Salts and polymorphs of tetracycline compounds

Abstract

This provides crystalline forms of tetracycline compounds, including salts and polymorphs, that are useful for the treatment or prevention of conditions and disorders such as bacterial infections and neoplasms. [Solution] The present invention provides a method for preparing a tosylate salt of an aminoalkyltetracycline compound represented by formula (1) and a stable crystalline tosylate salt thereof. TIFF2026121464000022.tif27128

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Patent Application No. 61 / 128,712, filed on May 23, 2008, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background of the Invention The development of tetracycline antibiotics was a direct result of a systematic screening of soil specimens collected from many regions of the world in search of microorganisms capable of producing bactericidal and / or bacteriostatic compositions. The first of these novel compounds was introduced in 1948 under the name chlortetracycline. Two years later, oxytetracycline became available. The elucidation of the chemical structures of these compounds confirmed their similarity and provided an analytical basis for the production of tetracycline, the third member of this group, in 1952. A new family of tetracycline compounds without the ring-attached methyl groups present in the early tetracyclines was prepared in 1957 and became commercially available in 1967; minocycline began to be used in 1972.

[0003] In recent years, research efforts have been concentrated on developing novel tetracycline antibiotic compositions that are effective under various treatment conditions and routes of administration. Novel tetracycline analogs are also being studied, and these may prove to be equal to or more effective than the initially introduced tetracycline compounds. Examples include U.S. Patent No. 2,980,584 (Patent Document 1); Patent No. 2,990,331 (Patent Document 2); Patent No. 3,062,717 (Patent Document 3); Patent No. 3,165,531 (Patent Document 4); Patent No. 3,454,697 (Patent Document 5); Patent No. 3,557,280 (Patent Document 6); Patent No. 3,674,859 (Patent Document 7); Patent No. 3,957,980 (Patent Document 8); Patent No. 4,018,889 (Patent Document 9); Patent No. 4,024,272 (Patent Document 10); and Patent No. 4,126,680 (Patent Document 11). These patents represent the range of pharmaceutically active tetracycline and tetracycline analog compositions.

[0004] Historically, shortly after its initial development and introduction, tetracycline was found to be highly pharmacologically effective against rickettsia; numerous Gram-positive and Gram-negative bacteria; and the causative agents of venereal lymphogranuloma, inclusion conjunctivitis, and psittacosis. Thus, tetracycline became known as a "broad-spectrum" antibiotic. Its subsequent establishment of in vitro antibacterial activity, efficacy in experimental infections, and pharmacological properties led to the rapid and widespread use of tetracycline as a class for therapeutic purposes. However, the extensive use of tetracycline for both major and minor diseases and disorders led to the emergence of resistance to these antibiotics directly, even in highly susceptible bacterial species, both commensal and pathogenic (e.g., Streptococcus pneumococcus and Salmonella). The emergence of tetracycline-resistant organisms has generally led to a decline in the use of tetracycline and tetracycline analog compositions as the preferred antibiotic.

[0005] Each pharmaceutical compound has an optimal therapeutic blood concentration and a lethal concentration. The bioavailability of a compound determines the strength of the dosage in the drug formulation required to obtain the ideal blood level. If a drug can crystallize as two or more polymorphs with different bioavailability, the optimal dose will depend on the polymorphs present in the formulation. Some drugs exhibit a narrow range between therapeutic and lethal concentrations. For example, chloramphenicol-3-palmitate (CAPP) is a broad-spectrum antibiotic known to crystallize in at least three polymorphic forms and one amorphous form. The most stable form, A, is commercially available. The difference in bioactivity between this polymorph and another stable form, B, is eightfold, and therefore, if administered unintended as form B due to changes during processing and / or storage, there is a possibility of a lethal overdose of the compound. For this reason, regulatory authorities such as the U.S. Food and Drug Administration have begun to strictly control the polymorphic content of active ingredients in solid dosage forms. Generally, with respect to drugs that exist in polymorphic forms, if substances other than the pure, thermodynamically preferred polymorph are marketed, regulatory authorities may require batch-by-batch monitoring. Therefore, producing and marketing a pure drug in its most thermodynamically stable polymorphic form, substantially free of other kinetically preferred polymorphs, is important for both medical and commercial reasons.

[0006] For example, the salt form of a compound, and the polymorphic form of a free compound or salt, are known in the pharmaceutical technology to affect not only solubility, dissolution rate, bioavailability, chemical and physical stability, flowability, fractability, and compressibility, but also the safety and efficacy of drugs based on the compound (see, for example, Knapman, Modern Drug Discovery, 2000, 3(2): 53 (Non-Patent Literature 1)).

