5,472,969; 5,525,616).
[0067] [00067] The compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt thereof) can be used in the methods described as a monotherapy, that is, as the only pharmaceutically active ingredient to be administered in order to to handle the indication.
[0068] [00068] Alternatively, the compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt thereof) can be used in the methods described as a combination therapy with other therapeutically active drugs known in the art. in order to treat the desired diseases or indications. The term "Coterapia" or "combination" or "combination therapy" or "coadministered" is used in the present invention interchangeably and means that the compound of Formula (I) or pharmaceutically acceptable salt thereof (including the hemitartrate salt) is administered before, after, or simultaneously with one or more other therapeutic agents. In one embodiment, a combination therapy is used to treat a lysosomal disease, such as Gaucher disease or Fabry disease. Alternatively, the compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt) is co-administered simultaneously (for example, at the same time) either as separate formulations or as a joint formulation. Alternatively, the agents can be administered sequentially, as separate compositions, within an appropriate period of time, as determined by the skilled clinician (e.g., sufficient time to allow for the overlap of the pharmaceutical effects of the therapies). The compound of Formula (I) or pharmaceutically acceptable salt thereof (including the hemitartrate salt) and one or more other therapeutic agents can be administered in a single dose or in multiple doses, in an order and on an appropriate schedule in order to achieve a desired therapeutic effect.
[0069] [00069] Effective therapeutic agents for the treatment of Gaucher disease include glucocerebrosidase, glycocerebrosidase analogs, glycosylceramide synthase inhibitors and molecular chaperones that bind to glucocerebrosidase and restore their correct conformation. Glycocerebrosidase or the analogs thereof can be derived from humans or mammals. Alternatively, glycocerebrosidase and the analogs thereof can be obtained recombinantly. Glucocerebrosidase analogs include truncated forms of the enzyme and / or enzymes with amino acid substitutions relative to the native amino sequence of the native enzyme, as long as biological activity is maintained. Examples of glycocerebrosidase analogs include imiglucerase (sold under the trademark Cerezima ®) by Genzima Corporation), Taliglucerase Alfa (to be marketed under the trademark Upliso ® and developed by Protalix Bioterapeutics, Inc.) and velaglucerase Alfa (developed by Shire PLC ), which are recombinant DNA analog produced from human β-glucocerebrosidase. Examples of molecular chaperones include isofagomine (under development under the trademark Plicera ® by Amicus Terapeutics, Cranbury, NJ). Isofagomine is also known as afegostat tartrate and contains the isofagomine tartrate salt form as its active ingredient. Examples of glycocerebrosidase inhibitors include miglustat (developed under the trademark of Zavesca ® by Actelion Pharmaceuticals Ltd. Allschwil, Switzerland).
[0070] [00070] Effective therapeutic agents for the treatment of Fabry disease include α-galactosidase A, galactosidase A analogs and molecular chaperones that bind to α-galactosidase A and restore their correct conformation. α-Galactosidase A or the analogs thereof can be derived from humans or mammals. Alternatively, α-galactosidase A and the analogs thereof can be obtained recombinantly. Analogs of α-galactosidase A include the truncated forms of the enzyme and / or enzymes with amino acid substitutions in relation to the native amino sequence of the native enzyme, as long as biological activity is maintained. Examples of α-galactosidase A analogs include agalsidase beta (a recombinant human α-galactosidase sold under the trade name Fabrazima ® as a lyophilized drug by Genzima Corporation) and agalsidase alfa (a recombinant protein sold under the trade name ® Replagal by Shire PLC ). Examples of molecular chaperones include migalastat (developed under the trademark Amigal® by Amicus Terapeutics, Cranbury, NJ as a drug containing migalastat hydrochloride as its active ingredient).
[0071] [00071] In one embodiment, combination therapy for the treatment of Gaucher or Fabry disease is carried out in two stages. In a first stage, an effective drug for the treatment of Gaucher disease or Fabry disease (typically, glycocerebrosidase of an analogue of the same for Gaucher disease and galactosidase A or an analogue of the same for Fabry disease) is used for stabilize the individual. For example, in Gaucher disease (or Fabry disease), one of these drugs is used to reduce the GL-1 storage load on visceral organs, such as the liver, spleen, lung and / or kidney. Once this has been achieved, the compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt) is used in the second phase, as a convenient maintenance therapy. The first stage typically lasts for one, two, three, or four weeks, or up to one, two, three, four, six, nine, or twelve months, or until the individual's platelet count is equal to or greater than 100,000 mm3; hemoglobin concentration is equal to or greater than 11 g / dl (female) or 12 g / dl (male), and / or the volume of the individual in the spleen is less than or equal to 10 multiples of normal volumes and the liver is less than or equal to 1.5 multiples of normal. The administration of the first phase is typically therapy terminated once the treatment with the compound of Formula (I) is initiated.
[0072] [00072] As used in the present invention, an "effective amount" refers to an amount effective to alleviate the individual's existing symptoms to be treated with the minimum of unacceptable side effects in the individual. The exact formulation, route of administration and dosage is chosen by the individual physician in view of the patient's condition. The dosage amount and interval can be individually adjusted to provide plasma levels of the active compound that are sufficient to maintain the desired therapeutic effects. In addition, the condition of the patient and the mode of administration, the dose administered will depend on the severity of the patient's symptoms and the patient's age and weight. An effective amount will typically result in plasma levels through the compound above at least 5 ng / ml. If the minimum plasma levels are less than 5 ng / ml after administering an effective amount of the compound, the dose to be administered to individuals that is changed to an "adjusted effective amount" such that the minimum levels of the compound is at least 5 ng / ml. Alternatively, if the minimum plasma levels of the compound are less than 5 ng / ml and / or the Cmax is above 100 ng / ml after administering an effective amount of the compound, the dose to be administered to the individual is changed to an "adjusted effective amount" such that the plasma levels of the compound is at least 5 ng / ml and the Cmax is below 100 ng / ml. Effective amounts can vary from 0.1 to 500 mg / day. Alternatively, the effective amount ranges from 50-300 mg / day. In another alternative, the effective amount ranges from 100 to 300 mg / day. The compound of the present patent application can be administered continuously or at specific time intervals. For example, the compound of the present patent application can be administered 1, 2, 3 or 4 times a day, such as, for example, a daily formulation or twice a day. Commercially available assays can be employed to determine optimal dose ranges and / or schedules for administration.
[0073] [00073] In one embodiment, an effective amount for the compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt described above) is (either as monotherapy or as a co-therapy), a daily dose from 25 milligrams to 300 milligrams (alternatively 25 milligrams to 150 milligrams; in another alternative from 50 milligrams to 300 milligrams, and in another alternative from 100 milligrams to 300 milligrams), such as 25, 50 , 100, 200 or 300 milligrams per day. In a specific embodiment, an effective amount of the Formula (I) compound or a pharmaceutically acceptable salt thereof (including Formula (I) Hemitartarate) is (either as monotherapy or as a co-therapy), a dose twice a day. 50 milligrams per day (for a total of 100 milligrams per day), 100 milligrams (for a total of 200 milligrams per day) or 150 milligrams (for a total of 300 milligrams per day). In an alternative embodiment, an effective amount for the Formula (I) compound or a pharmaceutically acceptable salt thereof (including Formula (I) Hemitartrate) is (either as a monotherapy or as a therapy), administered as a single dose. daily dose of 100 milligrams per day, 200 milligrams per day or 300 milligrams per day.
[0074] [00074] In another embodiment, an effective amount is determined by assuming the individual is a "slow P450 metabolizer" and then assessing plasma and / or Cmax levels. The amount administered to the subject is then changed to an adjusted effective value, as described below, if plasma levels are less than 5 ng / ml; or the minimum levels of the compound are below 5 ng / ml and / or the Cmax is above 100 ng / ml; or if the individual is determined to be an intermediate or extended / ultra-fast P450 metabolizer. An effective amount for slow P450 metabolizers is (either as monotherapy or as a co-therapy), usually between 100 to 200 mg per day, for example 100 or 200 milligrams, as a single daily dose or a twice daily dose.
[0075] [00075] Typically, the pharmaceutical compositions of the present patent application can be administered before or after a meal, or with a meal. As used in the present invention, "before" or "after" a meal is typically within two hours, preferably within an hour, preferably within thirty minutes, more preferably within ten minutes of beginning or ending a meal. meal, respectively.
[0076] [00076] It has now been found that the compound of Formula (I) and pharmaceutically acceptable salts thereof (including Formula (I) Hemitartarate) is metabolized via the liver, mainly by cytochrome P450 enzymes. Cytochrome P450s ("CYPs") are the main xenobiotic liver metabolizing enzymes. There are eleven P450 metabolizing xenobiotics expressed in a typical human liver (ie CYP1A2, CYP2A6, CYP2B6, CYP2C8 / 9/18/19, CYP2D6, CYP2E1 and CYP3A4 / 5). are the main cytochrome P450 isoforms that are responsible for detoxifying the Formula (I) compound and its pharmaceutically active salts, such as Formula (I) Hemitartarate. The level of activity of P450 enzymes differs according to the individual. For example, individuals can be classified as P450 slow, intermediate and long / ultra fast metabolizers. Because the lowest levels of P450 activity at which an individual can give rise to drug / drug interactions ("DDI"), another embodiment of the present invention is to determine whether the individual is a slow, intermediate and extensive / ultra-fast P450 metabolizer . If the individual is an intermediate or extensive / ultrafast metabolizer, then the dose administered to the individual which must be increased to an "adjusted effective dose", that is, the amount that results in plasma levels of the compound of at least 5 ng / ml; or the amount that results in minimum levels of the compound or at least 5 ng / ml and a Cmax of the compound below 100 ng / ml. The dose can be increased incrementally and the individual retested once, twice, three, four or as many times as necessary to achieve an effective adjusted dose.
[0077] [00077] For the 2D6 CYP gene there are four predicted phenotypes:
[0078] [00078] A "slow P450 metabolizer" carries two mutant alleles, which results in complete loss of enzyme activity.
[0079] [00079] An "intermediate P450 metabolizer" has reduced allele activity and a null allele.
[0080] [00080] An "extensive P450 metabolizer" has at least one and no more than two normal functional alleles.
[0081] [00081] A "P450 ultra-fast metabolizer" carries multiple copies (3 to 13) of functional alleles and produces excess enzyme activity.
[0082] [00082] An individual is usually assessed as a slow, intermediate and extensive / ultrafast P450 metabolizer by genotyping or by monitoring the plasma levels of a drug that is metabolized by a P450 enzyme, such as CYP2D6 or CYP3A4. Commonly, the minimum plasma and / or Cmax levels of the Formula (I) compound or a pharmaceutically acceptable salt thereof, including Formula (I) Hemitartarate, are monitored in the individual per week until one, two, three or four, or even for one, two, three, six, nine or twelve months or the most subsequent initiation of treatment with the compound. Dose adjustments are made, as necessary, to maintain levels within the limits described, that is, a trough plasma level at or above 5 ng / ml.
[0083] [00083] Individuals can become poor P450 metabolizers as a result of being treated with certain drugs that are inhibitors of the cytochrome P450 enzyme. Examples of such drugs include paroxetine, fluoxetine, quinidine, or ketoconazole. Alternatively, an individual is a slow P450 metabolizer as a result of the low expression of a cytochrome P450 enzyme. In such cases, low expression can be assessed by determining the expression of the P450 enzyme in the individual, that is, genotyping the individual to the cytochrome P450 enzyme. For example, CYP2D6 expression is commonly assessed by PCR (McElroy et al. "CYP2D6 Genotyping as an Alternative to Phenotyping for Determination of Metabolic Status in a Clinical Trial Setting", AAPS Pharmsi (2000) 2 (4): article 33 ( http://www.pharmsci.org/) or by pharmacogenomics-based microarray assays (background information, Roche Diagnostics "Te CYP450 Gene Famili and Drug Metabolism", Hoffmann-La Roche Ltd.), the teachings in their entirety which are incorporated by reference into the present invention. As such, the individual can be conveniently genotyped for P450 expression (for example, CYP2D6) prior to initiation of treatment and administered in an adjusted effective amount, if necessary. In the case of genotyping, before starting treatment, it is still advisable to monitor the minimum plasma and Cmax levels of the compound and adjust the dose as necessary.
