Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/20—Carbocyclic rings
  • C07H15/24—Condensed ring systems having three or more rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms

Definitions

• the present invention relates to stable elsamitrucin salts and related synthetic methods.
• the stable, solid elsamitrucin salts of the present invention are suitable for preparing solutions useful for parenteral administration in the treatment of neoplastic diseases.
• Elsamitrucin is a heterocyclic antineoplastic antibiotic isolated from the gram positive bacterium Actinomycete strain J907-21 as described in United States patent numbers (USPN) 4,518,589 and 4,572,895 which are incorporated herein by reference for all they disclose related to the natural history, chemical composition, methods of preparing and bioactivity of elsamitrucin.
• Elsamitrucin intercalates into DNA at guanine-cytosine (G-C)-rich sequences and inhibits topoisomerase I and II, resulting in single-strand breaks and inhibition of DNA replication.
• G-C guanine-cytosine
• Elsamitrucin possesses significant oncolytic activity against metastatic cancer of the breast, colon and rectum, non-small cell lung and ovary and in patients with relapsed or refractory non-Hodgkin's lymphoma.
• Elsamitrucin is known chemically as benzo(h)(1 )benzopyrano(5,4,3- cde)(1 )ebnzopyran-5,12-dione,10((2-O-(2-amino-2,6-dideoxy-3-O-methyl-alpha-D- galactopyranosyO- ⁇ -deoxy-S-C-methyl-beta-D-galactopyranosyOoxyJ- ⁇ -hydroxy-i - methyl, and has the structure generally depicted in Formula I.
• Elsamitrucin is also known as 10-O-elsaminosylelsarosylchartarin, BBM 2478A, BMY-28090, SPI-28090, BRN 5214813, elsamicin A, elsamitrucina, and elsamitrucine.
• Elsamitrucin is typically administered parenterally (generally intravenously) to animals, including humans and is supplied as a lyophilized powder that is reconstituted with sterile water for injection immediately prior to use.
• the prior art lyophilized elsamitrucin powder is provided as a 1 :1 succinate formed in situ by dissolving elsamitrucin base in an organic solvent and then adding sufficient aqueous succinic acid to form a 1 :1 solution of solubilized free base to acid.
• the resulting elsamitrucin-succinic acid suspension is then adjusted to a pH of between 3.5 and 4.5 and mixed with a bulking agent such as mannitol to enhance stability prior to lyophilization (see for example USPN 5,508,268).
• a bulking agent such as mannitol
• Stable elsamitrucin salts in powder form are not presently available thus all highly soluble elsamitrucin pharmaceutical compositions must be prepared in situ using the free base.
• the present invention provides water soluble, solid elsamitrucin salts useful for preparing stable parenteral solutions intended for use in anti-neoplastic therapeutic regimens. Additionally, methods for preparing the stable, elsamitrucin salts of the present invention are provided.
• the counter-ion of the stable, solid elsamitrucin salt is selected from the group consisting of, but not limited to, lactate, fumarate, maleate, succinate, tartrate, tosylate, methanesulfonate, benzoate, salicylate, hydrochloride, sulfate, phosphate, and others.
• the stable, solid elsamitrucin salts may be either crystalline or amorphous.
• a stable, solid Elsamitrucin tosylate salt is provided.
• a method for treating a neoplatic disease includes administrating an elsamitrucin parenteral formulation intravenously to a human wherein the elsamitrucin parenteral formulation includes at least one stable solid elsamitrucin salt selected from the group consisting of lactate, fumarate, maleate, succinate, tartrate, tosylate, methanesulfonate, benzoate, salicylate, hydrochloride, sulfate and phosphate.
• Still another embodiment of the present invention includes a method for treating neoplastic diseases in a human that includes providing a parenteral formulation consisting essentially of elsamitrucin tosylate, a pharmaceutically acceptable carrier and optionally at least one pharmaceutically acceptable excipient.
• the neoplastic disease being treated using the elsamitrucin parenteral formulations of the present invention is relapsed or refractory non-Hodgkin's lymphoma.