[0007] Therefore, identifying the salt form or free base of a compound with optimal physical and chemical properties will advance the development of tetracycline compounds as pharmaceuticals. The most useful physical and chemical properties of such compounds include: easy and reproducible preparation, crystalline, non-hygroscopic, water solubility, stability to visible and ultraviolet light, low degradation rate under accelerated stability conditions of temperature and humidity, low isomerization rate between isomer forms, and safety when administered to humans over long periods. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] U.S. Patent No. 2,980,584 [Patent Document 2] No. 2,990,331 [Patent Document 3] No. 3,062,717 [Patent Document 4] No. 3,165,531 [Patent Document 5] No. 3,454,697 [Patent Document 6] No. 3,557,280 [Patent Document 7] No. 3,674,859 [Patent Document 8] No. 3,957,980 [Patent Document 9] No. 4,018,889 [Patent Document 10] No. 4,024,272 [Patent Document 11] No. 4,126,680 [Non-patent literature]

[0009] [Non-Patent Document 1] Knapman, Modern Drug Discovery, 2000, 3(2): 53 [Overview of the Initiative]

[0010] In one embodiment, the present invention relates to compound 1, which is at least partially an aminoalkyltetracycline compound: TIFF2026121464000001.tif27128 This relates to the stable solid form, such as the crystalline form, of (4S,4AS,5AR,12AS)-4-7-bis(dimethylamino)-9{[(2,2-dimethylpropyl)amino]methyl}-3,10,12,12A-tetrahydroxy-1,11-dioxo-1,4,4A,5,5A,6,11,12A-octahydrotetracene-2-carboxamide (9-(2,2-dimethyl-propyl-aminomethyl)-minocycline).

[0011] In another embodiment, the present invention relates at least in part to an HCl salt of compound 1. In another embodiment, the present invention relates at least in part to a tosylate (p-toluenesulfonic acid) salt of compound 1. In another embodiment, the present invention relates at least in part to a mesylate salt of compound 1.

[0012] In another embodiment, the present invention relates at least in part to a stable crystalline form of compound 1.

[0013] In another embodiment, the present invention relates at least in part to stable crystalline forms of salts of compound 1. For example, the stable crystalline forms of the salt are stable crystalline forms of tosylate, hydrochloric acid, or mesylate salts of compound 1.

[0014] In another aspect, the present invention relates at least partially to polymorphs of compound 1.

[0015] In another aspect, the present invention relates at least partially to polymorphs of salts of compound 1.

[0016] For example, the present invention relates to a polymorph of the tosylate salt of compound 1. The present invention partially relates to a type 1 polymorph of compound 1. The present invention partially relates to a type 2 polymorph of compound 1. The present invention partially relates to a type 3 polymorph of compound 1.

[0017] For example, the Form 1 polymorph of the tosylate salt of Compound 1 has X-ray powder diffraction peaks at approximately 8.06, 13.02, and 18.83° 2θ when irradiated with Cu Kα radiation. In some embodiments, the Form 1 polymorph of the tosylate salt of Compound 1 has X-ray powder diffraction peaks at approximately 8.06, 11.41, 13.02, 18.83, 20.54, and 24.53° 2θ when irradiated with Cu Kα radiation. In some embodiments, the Form 1 polymorph of the tosylate salt of Compound 1 has X-ray powder diffraction peaks at approximately 5.60, 8.06, 8.57, 11.41, 13.02, 15.58, 18.83, 20.54, and 24.53° 2θ when irradiated with Cu Kα radiation.

[0018] For example, the Form 1 polymorph of the tosylate salt of Compound 1 is stable at temperatures in the range of about 0 °C to about 70 °C. In some embodiments, the Form 1 polymorph of the tosylate salt of Compound 1 is stable at temperatures in the range of about 5 °C to about 50 °C. In some embodiments, the Form 1 polymorph of the tosylate salt of Compound 1 is stable at temperatures in the range of about 20 °C to about 30 °C.

[0019] The Form 1 polymorph of the tosylate salt of Compound 1 can be obtained by crystallizing the tosylate salt of Compound 1 from isopropanol.

[0020] For example, the Form 2 polymorph of the tosylate salt of Compound 1 has peaks in the X-ray powder diffraction pattern at 7.82, 11.88, 16.12, and 21.46° 2θ when irradiated with Cu Kα radiation.

[0021] For example, the Form 3 polymorph of the tosylate salt of Compound 1 has peaks in the X-ray powder diffraction pattern at 5.11, 8.89, 10.34, 11.76, and 15.60° 2θ when irradiated with Cu Kα radiation.