[0084] [00084] The effective amounts for migalastat, B agalsidase, imiglucerase, isofagomine and miglustat are as described on the drug label or as performed in the clinical trials of each drug.
[0085] [00085] The compound of Formula (I) can be reacted with pharmaceutically acceptable acids to form a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl- sulfonic, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohmonohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formyl caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butino-1, 4-dioate, hexino-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, citrate, phenylbutyrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Pharmaceutical compositions including Formula (I) Hemitartarate
[0086] [00086] Suitable formulations and modes of administration for the compound of Formula (I) or a pharmaceutically acceptable salt thereof (including the hemitartrate salt thereof) include those described in US Patent No. 7,253,185, the teachings of which all of which are incorporated by reference in the present invention. A preferred formulation for the Formula (I) Hemitartarate is described in the following paragraphs.
[0087] [00087] One embodiment of the present invention is a pharmaceutical composition comprising Hemitartarate of Formula (I), at least one water-soluble filler, at least one water-insoluble filler, at least one binder, and at least a lubricant. Suitable water-soluble fillers may include, for example, anhydrous lactose, lactose monohydrate, mannitol, sodium chloride, powdered sugar, sucrose, sorbitol, pregelatinized starch and inositol. Suitable water-insoluble fillers can include, for example, microcrystalline cellulose, calcium phosphate and starch. Suitable binders can include, for example, alginate, pregelatinized starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, copolividone, gelatin, natural gums, starch paste, sucrose, corn syrup, glycols polyethylene and sodium. Suitable lubricants may include, for example, hydrogenated vegetable oil, calcium stearate, and glyceryl beenate. In one embodiment of the pharmaceutical composition, the water-soluble filler is selected from the group consisting of anhydrous lactose, lactose monohydrate, mannitol, sodium chloride, powdered sugar, sucrose, sorbitol, inositol and pregelatinized starch ; the water-insoluble filling agent is selected from the group consisting of microcrystalline cellulose, calcium phosphate and binder starch is selected from the group consisting of pregelatinized starch, sodium carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose methyl, polyvinyl pyrrolidone, copolividone, gelatin, natural gums, starch paste, sucrose, corn syrup, polyethylene glycols and sodium alginate; and the lubricant is selected from the group consisting of hydrogenated vegetable oil, calcium stearate, and glyceryl beenate.
[0088] [00088] The pharmaceutical formulation comprises between 8% by weight to 32% by weight, between 8% by weight to 24% by weight, between 12% by weight to 20% by weight, or between 14% by weight to 18% by weight. weight of the water-insoluble filling agent, on a dry solids basis.
[0089] [00089] The pharmaceutical formulation comprises between 26% by weight to 50% by weight, between 30% by weight to 46% by weight, between 34% by weight to 46% by weight, or between 38% by weight to 44% by weight. weight of the water-soluble filling agent, on a dry solids basis.
[0090] [00090] The pharmaceutical composition comprises between 30% by weight and 45% by weight, between 35% by weight and 40% by weight and 36% by weight to 39% by weight of Formula (I) Hemitartarate on a solid basis dry.
[0091] [00091] The pharmaceutical formulation typically comprises between 2 and 6% by weight of% by weight binder on a dry solids basis.
[0092] [00092] The pharmaceutical formulation typically comprises between 0.1% by weight and 2% by weight of binder, on a dry solids basis.
[0093] [00093] In a specific embodiment, the pharmaceutical formula comprises between 8% by weight to 32% by weight of water-insoluble filler, between 26% by weight to 50% by weight, of water-soluble filler, between 30 % by weight and 45% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a solid basis dry. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler agent is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0094] [00094] In a specific embodiment, the pharmaceutical formula comprises between 8% by weight to 32% by weight of water-insoluble filling agent, between 26% by weight to 50% by weight, water-soluble filling agent, between 35% by weight and 40% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a dry solids. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0095] [00095] In another specific embodiment, the pharmaceutical formula comprises between 8% by weight to 24% by weight of water-insoluble filler, between 30% by weight to 46% by weight, water-soluble filler, between 35% by weight and 40% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a dry solids. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0096] [00096] In another specific embodiment, the pharmaceutical formula comprises between 12% by weight to 20% by weight of water-insoluble filler, between 34% by weight to 46% by weight, water-soluble filler, between 35% by weight and 40% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a dry solids. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0097] [00097] In another specific embodiment, the pharmaceutical formula comprises between 14% by weight to 18% by weight of water-insoluble filler, between 38% by weight to 44% by weight, water-soluble filler, between 35% by weight and 40% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a dry solids. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0098] [00098] In another specific embodiment, the pharmaceutical formula comprises between 14% by weight to 18% by weight of water-insoluble filler, between 38% by weight to 44% by weight, water-soluble filler, between 36% by weight and 39% by weight of Formula (I) Hemitartarate, between 2% by weight and 6% by weight of binder and between 0.1% by weight and 2% by weight of binder, all on a dry solids. More specifically, the water-soluble filler is lactose monohydrate; and the water-insoluble filler is microcrystalline cellulose. Even more specifically, the water-soluble filler is lactose monohydrate; the water-insoluble filler is a microcrystalline cellulose; the binder is hydroxypropyl methylcellulose, and the lubricant is glyceryl beenate.
[0099] [00099] The present invention is illustrated by means of the following examples, which are not intended to be limiting in any way. EXPERIMENTAL Example 1: Preparation of Salts of Formula (I)
[0100] [000100] The Formula I hemitartrate salt is readily crystallized and exhibits many beneficial properties compared to other salts. For example, the following acids were used in the preparation of salts of the compound represented by Formula (I): citric acid (generating salts in 1: 1, 1: 2, 1: 3 and (salt: Formula I) ratio); L-malic (1: 1 and 1: 2); methane sulfonic acid (1: 1); fumaric acid (1: 1 and 1: 2); hydrochloric acid (1: 1); acetic acid (1: 1) and tartaric acid (1: 1 and 1: 2). Salts only generated by hydrochloric acid (1: 1); tartaric acid (1: 1) and tartaric acid (1: 2) were in solid form. Of these three salts of hydrochloric acid (1: 1) and tartaric acid (1: 1) were found to be hygroscopic and non-crystalline and therefore unacceptable for use in a pharmaceutical product. The hemitartrate (1 salt: 2 Formula I) of the compound represented by Formula I was found to be crystalline and non-hygroscopic. Preparation of Formula (I) Hemitartarate acetone
[0101] [000101] L-tartaric acid (6.02 g, 40.11 mmols, 0.497 equivalent) was dissolved in acetone (175 mL) by refluxing the solution and then cooling it to room temperature. Database of free formula (I) (32.67 g, 80.76 mmols) was dissolved in acetone (300 mL) at room temperature. The L-tartaric acid solution was added to the free base solution of Formula (I), at room temperature over 15 min. A white precipitate formed halfway through the addition. The mixture was stirred at room temperature for 0.5 hr and then briefly refluxed and heated to room temperature. After stirring at room temperature for 0.5 h, the white precipitate was filtered. The white solid was washed twice with acetone (2 x 130 ml). The solid was air dried and then vacuum dried at 55 to 60 ° C. The yield was 36.66g (95%). 5% methanol in preparation of Formula (I) Hemitartarate Acetone.
[0102] [000102] Free base of formula (I), 10 g / 24.7 mmols, was dissolved in 5% methanol / acetone at 120 ml or 240 ml. L-tartaric acid, 1.85 g / 12.3 mmol, was dissolved in 5% methanol / acetone at 60 ml or 120 ml (N or 2 N) by heating to 40 to 45 ° C, and this solution was added to the first solution. After 1 hour, without precipitation, 1 mg of Formula (I) Hemitartarate was added as a seed crystal. The precipitation occurred after 5 minutes, and the reaction continued to stir for an additional 30 minutes. The reaction was then heated to reflux for 5 minutes (the precipitate was completely soluble) and then cooled to room temperature in a water bath at 20 to 22 ° C. The precipitate formed and the reaction continued to stir for 3 hours. The final product was collected by filtration and was washed with acetone, 2 x 40 ml, and then dried in the vacuum oven at 55 to 60 ° C for 16 hours. The weight of the product was 8.72 g / 74% yield. 1% water in preparation of acetone of Formula (I) Hemitartarate. Free base of formula (I) (10 g / 24.7 mmols) was dissolved in 1% water / acetone at 120 ml or 240 ml at room temperature. L-tartaric acid, 1.85 g / 12.3 mmols, was dissolved in 1% water / acetone at 60 ml or 120 ml (N or 2 N) by heating to 40 to 45 ° C, and this solution was added to the first solution. After 1 hour, without precipitation, 1 mg of Formula (I) Hemitartarate was added as a seed crystal. The precipitation occurred after 5 minutes, and the reaction continued to stir for 30 minutes. The reaction was then heated to reflux for 5 minutes (the precipitate was not completely soluble) and then cooled to room temperature in a 20 to 22 ° C water bath. The precipitate formed and the reaction continued to stir for 3 hours. The final product was collected by filtration and was washed with acetone, 2 x 40 ml, and then dried in the vacuum oven at 55 to 60 ° C for 16 hours. The weight of the product was 8.62 g yield equal to 73%. 5% methanol in recrystallization from acetone of Formula (I) Hemitartarate.
[0103] [000103] Hemitartarate of Formula (I) (3.06 g) was dissolved in 116 ml of 5% methanol in reflux acetone. The solution was cooled to room temperature and stirred at room temperature for 2 h. The white precipitate was filtered and washed with 10 ml of 5% methanol in acetone and then with acetone (15 ml). After vacuum drying for 18 hours at 55 to 60 ° C, they received 2.38 g of Formula (I) Hemitartarate (78% recovery). 1% H2O in the recrystallization of acetone from Formula (I) Hemitartarate.
[0104] [000104] Formula (I) hemitartarate (3.05 g) was dissolved in 125 ml of 1% H2O in refluxing acetone. The solution was cooled to room temperature and stirred at room temperature for 2 h. The white precipitate was filtered and washed with 10 ml of 1% H2O in acetone and then with acetone (15 ml). After vacuum drying overnight at 55 to 60 ° C, 2.35 g of Formula (I) Hemitartarate (77% recovery) was obtained. Example 2: Preparation of Crystalline Formula (I) Hemitartarate
[0105] [000105] Formula (I) hemitartarate was crystallized by means of several methods. Batch 1 was prepared using ethyl acetate / acetone solvents and dried at room temperature. Batch 3 was prepared using ethyl acetate / acetone solvents and recrystallized from ethyl acetate. Batch 4 was recrystallized from acetone using the batch of material 1. Batch 5 was recrystallized from isopropanol. Batch 7 was prepared using ethyl acetate / acetone solvent similar to Batch 1, but on a large scale, Batch 8 was prepared using acetone only without further recrystallization. Batch 9 was prepared using acetone with only brief reflux, again no further recrystallization. Table 1: Summary of batch 1 to 9 batch polymorphism screening of Formula (I)
[0106] [000106] Crystalline forms of Formula (I) Hemitartarate have also been prepared using slow evaporation, slow cooling, rapid cooling and precipitation antisolvent with a variety of solvents.
[0107] [000107] Slow evaporation method. A heavy sample (usually 20 mg) was treated with solvent test aliquots. The aliquots were typically 100 to 200 µL. Between solvent additions, the mixture was stirred or sonicated. When the solids dissolved, as assessed by visual inspection, the solution was allowed to evaporate under ambient conditions, in an open flask covered with perforated aluminum foil with holes. Solubilities were estimated from these experiments based on the total solvent added in order to obtain a clear solution. Table 2: Approximate solubility of Formula (I) Hemitartarate at room temperature (20 to 25 ° C).