• Figure 1 Depicts Elsamitrucin tosylate re-crystallized from 1 :1 mixture of acetonitrile:water made in accordance with the teachings of the present invention.
• Analogue(s) include compounds having structural similarity to another compound.
• the anti-viral compound acyclovir is a nucleoside analogue and is structurally similar to the nucleoside guanosine which is derived from the base guanine.
• acyclovir mimics guanosine (is “analogous with” biologically) and interferes with DNA synthesis by replacing (competing with) guanosine residues in the viral nucleic acid and prevents translation/transcription.
• compounds having structural similarity to another (a parent compound) that mimic the biological or chemical activity of the parent compound are analogues.
• Analogues can be, and often are, derivatives of the parent compound (see “derivative" infra). Analogues of the compounds disclosed herein may have equal, less or greater activity than their parent compounds.
• a “derivative” is a compound made from (derived from), either naturally or synthetically, a parent compound.
• a derivative may be an analogue (see “analogue” supra) and thus may possess similar chemical or biological activity. However, as used herein, a derivative does not necessarily have to mimic the activity of the parent compound. There are no minimum or maximum numbers of elemental or functional group substitutions required to qualify as a derivative.
• the antiviral compound ganclovir is a derivative of acyclovir. Ganclovir has a different spectrum of anti-viral activity from that of acyclovir as well as different toxicological properties. Derivatives of the compounds disclosed herein may have equal, less, greater or no similar activity to their parent compounds.
• Elsamitrucin refers to an antineoplastic composition having a molecular weight of approximately 825.83 Da and is known chemically as benzo(h)(1 )benzopyrano(5,4,3-cde)(1 )ebnzopyran-5,12- dione,10((2-O-(2-amino-2,6-dideoxy-3-O-methyl-alpha-D-galactopyranosyl)-6-deoxy- 3-C-methyl-beta-D-galactopyranosyl)oxy)-6-hydroxy-1 -methyl, and has the structure generally depicted in Formula I.
• Elsamitrucin is also known as 10-O- elsaminosylelsarosylchartarin, BBM 2478A, BMY-28090, SPI-28090, BRN 5214813, elsamicin A, elsamitrucina, and elsamitrucine.
• USPNs 4,518,589 and 4,572,895 for methods of isolating and characterizing elsamitrucin from natural sources. See also Konishi M, Sugawara K, Kofu F, Nishiyama Y, Tomita K, Miyaki T, Kawaguchi H. 1986.
• Elsamicins new antitumor antibiotics related to chartreusin I. Production, isolation, characterization and antitumor activity. J. Antibiot. (Tokyo) Jun;39(6):784- 91 .
• composition refers to a pharmaceutically acceptable preparation comprising one or more of the elsamitrucin salts of the present invention and at least one pharmaceutically acceptable carrier such as, but not limited to water for injection, saline or phosphate buffered saline.
• pharmaceutical formulations of the present invention may also include stabilizers, preservatives, buffers or additional therapeutic agents.
• the pharmaceutical formulations of the present invention may be administered by any means known to those skilled in the art and are ideally suited for intravenous administration or infection into the skin, muscle or other tissues of the body.
• the pharmaceutical formulation may be intended for oral administration.
• Salt As used herein a “salt” or “salts” include any compounds that result from replacement of part or all of the acid hydrogen of an acid by a metal or a group acting like a metal: an ionic crystalline compound. In this case, the salt is a product of a free base and an organic acid that can exist as a stable solid and does not include pseudo salts, or salts made in situ, which only exist in the solution.
• Suitable salt form(s) As used herein, the term "suitable salt form(s)” means an elsamitrucin salt prepared in stable solid state either as amorphous or crystalline form.
• Solid or solid salt refers to an elsamitrucin salt existing in a solid state and having less than 30% residual moisture, preferably less than 10% residual moisture and more preferably less than 5% residual moisture.
• moisture refers to water or an organic solvent.
• solid is also used herein to differentiate the elsamitrucin salts of the present invention from salts formed in situ and exist primarily in the aqueous phase.