[0022] In yet another embodiment, the present invention includes a pharmaceutical composition comprising a crystalline form of Compound 1 and a pharmaceutically acceptable diluent, excipient, or carrier.

[0023] For example, the pharmaceutical compound compositions of the present invention include compositions comprising a polymorph of compound 1 and a pharmaceutically acceptable diluent, excipient, or carrier.

[0024] In another embodiment, the pharmaceutical composition compound of the present invention comprises a salt of compound 1 and a pharmaceutically acceptable diluent, excipient, or carrier. For example, the salt may be a hydrochloride salt, a tosylate salt, or a mesylate salt.

[0025] In one embodiment, the compounds of the pharmaceutical composition of the present invention include a polymorph of a salt of compound 1 and a pharmaceutically acceptable diluent, excipient, or carrier. For example, the polymorph may be a polymorph of a tosylate, hydrochloride, or mesylate salt of compound 1.

[0026] In some embodiments, the pharmaceutical composition comprises a polymorph of compound 1 or a salt thereof in its pure form.

[0027] In another embodiment, the pharmaceutical composition of the present invention comprises a polymorph of the tosylate salt of compound 1 and a pharmaceutically acceptable diluent, excipient, or carrier. For example, the polymorph may be a type 1, type 2, or type 3 polymorph of the tosylate salt of compound 1.

[0028] In some embodiments, the pharmaceutical composition comprises a polymorph of the tosylate, hydrochloride, or mesylate salt of compound 1 in its pure form.

[0029] In another aspect of the present invention, a salt of compound 1 is more stable than the free base of compound 1.

[0030] In another embodiment, the present invention includes a method for preparing a stable crystalline form of compound 1.

[0031] In another embodiment, the present invention includes a method for preparing a stable crystalline form of a salt of compound 1. For example, the stable crystals may be crystals of a tosylate, hydrochloric acid, or mesylate salt of compound 1.

[0032] In another embodiment, the present invention includes a method for preparing polymorphs of salts of compound 1. For example, the polymorphs may be polymorphs of tosylate, hydrochloric acid, or mesylate salts of compound 1.

[0033] In another aspect, the present invention includes a method for preparing polymorphs of the tosylate salt of compound 1. For example, the polymorphs may be type 1, type 2, or type 3 polymorphs of the tosylate salt of compound 1.

[0034] In one embodiment, the present invention includes a method for preparing one polymorph of a tosylate salt of compound 1, comprising the steps of miscible compound 1 with a solvent to produce a slurry, and adding p-toluenesulfonic acid. For example, the solvent may be an alcohol solvent such as isopropanol. p-toluenesulfonic acid is provided in an amount of 25 to 75% by weight relative to the amount of compound 1, for example, in amounts of 25 to 50%, 30 to 40%, or 33% by weight relative to the amount of compound 1. For example, p-toluenesulfonic acid is provided in the form of p-toluenesulfonic acid monohydrate.

[0035] For example, the slurry is heated before p-toluenesulfonic acid is added.

[0036] For example, the slurry is stirred before adding p-toluenesulfonic acid. For example, stirring is carried out at a temperature in the range of 20-25°C. For example, stirring is carried out for 10-24 hours.

[0037] For example, the slurry is dried. For example, the water content of the slurry supernatant is in the range of 0.2 to 1.0 mg / mL, or in the range of 0.4 to 0.8 mg / mL.

[0038] In another embodiment, the present invention includes a method for preparing a type 1 polymorph of the tosylate salt of compound 1, comprising the steps of preparing a solution of compound 1 in a solvent or a solvent mixture, and adding a p-toluenesulfonic acid solution to the solvent or solvent mixture.

[0039] For example, the solvent is an alcohol solvent such as methanol, ethanol, or isopropanol. For example, the solvent mixture contains an alcohol solvent. For example, the solvent mixture further contains a second alcohol solvent. For example, the solvent mixture contains ethanol and isopropanol. For example, the solvent mixture contains antisolvents such as ketones, ethers, and esters. For example, ethers include, but are not limited to, methyl t-butyl ether. For example, the solvent mixture contains an alcohol solvent and an antisolvent. For example, the solvent mixture contains methanol and methyl t-butyl ether.

[0040] For example, p-toluenesulfonic acid is provided in amounts of 25-75% by weight, 30-50% by weight, 35-45% by weight, or 40% by weight relative to the amount of compound 1. For example, p-toluenesulfonic acid is provided in the form of p-toluenesulfonic acid monohydrate.

[0041] For example, the solution is prepared at temperatures in the range of 0–60°C, 15–45°C, or 20–25°C.

[0042] For example, the solution is heated after preparation. For example, the solution is maintained at a temperature in the range of 20-50°C, or at approximately 45°C.