[0108] [000108] Slow / Fast Cooling Method. The hemitartrate of formula (I) was dissolved in a test solvent at 50 to 60 ° C. The resulting solution was then allowed to cool to room temperature (slow cooling). If no solids had been formed after a day, the vessels would be placed in a refrigerator. With regard to the rapid cooling experiments, the resulting solution was then allowed to cool in the refrigerator. The solids formed were collected by filtration in dry air. Table 4: Polymorphism synthesis using a slow cooling approach.
[0109] [000109] Anti-solvent method. The hemitartrate of formula (I) was dissolved in a solvent. An anti-solvent was added to the solution. The solids formed were collected by filtration in dry air. Table 6: Polymorphism synthesis using an anti-solvent approach
[0110] [000110] Differential exploratory calorimetry (DSC). DSC data was collected on a TA Q100 instrument using nitrogen as the purge gas. Approximately 2 to 5 mg of the sample was accurately weighed in an aluminum DSC pan. The tray was covered with a lid and punctured with forceps. The sample cell was equilibrated to 30 ° C and heated at a rate of 10 ° C per minute to a final temperature of 220 ° C.
[0111] [000111] Hot phase microscopy. Hot stage microscopy was performed using a hot Linkam stage (model FTIR 600) mounted on a Leica DM LP microscope equipped with a Sony DXC-970MD 3CCD camera for image collection. A 40x objective was used with polarized light to view the samples. Each sample was placed between two cover sheets. Each sample was observed visually as the phase was heated. The images were captured using links in version 2.27 (Linkam). The hot phase was calibrated with USP melting point standards.
[0112] [000112] The endothermic transition observed in the DSC profile was confirmed to be a melting transition at a temperature between 160 to 163 oC by means of hot phase microscopy. Example 4: X-ray powder diffraction of Formula (I) Hemitartarate
[0113] [000113] All X-Ray Powder Diffraction (XRPD) analyzes were performed at SSCI, Inc. (West Lafayette, IN 47906). XPRD analyzes were performed using a Shimadzu XRD-6000 X-ray powder diffractometer using Cu K α radiation. The instrument is equipped with a fine X-ray focusing of the tube. The tube voltage and amperage were set to 40 kV and 40 mA, respectively. The divergence and spreading slits were set at 1 ° and the receiving slit was set at 0.15 mm. Diffracted radiation was detected by means of a Nal scintillation detector. Continuous verification of theta-two theta at 3 ° / min (step 0.4 s / 0.02) from 2.5 to 40 ° 2θ was used. A silicon pattern was analyzed to check the alignment of the instrument. The data were collected and analyzed using XRD-6000 v 4.1. Example 5: Comparison of Formula (I) Hemitartarate to Formula (I) Free Base
[0114] [000114] The characterization of the free base solid and the hemitartrate salt are summarized in Table 7. The Formula (I) Hemitartarate has superior properties compared to Formula I free base. For example, Formula (I) Hemitartarate has a higher melting point (> 150 ° C), higher packaging energy (greater endothermic enthalpy), less variation in particle size, greater aqueous solubility (more than 300 mg / mL in water), the appropriate crystal form, and a higher bulk density compared to Formula I Free Base. Table 7: Synthesis of the solid state and the physical and chemical properties of Formula (I) of free base and Formula (I) Hemitartarate.
[0115] [000115] Activity of Formula (I) Hemitartarate in the inhibition of glycosphingolipid synthesis in vitro. Two assays were used in order to quantify the inhibitory activity of Formula (I) Hemitartarate for glycosylceramide synthase. Since glycosylceramide is the first and speed limiting step in glycosphingolipid biosynthesis, a flow cytometry assay that measured the levels of GM 1 and GM3 surface cells was used to indirectly assess the activity of the inhibitor in intact cells. Incubation of K562 or B16 / F10 cells for 72 h with increasing amounts of Formula (I) Hemitartarate (0.6 to 1000 nM) resulted in a dose-dependent reduction in cell surface levels of both GM1 and GM3. The IC50 value for the mean inhibition of GM1 cell surface presentation in K562 cells was 24 nM (range 14 to 34 nM) (Table 8) and that for GM3 in B16 / F10 cells was 29 nM ( range from 12 to 48 nM). No patent toxicity of the cell was observed in any cell line, even when tested at a higher dose.
[0116] [000116] An alternative assay for activity measured the inhibition of glycosylceramide synthase in microsomes derived from human cells. In this assay, microsomes were prepared from human A375 cell melanoma by sonication and centrifugation. The microsomal preparation was incubated with a fluorescent ceramide substrate (NBD-C6-ceramide), UDP-glucose, and increasing amounts of Formula (I) Hemitartarate (0 to 1000 nM) for one hour at room temperature. After incubation, labeled glycosylceramide by means of fluorescence and unreacted ceramide was separated and quantified by means of reverse phase HPLC and by means of fluorescence detection. In this assay, the IC50 value for glycosylceramide inhibition synthesis ranged from 20 to 40 nM. This value was similar to those obtained above for GM1 and GM3 and suggests that measurements of these cell surface glycolipids are substituted for good activities of Formula (I) Hemitartarate for glycosylceramide synthase.
[0117] [000117] Specificity of the inhibition of substrate synthesis by the Hemitartarate of Formula (I). The specificity of Formula (I) Hemitartarate was evaluated in a series of cell-based in vitro assays and cell-free assays. Intestinal glycosidase enzymes were tested in mouse tissue homogenates (see U. Andersson, et al., Biochem. Pharm. 59 (2000) 821 to 829, the teachings in full which are incorporated by reference in the present invention), and the glycogen disrupting enzyme was tested in a free cell assay as described (see U. Andersson, et al., Biochem. Pharm. 67 (2004) 697 to 705, the teachings in full which are incorporated by reference in the present invention). There was no detectable inhibition of intestinal glycosidases (lactase, maltase, sucrase), α-glycosidase I and II, and the cytosolic debranching enzyme (α-1, 6-glycosidase), was found in concentrations up to 2500 iM (Table 8) .
[0118] [000118] No lysosomal glycosylceramidase and lysosomal glycocerebrosidase were tested on intact human cells using C6-NBD-glycosylceramide as a substrate (see HS Overkleeft, et al. J. Biol. Chem. 273 (1998) 26,522 to 26,527, the teachings in full. which are incorporated by reference in the present invention). Conduritol epoxide β (a specific inhibitor of lysosomal glucocerebrosidase) was used to differentiate lysosomal versus non-lysosomal activity. Glucocerebrosidase activity was also measured using activated cell separation by fluorescence (FACS). K562 cells were cultured with increasing amounts of Formula (I) Hemitartarate in the presence of 1 µM 5 - (pentafluorobenzoylamino) -fluorescein di-β-D-glycopyranoside (PFB-FDGlu, Molecular Probes / Invitrogen Carlsbad, CA.) by 30 to 60 min. The cells were immediately cooled on ice and the fluorescence is quantified above. No glycosylceramidase -lysosomal was weakly inhibited with an IC 50 of 1600 µM. There was no inhibition of lysosomal glycocerebrosidase, the enzyme that is deficient in Gaucher disease, up to the highest concentration of 2500 iM (Table 8). Thus, a difference of approximately 40,000 in concentration was necessary to inhibit glycosylceramide synthase compared to any of the other enzymes tested. Table 8: Biochemical activities of Formula (I) Hemitartarate in vitro
[0119] [000119] To determine whether the combined use of both enzyme replacement therapy (ERT) and substrate reduction therapy (TRS) can keep the enzyme in bulk or provide additional benefits, the relative efficacies of separate and combined therapies in one murine model of Fabry disease (Fabry-Rag) were compared. The parental Fabry mice are described in Wang, AM et al. I am. Hum J..Genet. 59: A208 (1996). The Fabry Rag is crossed with a mouse RAG-1 and does not develop mature lymphocytes or T cells (immunocompromised). Animal studies
[0120] [000120] For monotherapy studies, Fabry mice were studied in the first month of age (prevention model). The treatment groups received Formula (I) Hemitartarate (Genzima Corp, Cambridge, MA) as a component of the diet pellet. The drug was formulated in 0.15% (w / w) in the standard of 5053 murines (TestDiet, Richmond, IN) and supplied ad libitum. This formulation provided 300 mg / kg of Formula (I) Hemitartarate per day in a 25 g mouse.
[0121] [000121] For combination therapy studies, Fabry-Rag mice were placed in the study, 3 months old (treatment model). Group A mice received intravenous injections of recombinant human alpha galactosidase A (Genzima Corp) at a dose of 1mg / kg for 2 months (ie, 3, 5, 7 and 9 months of age). Group B received the same intravenous doses of enzyme plus they received Formula (I) Hemitartarate (Genzima Corp, Cambridge, MA) as a sediment component of the diet. The drug was formulated in 0.15% (w / w) in the 5053 murine standard (TestDiet, Richmond, IN) and provided ad libitum. This formulation provided 300 mg / kg of Formula (I) Hemitartarate per day in a 25 g mouse. Group C received enzyme injections every 4 months (that is, 3 and 7 months of age) and was on the drug diet in the same group B diet. Group D received only the drug diet in the food (the same as the groups B and C). Group E was treated with Fabry Rag mice and group F was wild-type controls. See figure 10. Quantification of globotriaosylceramide tissue levels (GL-3, Gb3)
[0122] [000122] The quantitative determination of GL-3 was by Tandem Mass Spectrometry essentially as for GL-1.
[0123] [000123] Hot plate assay was performed as previously described (Ziegler, RJ et al. Moles. Ter. 15 (3), 492-500 (2007). Results of Monotherapy of Fabry mice with Formula (I) Hemitartarate
[0124] [000124] LRF was evaluated in a mouse model of Fabry disease, which is caused by a deficiency of α-galactosidase A activity. Formula (I) Hemitartarate Therapy started with one-month-old Fabry mice and continued until the mice to reach one year of age. The animals were dosed with 300 Hemitartarate Formula mg / kg (I) in their diet each day. The behavioral tests (ie, the hot plate assay) and biochemical tests (ie, analysis of urine level in GL-3 in tissues / blood / urine) of the mice were performed bimonthly.
[0125] [000125] As shown in figure 7, administration of Formula (I) Hemitartarate to Fabry-Rag Mice over an 11-month period decreased the rate of lysosomal accumulation of globotriaosliceramide (GL-3) in somatic organs (liver, kidney, heart and spleen) by about 50%. This translated into a delay in the progression of the disease as evidenced by a later presentation of insensitivity to an aversive heat stimulus (see figure 8) and a prevention of deterioration of urine factors, for example, urine volume, creatinine and levels sodium (see figure 9). Thus, the measurement of Formula (I) Hemitartarate for the inhibition of glycosylceramide synthase that catalyzes the first step in the synthesis of glycosphingolipids, is not only advantageous in animal models of Gaucher disease, but also of Fabry disease, and can also have positive effects on other glycosphingolipidoses.
[0126] [000126] Combined therapy of Fabry mice with -galactosidase A and Hemitartarate of Formula (I)
[0127] [000127] The effectiveness of ERT alone and in combination with TSA using Formula (I) Hemitartarate was evaluated in five Fabry-Rag Mouse populations (n = 12 / group). Beginning at three months of age, the mice were subjected to a program of behavioral tests (ie, hot plate assay) and biochemical tests (ie, analysis of GL-3 level in tissues / blood / urine), as shown in figure 10. In mice submitted to ERT, 1 mg / kg of doses of -galactosidase A were administered in the schedule as shown in figure 10. In mice submitted to TSA, 300 mg / kg of doses of Formula (I) Hemitartarate were administered daily in the mouse diet.
[0128] [000128] As shown in figure 11, ERT reduces blood GL-3 levels in Fabry-Rag mice, while SRT does not. As shown in figure 12, the ERT / SRT combination is more effective in reducing GL-3 Levels in the liver and kidney of Fabry Rag mice.
[0129] [000129] As shown in figure 13, SRT reduces urine GL-3 levels in Fabry-Rag mice, whereas ERT does not. As shown in figure 14, SRT but not ERT delays the onset of heat insensitivity in Fabry-Rag mice.
[0130] [000130] In summary, Fabry-Rag mice treated with a combination of Fabrazima and Formula (I) Hemitartarate exhibited improvements in disease markers plus ERT or TSA alone in a treatment model in the following ways: significantly reduced liver and GL accumulation -3 in the kidney with combination therapy; GL-3 urine improved in SRT groups; peripheral and late neuropathy in SRT groups; GL-3 blood in ERT groups improved.
[0131] [000131] B. Gaucher disease. To determine whether sequential use of both enzyme replacement therapy (ERT) and substrate reduction therapy (TRS) can provide additional benefits, the relative efficacies of separate and sequential therapies in a murine model of Gaucher disease (D409V / null) were compared. Methods