• Stable refers to an elsamitrucin salt or a parenteral elsamitrucin salt-containing formulation (made by method other than in situ salt formation) wherein the elsamitrucin salt retains NMR data showing a near perfect 1 :1 salt ratio (thus indicating no decomposition in the solid state) during drying at elevated temperatures at 75°C for nine hours or more preferably 98°C overnight.
• stable refers to elsamitrucin salt-contained in a parenteral formulation that retains at least 90% of its anti-neoplastic activity as determined by in vitro growth inhibition testing (see Example 4) for at least 24 months in the solid form and for 18 months in the liquid form at a suitable storage temperature.
• the present invention provided water soluble, solid elsamitrucin salts useful for preparing stable pharmaceutical formulations intended for use in antineoplastic therapeutic regimens. Additionally, methods for preparing the stable, elsamitrucin salts have also been provided.
• the stable, elsamitrucin salts of the present invention are selected form the group consisting of, but not limited to, lactate, fumarate, maleate, succinate, tartrate, tosylate, methanesulfonate, benzoate, salicylate, hydrochloride, sulfate, phosphate, and others.
• the elsamitrucin salts of the present invention are stable solids and may be either crystalline or amorphous.
• Elsamitrucin and structurally related antibiotics bind to GC-rich tracts in DNA, with a clear preference for B-DNA over Z-DNA. They inhibit RNA synthesis and cause single-strand scission of DNA via the formation of free radicals. Elsamitrucin can also be regarded as the most potent inhibitor of topoisomerase Il reported so far and can inhibit the formation of several DNA-protein complexes. Elsamitrucin binds to the P1 and P2 promoter regions of the c-myc oncogene inhibits the binding of the Sp1 transcription factor, thus inhibiting transcription.
• Elsamitrucin has shown activity in patients with relapsed or refractory non- Hodgkin's lymphoma and in vivo activity against a wide range of murine neoplasmas including leukemia P388, leukemia L1210, and melanoma B16 and M5076, as well as against MX1 and HCT1 16 xenografts (see for example Raber MN, Newman RA, Newman BM, Gaver RC, Schacter LP1992 Phase I trial and clinical pharmacology of elsamitrucin. Cancer Res. Mar 15;52(6):1406-10).
• United States patent number 5,508,268 issued April 16, 1996 to Nassar et al. assigned to Bristol-Myers Squibb discloses parenteral formulations comprising elsamitrucin base, an organic acid, a stabilizer and a buffer.
• the elsamitrucin compositions disclosed therein were prepared using various organic acids including hydrochloric, L(+)-lactic, L-tartaric, D-glucuronic, methane-sulfonic, adipic and succinic with the succinic acid being preferred.
• the elsamitrucin compositions are prepared according to the teachings of an Example occurring at column 4 lines 5-30. In this example only the succinate salt is described.
• the elsamitrucin salt is formed in situ using an organic acid in combination with at least one reducing agent (preservative) and the pH adjusted to approximately 4. The resulting solution was filtered and retained in the liquid state for stability testing.
• the organic acid, elsamitrucin base, reducing agent and other suitable pharmaceutical excipients such as, but not limited to sugars, are admixed in solution and the resulting composition is lyophilized.
• the '268 patent does not disclose, discuss or teach stable solid elsamitrucin salts.
• the present inventors have discovered methods that provide stable solid elsamitrucin salts made using elsamitrucin base and selected organic acids.
• the resulting compositions made in accordance with the teachings of the present invention are solid, dry or partially dried elsamitrucin salt powders, as opposed to lyophilizates described in the '268 patent.
• the elsamitrucin salt compositions of the present invention are true salts in solid state, not in situ solutions containing a solubilized base and organic acid admixture.
• the present invention offers numerous advantages over in situ formed admixtures as described in the '268 patent.
• the elsamitrucin salts made in accordance with the teachings of the present invention can be carefully analyzed for impurities and refined as needed to meet exceedingly high governmental regulations.
• the true salts of the present invention can be precisely weighed and dissolved in suitable pharmaceutical carriers such as Water for Injection.
• suitable pharmaceutical carriers such as Water for Injection.