[0043] For example, the method further includes the step of adding a seed crystal of the monotosylate salt of compound 1 to produce a slurry. The slurry may be stirred for 10 to 24 hours, or about 22 hours. The slurry may be stirred at a temperature in the range of 15 to 45°C, or about 20°C. The slurry may be dried. For example, the water content of the slurry is in the range of 1 to 10% by weight, 2 to 6% by weight, or about 3% by weight.

[0044] In another embodiment, the present invention includes a method for preparing a type 1 polymorph of the tosylate salt of compound 1, comprising: dissolving the free base of compound 1 in a first solvent or solvent mixture to form a first solution; dissolving p-toluenesulfonic acid in a second solvent or solvent mixture to form a second solution; and mixing the first and second solutions to form a third solution.

[0045] In one embodiment, the first and second solvents or solvent mixtures may be the same or different. In another embodiment, the solvent may be an alcohol solvent such as methanol, ethanol, and isopropanol. In another embodiment, the solvent mixture is a mixture of two alcohol solvents, including but not limited to ethanol and isopropanol. In a preferred example, the volume-to-volume ratio of ethanol to isopropanol is 2:1. In yet another embodiment, the solvent mixture may include, but not limited to, a combination of an alcohol solvent and an antisolvent (e.g., ketones, ethers, esters, etc.). For example, the solvent mixture may include, but not limited to, methanol and methyl-t-butyl ether. In a preferred example, the volume-to-volume ratio of methanol to methyl-t-butyl ether is 1:1.2.

[0046] In another embodiment, the method further includes the step of adding a type 1 polymorph tosylate salt of compound 1 to the third solution to form a fourth solution. For example, the type 1 polymorph tosylate salt is a seed crystal. In some embodiments, the fourth solution is stirred to form a slurry. The slurry may be washed with the first solvent or solvent mixture, or a solvent or solvent mixture which may be the same as or different from the second solvent or solvent mixture. The slurry may be dried.

[0047] In another embodiment, the present invention relates to a pure composition comprising compound 1, which is about 90-100%, preferably 95-100%, more preferably 98-100% (weight / weight) or 99-100% (weight / weight) pure, and contains, for example, less than about 10%, less than about 5%, less than about 2%, or less than 1% impurities. Such impurities include, for example, decomposition products, oxidation products, epimers, solvents, and / or other undesirable impurities.

[0048] In another embodiment, the present invention includes a method for treating the tetracycline response state in a subject by administering an effective amount of compound 1 in crystalline form to the subject. For example, the subject is a human subject.

[0049] In another embodiment, the present invention includes a method for treating a tetracycline response state in a subject by administering an effective amount of a stable salt of compound 1 to the subject. For example, the stable salt is a tosylate, hydrochloric acid, or mesylate salt of compound 1.

[0050] In another embodiment, the present invention includes a method for treating the tetracycline response state in a subject by administering an effective amount of a polymorph of compound 1 to the subject.

[0051] In another embodiment, the present invention includes a method for treating a tetracycline response state in a subject by administering an effective amount of a polymorph of a salt of compound 1 to the subject. For example, the polymorph may be a polymorph of the tosylate, hydrochloric acid, or mesylate salt of compound 1.

[0052] In another embodiment, the present invention includes a method for treating a tetracycline response state in a subject by administering an effective amount of a polymorph of the tosylate salt of compound 1 to the subject. For example, the tosylate polymorph may be type 1, type 2, or type 3 polymorph of the tosylate salt of compound 1.

[0053] For example, a tetracycline response is a bacterial infection. Bacterial infections can be associated with Gram-positive or Gram-negative bacteria. In some embodiments, bacterial infections are associated with Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), or Enterococcus faecalis (E. faecalis).