[0132] [000132] Animal studies. The procedures involving animals were analyzed and approved by the Institutional Animal Care and Use Committee (IACUC) at Genzima Corporation following the guidelines issued by the Association for Evaluation and Accreditation of the Animal Care Laboratory (AAALAC). The Gaucher mouse (D409V / null) is a type 1 Gaucher disease model exhibiting accumulation of glycosylceramide in the spleen, liver and lungs, but it lacks bone or brain pathology (see YH. Xu et al., Am. J Patol. 163, 2003, 2093-2101, the teachings in their entirety which are incorporated by reference in the present invention). Animals of both sexes with 3 months of age were placed in study as previous experiences indicated that there was no difference in the response between males and females of recombinant glycocerebrosidase or Formula (I) Hemitartarate. The study had 6 groups of mice with group A being sacrificed after 2 weeks to provide baseline levels of tissue glycosylceramide. Groups B, C, D and all received recombinant human glycocerebrosidase (Genzima Corp, Cambridge, MA) (10mg / kg) intravenously via a tail vein (100 uL) every 2 days for a total of 8 injections. Group B was sacrificed at the end of this scheme (at the same time as group A) to provide enzyme-reduced levels of tissue glycosylceramide. Groups D and E were fed both Formula (I) Hemitartarate (Genzima Corp, Cambridge, MA) as a component of the pellet diet. The drug was formulated at 0.075% (w / w) in the 5053 murine standard (TestDiet, Richmond, IN) and provided ad libitum. This formulation provided 150 mg / kg of Formula (I) Hemitartarate per day in a 25 g rat. Group F received no treatment and was sacrificed together with groups C, D and E 12 weeks after the start of the study. The mouse's food intake and weight were monitored three times a week to determine drug consumption and the potential impact of the drug on overall health. The animals were sacrificed by inhaling carbon dioxide and their tissues harvested immediately. Half of each tissue was frozen on dry ice and stored at -80 ° C until ready for further processing. The other half was processed for histological analysis.
[0133] [000133] Quantitative determination of tissue glycosylceramide levels. Glycosylceramide levels were quantified by mass spectrometry as previously described (see K. McEachern, et al., J. Med. Gene 8 (2006) 719-729; T. Doering, J. Biol Chem 274 (1999) 11,038 a 11.045, the teachings in their entirety which are incorporated by reference in the present invention). A known mass of tissue was homogenized in 2: 1 (v / v) chloroform: methanol and incubated at 37 ° C for 15 min. The samples were centrifuged and the supernatants were extracted with 0.2 volume of water overnight at 4 ° C. The samples were centrifuged, the aqueous phase was discarded, and the organic phase was dried down to a film under nitrogen. For ionization mass spectrometry (ESI / MS), analysis of tissue samples were reconstituted to the equivalent of 50 ng of original tissue weight in 1 ml of chloroform: methanol (2: 1, v / v) and vortexed for 5 min. Aliquots (40 µl) of each sample were delivered to the full recovery vials and 50 µl of an internal standard 10 µg / ml d3-C16-GL-1 (Matreya, Inc., Gap Pleasant, PA) was added. The samples were dried under nitrogen and reconstituted with 200 µL of 1: 4 (v / v) DMSO: methanol. ESI / MS analysis of glycosylceramides of different lengths of carbon chain was performed on an HPLC (Separation Module 2695) coupled to a Quattro Micromass Micro system equipped with an electrospray ion source. Samples of lipid extract (20 µL) were injected into a C8 column (4 mL X 3 mm di; Phenomenex, Torrance, CA) at 45 ° C and eluted with a gradient of 50 to 100% acetonitrile (ammonium acetate a 2mM, 0.1% formic acid) at 0.5 mL / min. The first 0.5 min was performed in 50% organic and then quickly moved to 100% for the final 3.5 min. The source temperature was kept constant at 150 ° C and nitrogen was used as a desolvation gas, with a flow rate of 670 L / h. The capillary voltage was maintained at 3.80 kV with a cone voltage of 23 V, while the residence time for each ionic species was 100 ms. The spectra were acquired by the MRM method to monitor eight dominant isoforms (C16: 0, C18: 0, C20: 0, C22: 1, C22: 0, C22: 1-OH, C24: 1, and C24: 0). The quantitative determination of glycosylceramide was based on the sum of these eight isoforms in relation to the internal standard, with a calibration curve ranging from 0.1 to 10 ug / mL.
[0134] [000134] Histology. For histological analysis, the tissues were fixed in zinc formalin (Electron Sciences Microscopy, Hatfield, PA) at room temperature for 24 h, then stored in PBS at 4 ° C until ready for further processing. All samples were dehydrated in ethanol, cleaned in xylenes, infiltrated and embedded in Surgipat R paraffin (Surgipat, Richmond, IL). Five micron slices were cut with a rotary microtome and dried in a 60 ° C oven before staining. The cuts were deparaffinized in Hemo-De (Scientific Safety Solvents, Keller, TX) and rehydrated in descending ethanol concentrations, followed by a PBS wash. The sections were stained with Hematoxylin and Eosin (H & E) and labeled with a monoclonal anti-mouse-CD68 mouse (Serotec, Raleigh, NC) to identify the macrophages. After washing for 5 min in PBS, the slides were dehydrated in ethanol and cleaned in Hemo-De before assembly with Shur / Mount ® medium mounting plate (TBS, Durham, NC). The percentage of CD68 immunopositivity area in the liver was quantified using MetaMorph (MDS Analitical Technologies, Toronto, Canada) by analyzing ten 400x images per tissue section. A veterinary pathologist certificate board blinded the group designation examined in all sections. Results of
[0135] [000135] Glucocerebrosidase regimen dosage for the release of GL1 accumulated in the liver, spleen and lung of 3-month-old Gaucher mice. To investigate the relative merits of combination and monotherapy with enzyme or substrate reduction therapy, the enzyme regimen that maximally depleted GL1 levels in Organs visceral organs of Gaucher mice was determined. Gaucher mice with 3 months of age (D409V / null) were administered intravenously 2, 4 or 8 doses of 10 mg / kg recombinant human glycocerebrosidase. Mice that were treated with 2 or 4 doses of the enzyme received drug infusions every 3 days while those that were treated with 8 doses received the enzyme every 2 days. The use of a short time interval between infusions in animals that received 8 treatments was designed to minimize the potential impact of any immune response to the administered human enzyme. The animals were sacrificed 7 days after the last enzyme infusion and the amount of GL1 remaining in their livers and spleens and the lungs were measured.
[0136] [000136] Treatment with 2 doses of glucocerebrosidase reduced GL1 levels in the liver by 50%. Increasing the number of enzyme infusions to 4, or 8, as expected, reduced liver GL1 levels to a greater extent (by approximately 75%). Less than complete lowering of GL1 levels, even at doses of 8, is consistent with experience in Gaucher subjects who show that hepatosplenomegaly is reduced only after a prolonged period of treatment (see GA Grabowski, et al., Ann. Int Med. 122 (1995) 33-39, the teachings in their entirety which are incorporated by reference in the present invention). The substrate levels in the spleens of Gaucher mice were more refractory to the enzyme treatment. The administration of 2 doses of glucocerebrosidase did not significantly alter the levels of GL1 as observed in untreated controls. By increasing the number of enzyme infusions to 4, or 8, the levels of splenic GL1 were reduced by about 50%. In the lung, a reduction of approximately 60% in the untreated control was observed after 8 doses. The slightly lower extent of substrate reduction in the lung was probably due to a degradation in the accessibility of the infused enzyme to the alveolar-loaded lipid macrophages. The observation of greater GL1 clearance in the liver, when compared to the spleen and lung, probably reflects the enzyme's biodistribution after systemic infusion (see Van Patten SM, et al. Glicobiology 17 (2007) 467-478, the teachings of which are whole incorporated by reference). Based on these results, the treatment regimen consisting of 8 consecutive doses of 10 glucocerebrosidase mg / kg administered at 2-day intervals was used for subsequent studies.
[0137] [000137] Relative enzyme and substrate capabilities of reduction therapy in order to reduce the levels of GL1 in the liver of Gaucher mice. Sections of 3-month-old Gaucher mice were treated with recombinant glycocerebrosidase or Formula (I) hemitartarate separately or sequentially. Mice in groups B, C and D were given doses of 8 enzyme as described above (over a period of 2 weeks) to clear accumulated GL1. Different groups were then fed with Formula feed or normal feed containing (I) Hemitartarate (150 mg / kg / day) for an additional period of 10 weeks with group F that received no treatment and serving as a naive control. Regardless of the Chow formulation, the mice ate comparable amounts of food and there were no discernible differences in weight gain. Approximately 80% of the stored GL1 levels were cleared from the liver after 2 weeks of enzyme therapy alone. When these animals were allowed to progress without further treatment for 10 weeks, their liver GL1 levels increased, indicating that substrate re-accumulation had occurred during the intervention period (figure 2, column C). These levels were not significantly different from those of untreated controls (figure 2, column F). However, if the rats were treated with enzyme and then Formula (I) Hemitartarate in their food over a 10-week period, their liver GL1 levels were significantly lower than the untreated controls (figure 2 , column D and F). This result suggests that the additional treatment with Hemitartarate Formula (I) had slowed the re-accumulation of the substrate. Interestingly, mice treated with Gaucher with Formula (I) Hemitartarate alone during the study period (12 weeks) also showed lower levels of GL-1 (figure 2, column E) when compared to untreated, similar age controls (figure 2, column F), although the difference was not significant. The ability of SRT alone to reduce GL1 levels in this animal model is consistent with our previous report (see KA McEachern, et al., Mol. Genet. Metab. 91 (2007) 259-267, the teachings in their entirety the which are incorporated by reference in the present invention) and probably reflects the fact that Gaucher mice (D409V / null) retain residual enzymatic activity (see YH. Xu et al., Am. J. Patol. 163, 2003, 2093- 2101, the teachings in their entirety which are incorporated by reference into the present invention).
[0138] [000138] Relative enzyme and substrate capabilities in reduction therapy to reduce GL1 levels in the spleen of Gaucher mice. Treatment of 3-month-old Gaucher mice with recombinant glycocerebrosidase alone for 2 weeks reduced splenic GL1 levels by about 60% (figure 3, column B). When these animals were allowed to age for an additional 10 weeks without any further intervention, the substrate levels returned to those observed at the beginning of the study (figure 3, column C) and were not significantly different from the untreated control (figure 3, column F). This suggests that the rate of GL1 re-accumulation in the spleen was higher than in the liver. This assumption was also supported by observing higher baseline levels of the substrate in the spleen (~ 1500 mg tissue / g; Figure 2, column A) than in the liver (~ 500 mg tissue / g; Figure 3, the column THE) . The animals that had been treated with the enzyme and then the Formula (I) Hemitartarate for the next 10 weeks showed the greatest reduction in the spleen levels GL1 (figure 3, column D) and these were significantly lower than those in the spleens untreated control units (figure 3, column F). This indicates that the implantation of SRT not only delayed the new accumulation of substrate, but also acted to reduce the storage load in this organ. It appears that, at least in the present example, the net effect of the residual endogenous enzyme and substrate reduction led to a further reduction in substrate levels in general. The observation of lower splenic levels of GL1 in mice treated with Formula (I) Hemitartarate alone for 12 weeks (figure 3, column E) than in untreated controls (figure 3, column F) is consistent with this concept , although the difference was not significant. Thus, in the Gaucher type, take 1 patients with high residual enzyme activity, ERT treatment followed by SRT could accelerate the rhythm and perhaps even the degree of substrate clearance.
[0139] [000139] Relative enzyme and substrate capabilities in reduction therapy to reduce the levels of GL1 in the lungs of Gaucher mice. As noted earlier, pulmonary GL1 levels were less effectively ascertained through intravenous administration of recombinant glucocerebrosidase. Treatment of 3-month-old Gaucher mice with enzyme for 2 weeks resulted in only a 30% reduction in substrate levels in the lung (Figure 4, column B). The group of animals fed normal ration for the next 10 weeks showed, as expected, a re-accumulation of GL1 and were not significantly different from the untreated levels (figure 4, column C and F). In contrast, animals fed a diet containing Formula (I) Hemitartarate during the same period showed a reduction in substrate levels below those administered with enzyme alone (figure 4, column D) and were significantly lower than those in untreated controls (figure 4, column F). Again, this suggests that in the lung, as in the spleen, the net effect of Formula (I) Hemitartarate (in the presence of endogenous residual enzyme activity), not only delayed the re-accumulation of GL1 but also acted to further reduce the starting levels. As with other visceral organs, treatment by Formula (I) Hemitartarate alone was effective in reducing pulmonary GL1 levels (figure 4, column E) when compared to untreated controls (figure 4, column F).