• the selected salts themselves are extremely stable when stored in the solid state and have extended shelf lives as do their corresponding solubilized solutions.
• parenteral solutions can be prepared using the elsamitrucin salts of the present invention and stored for extended periods of time.
• elsamitrucin salts were prepared prior to optimization and scale up.
• Eight counter ions based on organic acids were selected, these included lactic acid, maleic acid, succinic acid, L-tartaric acid, p-toluenesulfonic (also referred to herein as p-TSA or tosylate), , benzoic acid, salicylic acid, and sulfuric acids.
• Three solvents were selected based on previous screen methods known to those skilled in the art of pharmaceutical chemistry, the selected solvents included dioxane, dimethylformamide (DMF), and acetic acid (AcOH). An additional combination of p-TSA/MeOH was included for a total of twenty-five variations reactions.
• API elsamitrucin base.
• the initial temperature was held for ten minutes and then ramped down to room temperature at a rate of 20°C/hour for DMF and AcOH, 30°C/hour for dioxane and 25°C/hour for MeOH.
• Solids were formed in the vials with dioxane/L-tartaric acid, dioxane/p-TSA, dioxane/sulfuric acid, and AcOH/sulfuhc acid. Solids were collected by filtration and dried in vacuo at 50°C and 30 in. Hg. Vials were solids did not form were concentrated to dryness with a stream of nitrogen and dried in vacuo at 50 0 C and 30 in. Hg.
• Crystalline solids were obtained from dioxane/sulfuric acid and from AcOH/sulfuric acid; semi-crystalline solids were obtained from dioxane/L-tartaric acid, dioxane/p- TSA, DMF/lactic acid, DMF/maleic acid, DMF/L-tartaric acid, DMF/benzoic acid, DMF/sulfuric acid, AcOH/lactic acid, AcOH/ p-TSA, and AcOH/benzoic acid. All other solids were found to be amorphous by XRPD.
• Example 2 Three elsamitrucin salts made in accordance with the teaching of Example 1 were selected for scale-up development.
• the salts selected were elsamitrucin tartrate, elsamitrucin sulfate and elsamitrucin tosylate. These were selected because each provided crystalline or semi-crystalline solids that precipitated during the cooling process, which can allow better isolation and purification (if necessary) of the salt and thus lends them more suitable for larger scale manufacturing techniques.
• their selection for purposed of Example 2 should not be considered a limitation.
• the initial temperature was held for ten minutes and then ramped down to room temperature at a rate of 30°C/hour for dioxane.
• Solids were formed upon addition of acid to the vials with dioxane/L-tartahc acid and dioxane/sulfuhc acid and for dioxane/p-TSA precipitation occurred during the cooling process. After filtration the solids were dried in vacuo at 50°C and 30 inch Hg. Samples were analyzed by XRPD, DSC, and TGA to determine the crystallinity (Table 3), and other physical properties.
• Example 2 As Table 3 shows, all solids in Example 2 were semi-crystalline, contained up to about 5% of the residual solvent and were pasty in constancy due to the high amount of solvent that was retained in the solids due to a rapid precipitation.
• the elsamitrucin salts of the present invention were prepared using a slower precipitation method than described above. Reaction containers were charged with 7.6 x 10 ⁇ 5 mol of elsamitrucin base and 5 ml. of dioxane at 80°C. After the mixture was stirred for five minutes to ensure dissolution of the base, 400 ⁇ l_ of a 0.2 M aqueous solution of tartaric acid corresponding to 1 .05 equivalents was added to the dissolved elsamitrucin base. The temperature was held at 80 c C for ten minutes and then the vials were cooled to room temperature at a rate of 30°C/hour. During the cooling phase precipitation occurred.
• the solids were collected by filtration and dried in vacuo at 50°C and 30 inches Hg. Samples were analyzed by XRPD, DSC, and TGA to determine physical properties [Table 3, OVL-A-55(1 ) and OVL-A-55(2)].
• the first sample [dioxane/sulfuric acid, OVL-A- 55(1 )] was crystalline by XRPD, but it contained 3.6% residual solvent according to TGA analysis and three endothermic peaks on the DSC curve.