[0054] In some embodiments, bacterial infections are resistant to other tetracycline antibiotics, including but not limited to tetracycline, minocycline, doxycycline, sancycline, chlortetracycline, demeclocycline, oxytetracycline, kelocardin, lolitetracycline, rimecycline, metacycline, apicycline, chromocycline, pipacycline, mepircycline, meglucycline, guamecycline, penimocycline, and etamocycline. [Invention 1001] Crystal form of compound 1 below: TIFF2026121464000002.tif27128. [Invention 1002] Tosylate salt of compound 1 below: TIFF2026121464000003.tif27128. [Invention 1003] The crystalline form of the salt according to the present invention 1002. [Invention 1004] A polymorph of the crystalline form of the present invention 1003, characterized by an X-ray powder diffraction pattern substantially similar to the pattern shown in Figure 8. [Invention 1005] A polymorph of the present invention 1004, characterized by an X-ray powder diffraction pattern including peaks at approximately 8.06, 13.02, and 18.83°2θ when irradiated with Cu Kα rays. [Invention 1006] A polymorph of the present invention 1004, characterized by an X-ray powder diffraction pattern including peaks at approximately 8.06, 11.41, 13.02, 18.83, 20.54, and 24.53°2θ when irradiated with Cu Kα rays. [Invention 1007] A polymorph of the present invention 1004, characterized by an X-ray powder diffraction pattern including peaks at approximately 5.60, 8.06, 8.57, 11.41, 13.02, 15.58, 18.83, 20.54, and 24.53°2θ when irradiated with Cu Kα rays. [Invention 1008] Polymorph of the present invention 1004, obtained by crystallizing the tosylate salt of compound 1 below from isopropanol: TIFF2026121464000004.tif27129. [Invention 1009] The first step is to dissolve the free base of compound 1 in a first solvent or a mixture of solvents to form a first solution; The step of dissolving p-toluenesulfonic acid in a second solvent or a mixture of solvents to form a second solution; and The step of mixing the first solution and the second solution to form a third solution. A method for preparing a stable crystalline tosylate salt of compound 1, including the following: TIFF2026121464000005.tif27130. [Invention 1010] The method of the present invention 1009, wherein the first solvent or solvent mixture and the second solvent or solvent mixture are the same or different. [Invention 1011] The method of the present invention 1009, wherein the first solvent mixture and the second solvent mixture are each independently mixtures of alcohol solvents. [Invention 1012] The method of the present invention 1011, wherein each solvent mixture is independently a mixture of two different alcohol solvents. [Invention 1013] The method of the present invention 1012, wherein the two alcohol solvents are ethanol and isopropanol. [Invention 1014] The method of the present invention 1013, wherein the volume-to-volume ratio of ethanol to isopropanol is 2:1. [Invention 1015] The method of the present invention 1009, wherein each solvent mixture contains an alcohol solvent and an antisolvent. [Invention 1016] The method of the present invention 1015, wherein the alcohol solvent is methanol. [Invention 1017] The method of the present invention 1015, wherein the antisolvent is selected from ketones, ethers, and esters. [Invention 1018] The method of the present invention 1017, wherein the ether is methyl-t-butyl ether. [Invention 1019] The method of the present invention 1009, wherein each solvent miscible comprises methanol and methyl-t-butyl ether. [Invention 1020] The method of the present invention 1019, wherein the volume-to-volume ratio of methanol and methyl-t-butyl ether is 1:1.2. [Invention 1021] The method of the present invention 1009, wherein p-toluenesulfonic acid is provided in an amount of 25 to 75% by weight relative to the amount of compound 1. [Invention 1022] The method of the present invention 1009, wherein p-toluenesulfonic acid is provided in the form of p-toluenesulfonic acid monohydrate. [Invention 1023] The method of the present invention 1009 further comprises the step of adding a tosylate salt of the type 1 polymorph of compound 1 to a third solution to form a fourth solution. [Invention 1024] The method of the present invention 1023, wherein the fourth solution forms a slurry upon stirring. [Invention 1025] The method of the present invention 1024, further comprising the step of drying the slurry. [Invention 1026] Polymorphs of the tosylate salt of compound 1 below, obtained according to the method of the present invention 1009: TIFF2026121464000006.tif27128. [Invention 1027] A pharmaceutical composition comprising the crystalline salt of the present invention 1003 and a pharmaceutically acceptable diluent, excipient, or carrier. [Invention 1028] A pharmaceutical composition comprising a polymorph of the present invention 1004 and a pharmaceutically acceptable diluent, excipient, or carrier. [Invention 1029] A pharmaceutical composition of the present invention 1028, wherein polymorphic bodies exist in their pure form. [Brief explanation of the drawing]

[0055] [Figure 1] This provides the X-ray powder diffraction pattern at 25°C for a sample containing crystalline compound 1. [Figure 2] The following provides X-ray powder diffraction patterns at 25°C for the starting material (E00285), the type 1 tosylate salt, type 2 tosylate salt, type 3 tosylate salt of compound 1, and the amorphous tosylate salt form. [Figure 3] This provides a comparison of the X-ray powder diffraction patterns at 25°C between a crystalline compound 1 (E00285) and a sample containing a type 1 tosylate salt obtained from the recrystallization of the amorphous tosylate salt of compound 1 in IPA. [Figure 4] This provides a comparison of X-ray powder diffraction patterns at 25°C for samples containing type 1, type 2, and type 3 tosylate salts, which were dried overnight under vacuum. [Figure 5] This provides X-ray powder diffraction analysis of samples containing type 2 tosylate salts at various temperatures. [Figure 6] This provides X-ray powder diffraction analysis of samples containing type 3 tosylate salts at various temperatures. [Figure 7] This provides X-ray powder diffraction analysis at various temperatures for samples containing a slurry of a 50:50 mixture of type 1 and type 3 tosylate salts in IPA. [Figure 8] This provides a high-resolution X-ray powder diffraction pattern of a sample containing the type 1 tosylate salt of compound 1. [Figure 9] This provides a high-resolution X-ray powder diffraction pattern of a sample containing the type II tosylate salt of compound 1 (93.2% HPLC purity). [Figure 10] This provides a high-resolution X-ray powder diffraction pattern of a sample containing the type 3 tosylate salt of compound 1 (96.7% HPLC purity). [Modes for carrying out the invention]