[0140] [000140] Histopathological analysis of the liver of Gaucher mice after enzyme and substrate reduction treatment. To visualize the effects of different therapeutic regimes on the liver, tissue sections were stained for CD68, a macrophage marker. Analysis of liver cuts from untreated 3-month-old Gaucher mice showed the presence of large numbers of lipid-engorged, Gaucher CD68-positive cells that remained essentially unchanged when analyzed 12 weeks later. Consistent with the biochemical data above, livers from animals administered recombinant glycocerebrosidase over a 2-week period showed substantial lipid clearance in these abnormal macrophages. If these animals were allowed to age an additional 10 weeks without further treatment, there was evidence of GL1 re-accumulation as indicated by the re-emergence of Gaucher cells. However, this increase in Gaucher cells was negated if the mice received the substrate reduction therapy with the Formula (I) Hemitartarate during the same intervention period. As noted earlier, the mice that received Gaucher Formula (I) Hemitartarate alone also showed reduced accumulation of the substrate, although not to the same degree as those that received a combination of ERT and TSA. The extent of CD68-positive staining on the various sections was also quantified using MetaMorph software (figure 18). The degree of staining in these sections mirrored the amounts of liver. The biochemically determined GL1 levels (figure 15) continue to support suggestions about the relative merits of different therapeutic regimens. Example 8: Efficacy of Formula (I) Hemitartarate in a mouse model of Gaucher disease
[0141] [000141] Animal studies. Procedures involving animals have been reviewed and approved by Institutional Animal Care and Use Commitee (IACUC) according to the Association for the Evaluation and Accreditation of the Animal Care Laboratory (AAALAC),
[0142] [000142] State and federal guidelines. he Gaucher gbaD409V / null mice (see Y.-H. Xu. Et al., Am. J. Patol. 163 (2003) 2093-2101, the teachings in their entirety which are incorporated by reference into the present invention) have been permitted according to the needs of the study. There was no difference in phenotype or response to Formula (I) Hemitartarate between males and females, so both sexes were used in the studies. The delivery of Formula (I) Hemitartarate was for a single daily oral gavage in a volume of 10 mL / kg. The animals were acclimated to oral gavage with a similar volume of water for one week before the start of treatment. Formula (I) hemitartarate was dissolved in water for injection (WFI; VWR, West Chester, PA) and administered at a dose increase of 75 mg / kg / day to 150 mg / kg / day over nine days, with three days in each dose and increments of 25 mg / kg / day. The mice were weighed three times a week to monitor the drug's potential impact on their overall health. The animals were sacrificed by inhaling carbon dioxide and their tissues harvested immediately. Half of each tissue was frozen on dry ice and stored at -80 ° C until ready for further processing. The other half was collected for histological analysis.
[0143] [000143] The quantification of glycosylceramide levels in tissues by means of thin layer chromatography. Analysis of thin layer high performance chromatography (HP-TLC) was as described (A. Abe, et al., J. Clin Inv 105 (2000) 1563-1571H. Zhao, et al. Diabetes 56 (2007) 1341-1349; and SPF Miller, et al J. Lab. Clin Med 127 (1996) 353-358, the entire teachings of each are incorporated into this invention by reference). Briefly, a total lipid fraction was obtained by homogenizing the tissue in cold PBS, extracting with 2: 1 (v / v) chloroform: methanol. and sonicating in a water bath sonicator. The samples were centrifuged to separate the phases and the supernatant was recovered. The pellets were resonated in chloroform: methanol: saline, centrifuged and the resulting supernatant was collected and the second combined with the first. A 1: 1 (v / v) mixture of chloroform: saline mixture was aided in the combined supernatants, vortexed, and centrifuged. After removing the upper aqueous layer, methanol: saline was added, vortexed and recentrifuged. The organic phase was taken and dried under a nitrogen atmosphere, dissolved in 2: 1 (v / v) chloroform: methanol at 1 mL per 0.1 g of original tissue weight and stored at - 20 ° C.
[0144] [000144] A portion of the lipid extract was used to measure the total phosphate, (see BN Ames, Methods Enzymol. 8 (1966) 115-118, the teachings in their entirety which are incorporated by reference in the present invention), i.e. , the phospholipid content to use as an internal standard. The remainder was subjected to alkaline methanolysis in order to remove the phospholipids that migrate with glycosylceramide on the HP-TLC plate. Extract aliquots containing equivalent amounts of total phosphate were stained on an HP-TLC plate, along with known glycosylceramide standards (Matreya inc. Gap Pleasant, PA). The lipids were resolved and visualized with 3% cupric acetate monohydrate (w / v). Phosphoric acid 15% (v / v) followed by cooking for 10 min at 150 ° C. The lipid bands were digitized using a densitometer (GS-700, Bio-Rad, Hercules, CA) and analyzed using the Quantity One software (Bio- Rad).
[0145] [000145] Quantitative determination of tissue glycosylceramide levels by mass spectrometry. Glycosylceramide was quantified by mass spectrometry, as described. (see K. McEachern, et al J. Med. Gene 8 (2006) 719-729; T. Doering, et al, J. Biol Chem 274 (1999) 11.038-11.045, the entire teachings of each are in the present invention incorporated by reference). The tissue was homogenized in 2: 1 (v / v) chloroform: methanol and incubated at 37 ° C. The samples were centrifuged and the supernatants were extracted with 0.2 volume of water overnight. The samples were centrifuged again, the aqueous phase was discarded, and the organic phase was dried down to a film under nitrogen.
[0146] [000146] For ionization mass spectrometry (ESI / MS), analysis of tissue samples were reconstituted to the equivalent of. 50 ng weight of the original tissue in 1 ml of chloroform / methanol (2: 1, v / v) and vortexed for 5 min. Aliquots of each sample (40 µl) were delivered to 50 µl full recovery vial waters and 10 µg / mL d3-C16-GL-1 internal standard (Matreya, Inc., Gap Pleasant, PA) was added. The samples were dried under nitrogen and reconstituted with 200 μl of 1: 4 DMSO: methanol. ESI / MS analysis of glycosylceramides of different lengths of carbon chain was performed on an HPLC (Separation Module 2695) coupled to a Quattro Micromass Microequipped system with an ion source of electro-sprinkler. Twenty microliters of lipid extract samples were injected into a C8 column (4 ml x 3 mm id; Phenomenex, Torrance, CA) at 45 ° C and eluted with a 50-100% acetonitrile gradient (2 mM ammonium acetate, 0, 1% formic acid) at 0.5 ml / min. The first 0.5 min are performed at 50% organic and then quickly switched to 100% for the final 3.5 min. The source temperature was kept constant at 150 ° C and nitrogen was used as a desolvation gas, with a flow rate of 670 L / h. The capillary voltage was maintained at 3.80 kV with a cone voltage of 23 V, while the residence time for each ionic species was 100 ms. The spectra were acquired by the MRM method to monitor eight dominant isoforms (C16: 0, C18: 0, C20: 0, C22: 1 C22: .. 0 C22: 1-OH, C24: 1, and C24: 0). The quantitative determination of glycosylceramide is based on the sum of these eight isoforms for the internal standard, with a calibration curve range of 0.1-10 µg / mL.
[0147] [000147] Histology. For histological analysis, the tissues were fixed in zinc formalin (Electron Sciences Microscopy, Hatfield, PA) at room temperature for 24 h, then stored in PBS at 4 ° C until ready for further processing. All samples were dehydrated in ascending alcohol concentrations, cleaned in xylenes and infiltrated and embedded in Surgipat R paraffin (Surgipat, Richmond, IL). Five micron slices were cut with a rotary microtome and dried in a 60 ° C oven before staining. Cuts were deparaffinized in xylenes, and rehydrated in descending concentrations of alcohol followed by a wash with water. After washing in 1 min at 3% in acetic acid, the slides were stained for 40 min in 1% Alcian Blue 8GX (Science electron microscopy) in 3% acetic acid at pH 2.0. After rinsing in water and doxidation in 1% periodic acid for 1 min. slides were stained with Schiff's reagent (Surgipat) for 12 minutes. After washing for 5 min in hot water, the slides were dehydrated in alcohol and cleaned in xylenes before assembly with Shur / MountTM (TBS, Durham, NC). Gaucher cells morphologically identified in the liver were quantified using a cell counting manual for 10 high power fields (HPFS, 400x). Results of
[0148] [000148] Effect of the administration of Formula (I) Hemitartarate to D409V / null mice. The effect of administering Formula (I) Hemitartarate to D409V / null mice was evaluated. Approximately 7 months old mice were administered 150 mg / kg / day of Formula (I) Hemitartarate (a dose shown in preliminary studies to be effective in inhibiting glycosylceramide synthase) by oral gavage for 10 weeks. This treatment had no noticeable effect on the welfare or feeding habits of the rats. Measurements of body weight throughout the study showed no significant deviation from that of untreated rats, suggesting that Hemitartarate Formula (I) was well tolerated at a dose shown to be effective in inhibiting synthase.
[0149] [000149] Efficacy in the treatment of young Formula (I) Hemitartarate, pre-symptomatic Gaucher mice, Formula (I) Hemitartarate was evaluated for the reduction of the lysosomal accumulation of glycosylceramide and the appearance of Gaucher cells in young people (10 weeks of age) rat D409V / null. These young Gaucher mice exhibit low levels of GL-1 in the affected tissues. Ten-week-old animals were administered either 75 or 150 mg / kg / day of Formula (I) Hemitartarate by oral gavage for 10 weeks. Measurement of glycosylceramide levels showed a dose-dependent reduction when compared to age-matched treated vehicle controls. In the cohort that had been treated with 150 mg / kg / day, the levels of glycosylceramide were 60, 40 and 75% of the people in the controls, in the liver, lung and spleen, respectively (Figure 6). The statistically significantly lower levels of glycosylceramide observed in the liver and lung of treated D409V / null mice indicated that Formula (I) Hemitartarate was effective in reducing the accumulation of the present glycosphingolipids in these tissues.
[0150] [000150] Histopathological evaluation of livers from untreated D409V / null mice at the end of the study (20 weeks of age) showed the presence of Gaucher cells throughout the liver. Mice treated with 150 mg / kg / day of Formula (I) Hemitartarate for 10 weeks showed only the occasional presence of Gaucher cells that were also invariably smaller in size. The quantitative determination of these cells in a number of different sections confirmed that the frequency of Gaucher cells was significantly lower in the mice treated with Formula (I) Hemitartarate. Taken together, these biochemical and histological results suggested that daily oral administration of Formula (I) Hemitartarate to pre-symptomatic Gaucher mice was effective in decreasing the accumulation of glycosylceramide in the affected tissues and the consequent formation of Gaucher cells in the liver.
[0151] [000151] Effectiveness of treatment of Formula (I) Hemitartarate in older Gaucher mice with pre-existing pathology. The efficacy of Formula (I) Hemitartarate in arresting or reversing disease progression in older symptomatic Gaucher mice was also evaluated. Seven-month old mice of D409V / null were administered 150 mg / kg / day of Formula (I) Hemitartarate by oral gavage for 10 weeks. Analyzes of glycosylceramide levels in the liver, lung and spleen of mice treated at 5 and 10 weeks post-treatment showed that they had not increased beyond those seen at the beginning of the study. Alter 10 weeks of treatment, glycosylceramide levels were determined to be 60% lower in the liver, 50% lower in the lung and 40% lower in the spleen than in vehicle-treated mice. These results showed that Hermitartarate of Formula (I) was effective in inhibiting additional glycosylceramide accumulation in mice with an existing load of storage pathology.
[0152] [000152] Histopathological analysis of tissue sections showed a reduced number of Gaucher cells in the liver of treated D409V / null mice when compared to untreated controls. The quantitative determination of the number of Gaucher cells corroborated the biochemical results; the treated D409V / null mice exhibited Gaucher cell counts that were not significantly different from those at the start of treatment, both at 5 - and 10-week time points. The numbers of Gaucher cells at both these time points were significantly lower than those of untreated D409V / null mice. Taken together, these data demonstrate that Hermitartarate of Formula (I) effectively inhibited the additional accumulation of glycosylceramide and development of Gaucher cells in animals with pre-existing pathology. Discussion
[0153] [000153] Hermitardate of Formula (I) demonstrated a high degree of specificity for the enzyme glycosylceramide synthase. There was also no measurable inhibition of glucocerebrosidase activity at the effective dose, which is an important feature when it comes to type 1 Gaucher disease, patients, most of whom retain residual glucocerebrosidase activity. At an effective dose of 150 mg / kg / day, there were no observable gastrointestinal problems and there was no difference in body weights between treated and untreated control groups. Serum concentrations above IC50 (24 to 40 nm) were readily achievable with oral doses that were below the maximum tolerated level. Hermitardate of Formula (I) was also readily metabolized and cleaned: both the parent compound and metabolites effectively cleaned within 24 h as shown once and the repeat oral dose ADME studies with 14C-radiolabeled compound in mice and dogs.