• the second sample [dioxane/L-tartaric acid, OVL-A-55(2)] was semi-crystalline.
• elsamitrucin salts were prepared in an aqueous environment as follows. Reaction vials were charged with 100 mg of elsamitrucin base, 1 .05 equivalents of corresponding acid (p-TSA, succinic, and L-tartaric acid were added as solids; sulfuric acid was dissolved in 0.5 ml. of water) and water (10 ml. for p- TSA, succinic, and L-tartaric acid, 9.5 mL for sulfuric acid). The suspensions were heated to 80 0 C with stirring for ten minutes to form a clear solution and then ramped down to room temperature at a rate of 30°C/hour. After stirring overnight at room temperature precipitates were not formed in any of the experiments.
• the solvents used were isopropyl alcohol, methanol, ethanol, acetonithle, acetone, propylene glycol, tetrahydrofuran, dichloromethane and 1 :1 mixtures of isopropyl alcohol, methanol, ethanol, acetonitrile, acetone with water. Needle or rod-shaped crystals were observed under microscope in solvents - ethanol, methanol, propylene glycol, isopropyl alcohol, acetone, and all the water:solvent mixtures. Microscopic examination of the samples indicated that the Elsamitrucin tosylate salt became crystalline in at least one solvent.
• Elsamitrucin salts made in accordance with the teachings of the present invention were tested for stability. Two samples of the isolated p-TSA salt (40 mg each) were placed in a vacuum oven at 75°C for nine hours. After this exposure, sample #1 was taken out, the temperature was increased to 98°C and the second sample was dried overnight. The NMR data showed a perfect 1 :1 salt ratio, so there was no decomposition in the solid state during drying at elevated temperatures. The weight loss by TGA was approximately 2.5% for both samples. Karl Fischer analysis indicated the two lots still had water present: sample #1 had 4.0% water content and #2 had 4.6%.
• SK-MES-1 human non-small cell lung carcinoma, B16F10 murine melanoma cells, HCT 1 16 and HT29 human colon carcinomas were maintained in buffered RPMI 1640 supplemented with fetal calf serum, antibiotics and other appropriate growth factors such as glutamine.
• Test cells (1 ,500-2,000 cells/well) were seeded in a 96-well micro culture plate with a total volume of 100 ⁇ L/well. After overnight incubation in a humidified incubator at 37°C with 5% CO 2 and 95% air, elsamitrucin solutions were diluted to various concentrations with RPMI 1640, were added to each well in a 100 ⁇ l_ volume.
• the elsamitrucin base and the elsamitrucin tosylate solutions were prepared and stored in a -20°C freezer. The solutions were thawed not more than 10 times for the entire experiment.
• Top is the maximal percentage of control absorbance
• bottom is the minimal percentage of control absorbance at the highest agent concentration
• Y is the observed absorbance
• X is the agent concentration
• IC50 is the concentration of agent that inhibits cell growth by 50% compared to the control cells
• n is the slope of the curve.
• Table 4 demonstrates that elsamitrucin and elsamitrucin tosylate salt possess essentially the same anti-proliferative effect on the cell lines tested.
• the elsamitrucin salts made in accordance with the teachings of the present invention can be expected to have equivalent, or superior in vivo anti-neoplastic activity as therapeutic compositions made using elsamitrucin base alone.
• the elsamitrucin tosylate comprises an IC 50 that is within preferably about 20%, more preferably about 15% and most preferably about 10% of a similar amount of an elsamitrucin base.

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• 2007
  • 2007-05-24 MX MX2008015971A patent/MX2008015971A/es not_active Application Discontinuation
  • 2007-05-24 CN CNA2007800223402A patent/CN101472938A/zh active Pending
  • 2007-05-24 CA CA002654877A patent/CA2654877A1/en not_active Abandoned
  • 2007-05-24 WO PCT/US2007/069693 patent/WO2008143677A1/en active Application Filing
  • 2007-05-24 KR KR1020087030847A patent/KR20100014080A/ko not_active Application Discontinuation
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