[0056] Detailed description of the invention Tetracycline-type antibiotic compounds have long been known to have limited stability in their solid-phase free base form. One such amorphous tetracycline analog compound is (4S,4AS,5AR,12AS)-4-7-bis(dimethylamino)-9{[(2,2-dimethylpropyl)amino]methyl}-3,10,12,12A-tetrahydroxy-1,11-dioxo-1,4,4A,5,5A,6,11,12A-octahydrotetracene-2-carboxamide (compound 1; MW=556.66, MF=C 29 H 40 N4O7 exhibits limited stability in the solid phase when exposed to air, light, and / or moisture. TIFF2026121464000007.tif27128(4S,4AS,5AR,12AS)-4-7-bis(dimethylamino)-9{[(2,2-dimethylpropyl)amino]methyl}-3,10,12,12A-tetrahydroxy-1,11-dioxo-1,4,4A,5,5A,6,11,12A-octahydrotetracene-2-carboxamide

[0057] Specifically, compound 1 is a yellow amorphous solid that is unstable at temperatures above 0°C and when exposed to air. Compound 1 must be stored below 0°C, limiting its exposure to air, light, and moisture in the solid phase. Outside of these limited exposure conditions, compound 1 decomposes to produce decomposition products including air decomposition products 2, 3, and 4, as well as the 4-epiisomer 5. TIFF2026121464000008.tif62142

[0058] Prior to this disclosure, no stable crystalline form or stable crystalline acidic salt of compound 1 was known.

[0059] The present invention relates to a crystalline compound 1, a salt form of compound 1, a polymorphic form of compound 1, or a polymorphic form of a salt of compound 1; a pharmaceutical composition comprising the crystalline form, salt form, polymorphic form, or polymorphic form of a salt of compound 1; a method for preparing the crystalline form, salt form, polymorphic form, or polymorphic form of a salt of compound 1; and a method for using them to treat a tetracycline response state.

[0060] 1.Solid form compounds Compound 1 is a tetracycline compound. The term "tetracycline compound" includes many compounds that have a ring structure similar to tetracycline. Examples of tetracycline compounds include: tetracycline, chlortetracycline, oxytetracycline, demeclocycline, metacycline, suncycline, doxycycline, and minocycline.

[0061] The free base and certain pharmaceutically acceptable salts of compound 1 are described in U.S. Patent Application No. 10 / 786,881, corresponding to U.S. Patent Application Publication No. 2005 / 0026876 A1. As can be determined from the above-mentioned list of properties, there is no teaching or suggestion regarding the crystalline form of compound 1, nor is there any teaching or suggestion that any of the described salt forms is superior to others.

[0062] Thus, the present invention addresses the need for improved tetracycline compounds for manufacturing and bioavailability, and the need for improved solid-phase forms of tetracycline compounds.

[0063] Compound 1, which is a solid-phase form of a tetracycline compound, can be in crystalline form. The crystalline form of the compound can be a free base. Different salt crystalline forms of the compound can be formed from the free base. Examples of acids that can be used to convert the free base into a salt include, but are not limited to, hydrochloric acid, p-toluenesulfonic acid, trifluoroacetic acid, methylsulfonic acid, benzenesulfonic acid, and acetic acid.

[0064] The neutral form of the compound may be regenerated by contacting the salt with a base or acid and by isolating the parent compound as in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0065] As described herein, processes have been developed that can produce different crystalline forms of compound 1. More specifically, the inventors have shown that the resulting crystalline forms depend primarily on the properties of the solvent used in the process. For the purposes of this description, the term “crystalline form” refers without distinction to either polymorphic or amorphous forms. “Polymorphic form” refers to an organized structure that contains only solute molecules and has a characteristic crystalline signature.