[0154] [000154] Using a non-optimized regimen of administration of a single daily probe by oral route successfully prevented the accumulation of glycosylceramide in both young, pre-symptomatic mice and in older Gaucher mice that have already exhibited storage pathology. Young mice, 10-week-old mice, despite harboring high levels of glycosylceramide compared to wild-type controls, had not yet developed the engorged tissue macrophage characteristics, called Gaucher cells. Treatment with 150 mg / kg / day of Formula (I) Hemitararate halted all measurable disease progression and inhibited the development of Gaucher cells. In older mice exhibiting a higher level of lysosomal glycosylceramide and the number of Gaucher cells, there was no further increase in the levels of glycosphingolipids or in the number of storage cells after a week 5 or 10 weeks of treatment. As the main source of glycosylceramide in Gaucher cells is reported to be extracellular in origin, these results suggested that the inhibition of Formula (I) Hermitartarate from glycosylceramide synthase was systemic.
[0155] [000155] The observation that Formula (I) Hermitardate was effective in preventing further glycosylceramide accumulation suggests a therapeutic strategy that could further increase the treatment of Gaucher disease.
[0156] [000156] In summary, the data presented here demonstrate that Hermitartarate of Formula (I) is an active and specific inhibitor of glycosylceramide synthase exhibiting no evident adverse effects in a mouse model of Gaucher disease. He was able to prevent disease progression in both pre-symptomatic and older sick Gaucher mice by accumulating glycosylceramide inhibition and forming Gaucher cells. These findings suggest that Formula (I) Hermitartarate may represent yet another therapeutic option for both pediatric and adult type 1 Gaucher disease and, potentially, other glycosphingolipid storage disorders. Example 9: Phase 2 Hemitartarate Clinical Trial of Formula (I)
[0157] [000157] Methods. This clinical trial of Formula (I) Hemitartarate, gave 50 or 100 mg bid orally, treated with 26 adults with type 1 Gaucher disease (GD1) (16F: 10M; mean age 34 years, range 18-60 all Caucasian) at 7 locations in 5 countries. Patients should have splenomegaly (normal volume 10) and either thrombocytopenia (platelets 45,000-100,000 / mm3) or anemia (hemoglobin 8-10 g / dl, female; 8-11 g / dl, male). None received enzyme replacement or substrate reduction therapy in the previous 12 months. The primary end point of the compound's effectiveness is the globin level (+0.5 g / dl) or platelet count (+15%) after 52 weeks of treatment. Liver volume, chitotriosidase, glycosylceramide are also assessed. Patients continue to be treated and monitored for the long term.
[0158] [000158] Results. The 52-week data was available for up to 20 patients, another 4 withdrew prematurely and 2 were ongoing. The primary compound end point was received by 19 of the 20 patients. The changes in the medium (1DP) from the beginning to week 52 were: hemoglobin 1.6 (11.35) g / dL, platelet count 43.6% (137.59%); spleen and liver volume (multiples of normal) 40.2% (110.44%) and 15.8% (110.39%), respectively, and chitotriosidase 49.9% (120.75%). Plasma levels of glycosylceramide normalized after 4 weeks in all patients, Formula (I) Hemitartarate was well tolerated with an acceptable safety profile. Seven related adverse events in 6 patients were reported as related, all were mild and transient in nature. Example 10: Pharmaceutical composition of Formula (I) Hemitartarate, 100 mg capsules
[0159] [000159] Method of preparing 100 mg capsules: Formula (I) hemitartarate, microcrystalline cellulose, lactose monohydrate, and hypromellose, E15 were separately passed through a 20 mesh screen. Quantities of the sieved ingredients shown in Table 9 were mixed in a high shear granulator for nine to twelve minutes. Table 9. Pharmaceutical formulation for 100 mg capsules
[0160] [000160] The Ingredients were then wet granulated by adding purified Water (2.2 kg; 11.7% by weight of DRY Ingredients) to the granulator bowl until they were complete, as visually confirmed. The wet granulation discharged was from the bowl and passed through a rotary, sorting mill. The wet granulation was then dried and direct heating, static, solid, bed, oven, tray, dried at 50 ± 5 ° C at a moisture content not exceeding. 3.5%, as confirmed by the process of checking the dry granules which were then passed through a mill and screened. The granules were transferred to a V- mixer. Glycerol Beenate (0.2 kg) was added to the non-negligible V-mixer, and the final Mix was mixed until the mix was uniform, as determined by means of a uniformity test: Typically for ten to twenty minutes. The final Blend was then encapsulated in a # 2 pan size, using a Semi-Automatic Capsule Filling Agent to the appropriate weight of filler (270 mg of media), and the filled capsules were subjected to dedusting before being packaged. . Example 11A: Pharmaceutical composition of Formula (I) Hemitartarate, 10 mg capsules
[0161] [000161] 10 mg Capsule Preparation Method: The procedure of Example 10 was followed for the encapsulation step. To produce a 10 mg capsule, the final blend was encapsulated in a # 4 or # 5 size capsule using a capsule filling machine with the appropriate filling weight (average 27 mg), and the filled capsules were subjected to dedusting. before they are packed. Example 11B: Pharmaceutical composition of Formula (I) Hemitartarate, 50 mg capsules
[0162] [000162] 50 mg Capsule Preparation Method: The procedure of Example 10 was followed for the encapsulation step. To produce a 50 mg capsule, the final mixture was encapsulated in a size # 3 capsule, using a capsule filling machine with the appropriate filling weight (135 mg average), and the filled capsules were subjected to dedusting before be packaged. Example 11C: Pharmaceutical composition of Formula (I) Hemitartarate, 150 mg capsules
[0163] [000163] Method of preparing 150 mg capsules: The procedure of Example 10 was followed for the encapsulation step. To produce a 150 mg capsule, the final blend was encapsulated in a size # 0 capsule, using a capsule filling machine with the appropriate filling weight (405 mg average), and the filled capsules were subjected to dedusting before be packaged. Example 12: Pharmaceutical composition of Formula (I) Hemitartarate, 25 mg capsules
[0164] [000164] Method of preparing 25 mg capsules: The procedure of Example 10 was followed for the encapsulation step. To produce a 25 mg capsule, the final mixture was encapsulated in a size # 4 capsule, using a capsule filling machine with the appropriate filling weight (average 67.5 mg), and the filled capsules were subjected to dedusting. before they are packed. Example 13: Interactions of Medicinal Formula (I) Hermitardate - CYP2D6 Inhibitors
[0165] [000165] A study was conducted to assess the pharmacokinetics, safety and tolerability of multiple oral doses of Hermitartarate of Formula (I) (100 mg BID) administered with and without paroxetine (30 mg once daily), a potent inhibitor of CYP2D6. This was an open study, a fixed sequence study in 36 healthy subjects (17 men and 19 women). The secondary objectives were to evaluate paroxetine PK in combination with multiple doses of Formula (I) Hermitartarate (100 mg BID) in healthy subjects and to evaluate the sequence of Formula (I) PK Hermitartarate in multiple dose compared to a single dose of administration of Hermitartarate of Formula (I).
[0166] [000166] The mean pharmacokinetic parameters of the free base of Formula (I) Hermitardate as it exists in plasma were non-linear and revealed a 2-fold accumulation in AUC and Cmax with repeated administration (100 mg BID), compared to single dose administration. Concomitant administration of Formula (I) Hermitartarate and paroxetine resulted in a 7-fold increase in Cmax and a 9-fold increase in AUC compared to the administration of multiple doses of Formula (I) Hermitartarate alone. These results indicate that paroxetine can inhibit the metabolism of Hermitartarate of Formula (I) and increase the blood plasma concentrations of the drug. Similar effects would be expected with other potent CYP2D6 inhibitors (for example, fluoxetine and quinidine) and careful monitoring of plasma levels and potential dose adjustments are necessary when Formula (I) Hermitardate is co-administered with a drug known to be an inhibitor potent of CYP2D6. Paroxetine concentrations were about 1.5 to 2 times higher than expected, which suggests that Formula (I) Hermitardate or one of its metabolites may be a weak CYP2D6 inhibitor. Drug Interactions of Hermitardate of Formula (I) - CYP3A4 Inhibitors and P-glycoprotein (PGP) Inhibitors
[0167] [000167] A study was carried out to evaluate the pharmacokinetics, safety and tolerability of multiple doses of Hermitartarate of Formula (I) (100 mg twice daily) with and without multiple dose of ketoconazole (400 mg once daily), in healthy male and female individuals. This was an open and fixed study in 36 healthy subjects (18 men and women) consisting of 3 periods that included administration of 100-mg of a single dose of Formula (I) Hermitartarate, administration of multiple doses of Formula Hermitartarate (I), and concomitant administration of Hermitartarate of Formula (I) 100 mg (twice daily) with ketoconazole 400 mg (once daily). Repeated administration of Hermitartarate of Formula (I) and ketoconazole, a strong inhibitor of cytochrome P450 3A4 ("CYP 3A4") and p-glycoprotein, resulted in a 4-fold increase in the exposure of the free base of Formula (I) as Hermitartarate it exists in plasma at steady state. Thus, patients who already receive Formula Hermitate (I) may require a temporary dose reduction during concomitant treatment with potent CYP 3A4 inhibitors or p-glycoprotein. Example 15 - Stability Studies for the Formulation of Hermitartarate of formula (I)
[0168] [000168] The mixtures were prepared by mixing Hermitartarate of Formula (I) and excipients (Monohydrate encapsulation grade lactose, Avicel PH 301 (microcrystalline cellulose) and Metocel E15 LV Prem (Hydroxypropylmethylcellulose) in a bottle of scintillation in about one gram scale two water. 15.6% was added to the mixture and mixed to form wet granules. The wet granules were tested using a # 10 sieve (2000 micron aperture). The screened granules were then dried in an oven at 50 ° C for 2 hours. The dried granules were tested using a # 18 sieve (1000 micron aperture). The lubricant, glyceryl beenate, was added to the mixture and mixed to form the final mixture. are shown in the table below:
[0169] [000169] The seven formulation mixtures, which have different API: Lactose: avicel ratios, listed above were exposed to an elevated temperature at 85 ° C for 3 days (a forced degradation study condition) in order to understand the rate degradation and for the stability of each formulation. This accelerated condition was selected based on the results of the study that the extension of the degradation products of the drug 50mg after 24 months, similar to those obtained at 85 ° C for 3 days.
[0170] [000170] The forced degradation study was carried out using an HPLC reverse phase gradient method using a C18 column (Waters T3, 3μm, 100 x 4.6 mm), mobile phase consisting of water and 0.1% acetonitrile trifluoroacetic acid (TFA), UV detection at 280 nm, column temperature at 40oC, and a flow rate of 2 ml / min. The gradient started to hold on B 5% (acetonitrile and 0.1% TFA) for 0.5 minute, and then increasing the organic component on B 4.83% per minute for up to 15 minutes.
[0171] [000171] The total degradation products of each formulation mixture were added and depending on the ratio of API: Lactose: Avicel and the results are shown in figure 15. The results of the study suggest that, while keeping the proportion of API and lactose constant , decreasing the amount of avicel improves the stability of the formulation. When avicel is removed, the formulation has API: Lactose: Avicel 1: 2.1: 0 ratio, it is the most stable formulation. When lactose is removed, the formulation has an API: Lactose: Avicel Ratio of 1: 0: 2.1, and this formulation is not the most unstable compared to other ratios. The combined information suggests that lactose stabilizes the formulation, while avicel destabilizes the formulation. However, when both excipients are present, they interact with each other. The proportion must be adjusted to obtain a stable formulation.
[0172] [000172] For active pharmaceutical ingredients like Formula (I) hemihydrate that are soluble in water, microcrystalline cellulose helps to form granules during wet granulation as it is insoluble in water. If microcrystalline cellulose has not been used, a sudden change occurs from the granule phase to a paste form. The paste form was difficult to handle and the resulting particles after drying do not have adequate mechanical strength and particle size distribution. The pharmaceutical composition which has 37% by weight of a Formula (I) Hemitartarate, 41.0% by weight of a water-soluble filler; 16.7% by weight of an insoluble filler, 2% by weight to about 6% by weight of a binder, and about 0.1% by weight to about 2% by weight of a lubricant, all in a dry solids base has the best stability profile with respect to the amount of degradation products formed.
[0173] [000173] The teachings of all patents, published patent applications and references cited in the present invention are incorporated by reference in their entirety.
[0174] [000174] Although this has been particularly illustrated and described with references to the example modalities of the same, it will be understood by those who are versed in the technique that various changes in form and details can be made in it without departing from the scope encompassed by the claims in attachment.

权利要求:
Claims (2)
[0001]
Hemitartrate salt of a compound represented by the following structural formula:
[0002]
Pharmaceutical composition, characterized by the fact that it comprises the hemitartrate salt as defined in claim 1 and a pharmaceutically acceptable carrier or diluent.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US20210369672A1|2021-12-02|Inhibitors of glucosylceramide synthase

同族专利:

---

公开号 | 公开日

---

JP2013512252A|2013-04-11|

---

PT3133070T|2019-11-11|

---

EP2504332B2|2021-03-17|

---

AU2017265180B2|2019-08-01|

---

JP2015212291A|2015-11-26|

---

NZ625712A|2016-02-26|

---

PE20121337A1|2012-10-12|

---

ES2493940T5|2021-11-23|

---

KR20150125734A|2015-11-09|

---

ME02477B|2017-02-20|

---

TW201639562A|2016-11-16|

---

JP6452635B2|2019-01-16|

---

US20130137743A1|2013-05-30|

---

ES2493940T3|2014-09-12|

---

EA023923B1|2016-07-29|

---

LT3133070T|2019-11-11|

---

JP2019001809A|2019-01-10|

---

ES2586947T3|2016-10-19|

---

US20160166542A1|2016-06-16|

---

EP3599237B1|2021-03-24|

---

SG10201407881WA|2015-01-29|

---

EP2504332A1|2012-10-03|

---

DK3133070T3|2019-11-11|

---

LT2796457T|2016-09-12|

---

HRP20191647T1|2019-12-13|

---

CA2781676A1|2011-06-03|

---

CY1117996T1|2017-05-17|

---

PL3599237T3|2021-09-27|

---

AU2017265180A1|2017-12-14|

---

TN2012000237A1|2013-12-12|

---

CR20120277A|2012-09-05|

---

CN112521367A|2021-03-19|

---

EA029990B1|2018-06-29|

---

TW201737910A|2017-11-01|

---

PL2796457T3|2017-04-28|

---

PE20171255A1|2017-08-28|

---

BR112012012947B8|2021-05-25|

---

CL2016002589A1|2017-05-19|

---

AR079152A1|2011-12-28|

---

SMT201600273B|2016-08-31|

---

ES2754398T3|2020-04-17|

---

CN105777707B|2020-10-13|

---

WO2011066352A8|2011-08-11|

---

PL3133070T3|2020-01-31|

---

CN102712629B|2016-10-12|

---

SI3133070T1|2019-11-29|

---

SG10201800136QA|2018-02-27|

---

CA3075788A1|2011-06-03|

---

NZ600155A|2014-10-31|

---

EP3133070A1|2017-02-22|

---

IL260299D0|2018-08-30|

---

CY1115880T1|2017-01-25|

---

CA3140959A1|2011-06-03|

---

ES2875382T3|2021-11-10|

---

RS53503B1|2015-02-27|

---

SI2796457T1|2016-10-28|

---

TW201130822A|2011-09-16|

---

TWI606827B|2017-12-01|

---

SI2504332T2|2021-08-31|

---

WO2011066352A1|2011-06-03|

---

AU2016202591B2|2017-11-30|

---

US20210369672A1|2021-12-02|

---

UA113491C2|2017-02-10|

---

ECSP18063798A|2020-02-28|

---

CN112521366A|2021-03-19|

---

EA201270646A1|2013-01-30|

---

DK2504332T4|2021-06-07|

---

EP3896069A4|2021-10-20|

---

PL2504332T3|2014-10-31|

---

CA3075788C|2021-12-21|

---

MA33838B1|2012-12-03|

---

EP2796457B1|2016-05-18|

---

RS59543B1|2019-12-31|

---

RS54978B1|2016-11-30|

---

CN105777707A|2016-07-20|

---

HRP20140780T4|2021-08-06|

---

BR112012012947A2|2017-03-01|

---

DK2796457T3|2016-08-29|

---

EP3896069A1|2021-10-20|

---

PL2504332T5|2021-08-23|

---

CL2012001348A1|2012-08-24|

---

US20210393590A1|2021-12-23|

---

PT3599237T|2021-04-28|

---

NZ715108A|2017-06-30|

---

JP2016138124A|2016-08-04|

---

HUE045784T2|2020-01-28|

---

TWI656873B|2019-04-21|

---

AU2016202591A1|2016-05-19|

---

KR20180049255A|2018-05-10|

---

PT2796457T|2016-07-14|

---

DK2504332T3|2014-08-25|

---

HRP20161038T1|2016-10-21|

---

AU2010324810B2|2016-05-12|

---

CN105753846A|2016-07-13|

---

US10888547B2|2021-01-12|

---

EP3599237A1|2020-01-29|

---

JP6370264B2|2018-08-08|

---

DOP2012000141A|2012-09-30|

---

EP3133070B1|2019-08-14|

---

HK1172031A1|2013-04-12|

---

EA201890254A3|2019-02-28|

---

HK1203485A1|2015-10-30|

---

GT201200161A|2014-01-27|

---

RS53503B2|2021-07-30|

---

EA201890254A2|2018-10-31|

---

HRP20140780T1|2014-11-07|

---

NI201200096A|2012-10-30|

---

ECSP12011926A|2012-07-31|

---

HUE029371T2|2017-02-28|

---

CN102712629A|2012-10-03|

---

KR20200013105A|2020-02-05|

---

DOP2016000250A|2017-02-15|

---

IL283935D0|2021-07-29|

---

AU2010324810A1|2012-06-07|

---

CN105753846B|2019-11-15|

---

EP2504332B1|2014-06-04|

---

PH12015502514A1|2016-08-15|

---

IL219892D0|2012-07-31|

---

CA2781676C|2020-05-05|

---

SI2504332T1|2014-09-30|

---

EP2796457A1|2014-10-29|

---

PT2504332E|2014-09-02|

---

MX2012006083A|2012-06-19|

---

EA201592195A1|2016-04-29|

---

TWI586663B|2017-06-11|

---

MY160542A|2017-03-15|

---

US20160120842A1|2016-05-05|

---

KR102073207B1|2020-02-05|

---

MX358345B|2018-08-14|

---

KR20120115972A|2012-10-19|

---

JP2020189873A|2020-11-26|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB8311286D0|1983-04-26|1983-06-02|Searle & Co|Carboxyalkyl peptide derivatives|

---

DE3522475A1|1985-06-22|1987-01-02|Kali Chemie Pharma Gmbh|NEW AROMATIC COMPOUNDS, THEIR PRODUCTION AND USE|

---

US5041441A|1988-04-04|1991-08-20|The Regents Of The University Of Michigan|Method of chemotherapy using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol|

---

ZA929008B|1991-12-13|1993-05-21|Squibb Bristol Myers Co|Piperazinyl- and piperidinyl-cyclohexanols.|

---

US5302609A|1992-12-16|1994-04-12|The Regents Of The University Of Michigan|Treatment of diabetic nephropathy|

---

US5399567A|1993-05-13|1995-03-21|Monsanto Company|Method of treating cholera|

---

HUT73527A|1993-08-13|1996-08-28|Seikagaku Kogyo Co Ltd|Remedy for nervous diseases|

---

US6309823B1|1993-10-26|2001-10-30|Affymetrix, Inc.|Arrays of nucleic acid probes for analyzing biotransformation genes and methods of using the same|

---

DE69518627T2|1994-02-02|2001-01-11|Liposome Co Inc|PHARMACEUTICALLY EFFECTIVE COMPOUNDS AND LIPOSOME AND METHOD FOR THE PRODUCTION THEREOF|

---

CA2192555C|1994-06-10|2005-01-11|Jinichi Inokuchi|2-acylaminopropanol compound and medicinal composition|

---

FR2734819B1|1995-05-31|1997-07-04|Adir|NOVEL COMPOUNDS OF PIPERAZINE, PIPERIDINE AND 1,2,5,6-TETRAHYDROPYRIDINE, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM|

---

US20030073680A1|1995-09-20|2003-04-17|The Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

US6255336B1|1995-09-20|2001-07-03|The Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

US6916802B2|2002-04-29|2005-07-12|Genzyme Corporation|Amino ceramide-like compounds and therapeutic methods of use|

---

CA2378600A1|1999-07-09|2001-01-18|The Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

US5916911A|1995-09-20|1999-06-29|The Regents Of The University Of Michigan|Amino ceramide--like compounds and therapeutic methods of use|

---

EP1384719A1|1999-07-09|2004-01-28|The Regents of The University of Michigan|Inhibitors of the glucosyl ceramide synthase enzyme useful for the treatment of cancers, sphingolipidosis and microbial infections|