[0066] The terms “polymorph” and “polymorphic form,” as well as related terms herein, refer to the crystalline form of the same molecule, and different polymorphs may have different physical properties, such as melting temperature, heat of fusion, solubility, dissolution rate, and / or vibrational spectrum as a result of molecular alignment or conformation in the crystal lattice. Differences in physical properties exhibited by polymorphs affect pharmaceutical parameters such as storage stability, compressibility and density (important in the manufacture of formulations and products), and dissolution rate (an important factor in bioavailability). Differences in stability may also result from changes in chemical reactivity (e.g., discriminative oxidation, where one dosage form containing one polymorph fades more rapidly than one containing another), or mechanical properties (e.g., when a kinetically advantageous polymorph is converted to a thermodynamically more stable polymorph, the tablet may crumble during storage), or both (e.g., a tablet of one polymorph is more susceptible to decomposition at high humidity). As a result of differences in solubility / dissolution, in extreme cases, the same polymorph transition may lead to a lack of potency, or in other extreme cases, toxicity. In addition, the physical properties of the crystals may be important during processing; for example, one polymorph may be more prone to forming solvating compounds or may be more difficult to filter and wash to remove impurities (i.e., the distribution of particle shape and size may differ among polymorphs).

[0067] Polymorphs of molecules can be obtained by a number of methods, as are well known in the art. Such methods include, but are not limited to, melt-recrystallization, melt-cooling, solvent-recrystallization, solvent removal, rapid evaporation, rapid cooling, slow cooling, vapor diffusion, and sublimation.

[0068] Techniques for characterizing polymorphs include, but are not limited to, differential scanning calorimeter (DSC), X-ray powder diffraction (XRPD), single-crystal X-ray diffraction, vibrational spectroscopy, such as IR and Raman spectroscopy, solid-state NMR, hot-stage optical microscopy, scanning electron microscopy (SEM), electron crystallography, as well as quantitative analysis, particle size analysis (PSA), surface area analysis, solubility testing, and dissolution testing.

[0069] As used herein, the term "solvated compound" refers to the crystalline form of a substance containing a solvent. The term "hydrate" refers to a solvated compound in which the solvent is water.

[0070] Desolvated solvated compounds are crystalline forms of substances that can only be produced by removing the solvent from a solvated compound.

[0071] As used herein, the term "amorphous form" refers to the non-crystalline form of a substance.

[0072] As used herein, the term “pure” means a compound that is about 90–100%, preferably 95–100%, more preferably 98–100% (weight / weight), or 99–100% (weight / weight) pure; for example, it means that impurities are present in amounts less than about 10%, less than about 5%, less than about 2%, or less than 1%. Such impurities include, for example, decomposition products, oxidation products, epimers, solvents, and / or other undesirable impurities.

[0073] As used herein, a compound is considered "stable" if no significant amount of degradation products are observed over a period of four weeks under constant conditions of humidity, light exposure, and temperature above 0°C. A compound is considered unstable under certain conditions if degradation impurities appear or if the area percentage of existing impurities begins to grow. The rate of growth of degradation products as a function of time is important for determining the stability of a compound.

[0074] All ranges described herein are intended to encompass all values ​​and ranges, including not only the endpoints of the ranges shown, but also values ​​and ranges not specifically stated.

[0075] This invention relates to the crystalline form, salt form, and polymorphs of compound 1; compositions comprising the crystalline form, salt, and polymorphs alone or in combination with other active ingredients; methods for preparing the crystals, salt, and polymorphs; and methods for using them in modulating the tetracycline compound receiving state. While not intended to be bound by any particular operational theory, the storage stability, compressibility, density, or solubility properties of the crystalline form, salt, and polymorphs are beneficial for the production, formulation, and bioavailability of tetracycline compounds.

[0076] Preferred salts and polymorphs of the present invention are salts and polymorphs characterized by physical properties, such as stability, solubility, hygroscopicity, and dissolution rate, that are suitable for clinical and therapeutic dosage forms. Preferred polymorphs of the present invention are polymorphs characterized by physical properties, such as crystalline morphology, compressibility, and hardness, that are suitable for the manufacture of solid dosage forms. Such properties can be determined using techniques described herein and known in the art, such as X-ray diffraction, microscopy, IR spectroscopy, thermal analysis, and hygroscopic analysis.

[0077] 1.1 Salt of Compound 1 In one aspect, the present invention provides crystalline forms of specific pharmaceutically acceptable salts of compound 1. This aspect of the present invention provides crystalline forms of hydrochloric acid, mesylate, and tosylate salts of compound 1: TIFF2026121464000009.tif27128(4S,4AS,5AR,12AS)-4-7-bis(dimethylamino)-9{[(2,2-dimethylpropyl)amino]methyl}-3,10,12,12A-tetrahydroxy-1,11-dioxo-1,4,4A,5,5A,6,11,12A-octahydrotetracene-2-carboxamide.