---

JP3993908B2|1995-12-08|2007-10-17|生化学工業株式会社|Amino alcohol derivative and method for producing the derivative|

---

NO965193L|1995-12-08|1997-06-09|Seikagaku Kogyo Kk Seikagaku C|Aminal alcohol derivative and process for its preparation|

---

JP4140984B2|1995-12-20|2008-08-27|生化学工業株式会社|Drug with differentiation-inducing action|

---

US5972928A|1997-05-21|1999-10-26|Johns Hopkins University|Methods for treatment of conditions associated with lactosylceramide|

---

JP4036500B2|1997-05-23|2008-01-23|生化学工業株式会社|Amino alcohol derivatives and pharmaceuticals containing the same|

---

JP4176170B2|1997-06-06|2008-11-05|生化学工業株式会社|Medicine containing amino alcohol derivative and therapeutic agent for abnormal proliferative disease|

---

US6465488B1|1997-12-11|2002-10-15|Chancellor, Masters & Scholars Of The University Of Oxford|Inhibition of glycolipid biosynthesis|

---

EP1098642B1|1998-07-27|2007-04-04|Johns Hopkins University|Diamino-propanol-compounds for treating ischemia|

---

US6610703B1|1998-12-10|2003-08-26|G.D. Searle & Co.|Method for treatment of glycolipid storage diseases|

---

WO2001004108A1|1999-07-09|2001-01-18|Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

MXPA02006660A|2000-01-07|2002-12-13|Transform Pharmaceuticals Inc|Highthroughput formation, identification, and analysis of diverse solidforms.|

---

US20040252299A9|2000-01-07|2004-12-16|Lemmo Anthony V.|Apparatus and method for high-throughput preparation and spectroscopic classification and characterization of compositions|

---

DE04010651T1|2000-04-24|2005-09-01|Teva Pharmaceutical Industries Ltd.|Micronized zolpidem hemitartrate|

---

US6436987B1|2000-06-08|2002-08-20|Pfizer Inc.|Crystalline forms of -2-[3,4-dihydro-4-hydroxy-3--2H-1-benzopyran-7-yl]-4--benzoic acid|

---

US20060217560A1|2002-04-29|2006-09-28|Shayman James A|Amino ceramide-like compounds and therapeutic methods of use|

---

US7148251B2|2001-01-10|2006-12-12|The Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

US20040260099A1|2001-01-10|2004-12-23|The Regents Of The University Of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

EP1358172B1|2001-01-10|2009-11-18|The Regents of The University of Michigan|Amino ceramide-like compounds and therapeutic methods of use|

---

US20020198240A1|2001-01-10|2002-12-26|Shayman James A.|Amino ceramide - like compounds and therapeutic methods of use|

---

BR0116803A|2001-01-18|2004-02-17|Merck Patent Gmbh|Bifunctional fusion proteins with glucocerebrosidase activity|

---

PT2067775E|2001-07-16|2012-07-16|Genzyme Corp|A n-acylsphingosine glucosyltransferase inhibitor|

---

EP1281755A3|2001-07-31|2003-06-18|Pfizer Products Inc.|Variants of the human cyp2d6 gene|

---

US7893101B2|2002-03-20|2011-02-22|Celgene Corporation|Solid forms comprising -2-[1--2-methylsulfonylethyl]-4-acetylaminoisoindoline-1,3-dione, compositions thereof, and uses thereof|

---

AU2003251829B2|2002-07-09|2009-12-10|Radical Therapeutix|Method to inhibit ischemia and reperfusion injury|

---

US20050032070A1|2003-08-05|2005-02-10|Sebastian Raimundo|Polymorphisms in the human gene for CYP2D6 and their use in diagnostic and therapeutic applications|

---

EP1709053B1|2004-01-27|2011-04-06|Synthon B.V.|Stable salts of olanzapine|

---

JP2008514731A|2004-09-30|2008-05-08|ヴァンダファーマシューティカルズインコーポレイテッド|How to administer iloperidone|

---

ES2463681T3|2004-10-13|2014-05-28|Pharmacia & Upjohn Company Llc|Crystalline forms of 3- [5-Chloro-4 - [ oxy] -6-oxopyrimidin-1 -yl] -N- -4-methylbenzamide|

---

US8003617B2|2004-11-10|2011-08-23|Genzyme Corporation|Methods of treating diabetes mellitus|

---

MX2007016179A|2006-04-17|2008-03-11|Teva Pharma|Polymorphic forms of tegaserod maleate.|

---

US20070088082A1|2005-09-28|2007-04-19|Judith Aronhime|Polymorphic forms of ladostigil tartrate|

---

US20080293946A1|2005-10-03|2008-11-27|Cheng Brian K|Process for Preparing Zolpidem Hemitartrate and Tartrate Polymorphs|

---

EP2032134B1|2006-05-09|2015-06-24|Genzyme Corporation|Methods of treating fatty liver disease comprising inhibiting glucosphingolipid synthesis|

---

KR101271225B1|2006-10-31|2013-06-03|삼성디스플레이 주식회사|Method for manufacturing light emitting diode chip and light emitting diode light source module|

---

TWI314226B|2006-12-07|2009-09-01|Ind Tech Res Inst|Piezoelectricity-driving optical lens module|

---

EP1961765A1|2006-12-08|2008-08-27|Zealand Pharma A/S|Truncated PTH peptides with a cyclic conformation|

---

CA2682441A1|2007-03-30|2008-10-09|Amicus Therapeutics, Inc.|Method for the treatment of fabry disease using pharmacological chaperones|

---

EP2150254A4|2007-04-26|2010-11-10|Amicus Therapeutics Inc|Dosing regimens for the treatment of lysosomal storage diseases using pharmacological chaperones|

---

EP2594565B1|2007-05-31|2018-10-24|Genzyme Corporation|2-acylaminopropanol-type glucosylceramide synthase inhibitors|

---

DE102007029581B4|2007-06-26|2020-04-09|GM Global Technology Operations LLC |Motor vehicle roof and vehicle body|

---

US20090307180A1|2008-03-19|2009-12-10|Brandon Colby|Genetic analysis|

---

WO2009117150A2|2008-03-20|2009-09-24|Genzyme Corporation|Method of treating lupus with ceramide derivatives|

---

FR2953139B1|2009-11-27|2012-04-13|Servier Lab|PHARMACEUTICAL COMPOSITION COMPRISING STRONTIUM SALT, VITAMIN D AND CYCLODEXTRIN|

---

EP3646859A1|2009-11-27|2020-05-06|Boehringer Ingelheim International GmbH|Treatment of genotyped diabetic patients with dpp-iv inhibitors such as linagliptin|

---

SI3415515T1|2009-11-27|2020-03-31|Adverio Pharma Gmbh|Method for the preparation of methyl--1h-pyrazolopyridin-3-yl)pyrimidin-5-yl)methylcarbamate|

---

DK2796457T3|2009-11-27|2016-08-29|Genzyme Corp|Genz 112 638 for the treatment of Gaucher disease or Fabry of combination therapy|

---

EP2723733A1|2011-06-21|2014-04-30|Novartis AG|Polymorphs of -pyrrolidine-1,2-dicarboxylic acid 2-amide 1--pyridin-4-yl]-thiazol-2-yl}-amide|

---

JO3316B1|2013-05-30|2019-03-13|Lilly Co Eli|3,4-dihydroisoquinolin-2-yl compounds|

---

JP6728842B2|2016-03-24|2020-07-22|オムロン株式会社|Optical measuring device|

---

CN107445938B|2016-05-31|2020-04-03|北京启慧生物医药有限公司|Crystalline form of eliglutacoside hemitartrate, process for its preparation and pharmaceutical compositions containing it|

---

WO2018193090A2|2017-04-21|2018-10-25|Amneal Pharmaceuticals Company Gmbh|Process for preparation of eliglustat hemitartrate and intermediates thereof|WO2006007560A2|2004-07-01|2006-01-19|University Of Pennsylvania|Targeted protein replacement for the treatment of lysosomal storage disorders|

---

DK2796457T3|2009-11-27|2016-08-29|Genzyme Corp|Genz 112 638 for the treatment of Gaucher disease or Fabry of combination therapy|

---

CN103764824B|2011-06-20|2018-04-24|西奈山医学院|The anti-tnf-alpha therapy of mucopolysaccharidosis and other lysosome illnesss|

---

DK2753346T3|2011-09-07|2020-05-25|Sinai School Medicine|CERAMIDASE AND CELL DIFFERENTIATION|

---

WO2013078413A1|2011-11-22|2013-05-30|The United States Of America, As Represented By The Secretary, Department Of Health And Human Services|Modulators of lipid storage|

---

CA2874146A1|2012-06-01|2013-12-05|Icahn School Of Medicine At Mount Sinai|Ceramide levels in the treatment and prevention of infections|

---

JP6832158B2|2013-03-14|2021-02-24|アイカーン スクール オブ メディシン アット マウント サイナイ|Therapeutic acidic ceramidase composition and how to make and use it|

---

WO2016001885A2|2014-07-03|2016-01-07|Dr. Reddy’S Laboratories Limited|Amorphous form of eliglustat hemitartarate|

---

EP3283483B1|2015-04-14|2018-12-19|Sandoz AG|Crystalline eliglustat hydrochloride|

---

GB201508025D0|2015-05-11|2015-06-24|Ucl Business Plc|Fabry disease gene therapy|

---

EA201891170A1|2015-11-18|2018-11-30|Джензим Корпорейшн|BIOMARKER OF POLYCYSTIC KIDNEY DISEASE AND OPTIONS FOR ITS APPLICATION|

---

CN107445938B|2016-05-31|2020-04-03|北京启慧生物医药有限公司|Crystalline form of eliglutacoside hemitartrate, process for its preparation and pharmaceutical compositions containing it|

---

CN106349210A|2016-08-24|2017-01-25|北京阳光诺和药物研究有限公司|Method for preparing tartrate EGS|

---

EP3318277A1|2016-11-04|2018-05-09|Institut du Cerveau et de la Moelle Epiniere-ICM|Inhibitors of glucosylceramide synthase for the treatment of motor neuron diseases|

---

WO2018193090A2|2017-04-21|2018-10-25|Amneal Pharmaceuticals Company Gmbh|Process for preparation of eliglustat hemitartrate and intermediates thereof|

---

WO2018225085A1|2017-06-05|2018-12-13|Cipla Limited|Stable solid dispersions of eliglustat hemitartrate|

---

WO2018232235A1|2017-06-16|2018-12-20|Beta Pharma, Inc.|Pharmaceutical formulations of n-ethoxy)-4-methoxy-5-pyrimidin-2-yl)amino)phenyl)acrylamide and salts thereof|

---

WO2019030645A1|2017-08-08|2019-02-14|Kashiv Pharma Llc|Pharmaceutical composition comprising eliglustat|

---

WO2019118707A1|2017-12-15|2019-06-20|Genzyme Corporation|Methods for treating gaucher disease|

---

WO2019123476A1|2017-12-20|2019-06-27|Sarudbhava Formulations Private Limited|Stable amorphous eliglustat premix and process for the preparation thereof|

---

US20210300887A1|2018-05-02|2021-09-30|Kashiv Biosciences, Llc|Pro-drugs of eliglustat|

---

CN110878079A|2018-12-31|2020-03-13|北京启慧生物医药有限公司|Preparation method of high-purity eliagliptat|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-05-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-01-28| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2020-07-21| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2021-01-26| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-04-06| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 06/04/2021, OBSERVADAS AS CONDICOES LEGAIS. |

---

2021-05-25| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/11/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

---

申请号 | 申请日 | 专利标题

---

US26474809P| true| 2009-11-27|2009-11-27|

---

US61/264,748|2009-11-27|

---

PCT/US2010/057952|WO2011066352A1|2009-11-27|2010-11-24|An amorphous and a crystalline form of genz 112638 hemitartrat as inhibitor of glucosylceramide synthase|

---