[0078] Each salt of the present invention may be prepared from a preparation of compound 1. Compound 1 may be synthesized or obtained by any method obvious to those skilled in the art. In a preferred embodiment, compound 1 is prepared by a method described in detail in the following examples. See, for example, U.S. Patent Application Publication 2005 / 0026876 A1, the entirety of which is incorporated herein by reference.

[0079] Alternatively, compound 1 may be prepared by isolating a specific salt of compound 1 and converting such salt of compound 1 to a neutral form by treatment with a suitable base. For example, compound 1 may be prepared by isolating the hydrochloride salt of compound 1 by filtration and then converting it to a neutral form by treatment with monobasic sodium carbonate or other suitable base in ethyl acetate.

[0080] Compound 1, prepared by any method, can be brought into contact with a suitable acid, either undiluted (i.e., unmixed or undiluted) or in a suitable inert solvent or a combination of solvents, to produce the salt form of the present invention. For example, compound 1 can be brought into contact with p-toluenesulfonic acid to produce the tosylate salt form of the present invention.

[0081] Stability tests were performed on compound 1 (free base) and its amorphous dihydrochloride salt. The salt was formed by dissolving the compound in an aqueous solution, adjusting the solution's pH to approximately 4.2, and then freeze-drying. The free base decomposed in less than one month at 40°C and in approximately three months at 4°C. In contrast, the dihydrochloride salt of compound 1 was stable for six months at 40°C and for two years at room temperature (25°C).

[0082] As will be shown in detail in the following example, the tosylate salt of compound 1 and its polymorphs exhibit desirable properties.

[0083] 1.2 Polymorphs of Compound 1 The present invention also provides polymorphs of compound 1. In one aspect, the polymorph of the present invention is a polymorph of the tosylate salt of compound 1.

[0084] Each polymorph of the present invention can be prepared from a preparation of compound 1. After dissolving solid compound 1, crystallization from the solvent mixture described below can produce the polymorph forms of the present invention. In certain embodiments of the present invention, after dissolving the tosylate salt of compound 1, crystallization from the solvent mixture described below can produce certain polymorph forms of the present invention. In some embodiments of the present invention, after dissolving the free base of compound 1, an acid is added to form a crystalline salt of compound 1.

[0085] In one embodiment, the present invention provides polymorphs of the tosylate salt of compound 1.

[0086] In a further embodiment, the present invention provides a type 1 polymorph of a tosylate salt of compound 1 having an X-ray powder diffraction pattern similar to the pattern in Figure 8, the characteristics of which are all shown in Table 1. For example, a particular type 1 polymorph of the present invention has peaks in the X-ray powder diffraction pattern at 5.60, 8.06, 8.57, 11.41, 13.02, 15.58, 18.83, 20.54, and 24.53°2θ when irradiated with Cu Kα rays. For example, a particular type 1 polymorph of the present invention has peaks in the X-ray powder diffraction pattern at 8.06, 11.41, 13.02, 18.83, 20.54, and 24.53°2θ when irradiated with Cu Kα rays. For example, a particular type 1 polymorph of the present invention has peaks in the X-ray powder diffraction pattern at 8.06, 13.02, 18.83, and 24.53°2θ. For example, a particular type 1 polymorph of the present invention has major X-ray powder diffraction pattern peaks at 8.06 and 18.83°2θ.

[0087] [Table 1]

[0088] In another embodiment, the present invention provides a type II tosylate salt of compound 1. In one embodiment, a type II polymorph of the tosylate salt of compound 1 has an X-ray powder diffraction pattern similar to the pattern in Figure 9, the features of which are all shown in Table 2. For example, a particular type II polymorph of the present invention has X-ray powder diffraction pattern peaks at 7.82, 11.88, 12.68, 16.12, 18.63, 21.46, and 23.74°2θ when irradiated with Cu Kα rays. For example, a particular type II polymorph of the present invention has major X-ray powder diffraction pattern peaks at 7.82, 11.88, 16.12, and 21.46°2θ. For example, a particular type II polymorph of the present invention has X-ray powder diffraction pattern peaks at 11.88 and 16.12°2θ.

[0089] [Table 2]

[0090] In another embodiment, the present invention provides three types of compound 1. In a further embodiment, the three polymorphs of the tosylate salt of compound 1 have an X-ray powder diffraction pattern similar to the pattern in Figure 10, the features of which are all shown in Table 3. For example, a particular three polymorph of the present invention has X-ray powder diffraction pattern peaks at 5.11, 8.89, 10.34, 11.76, 13.70, 14.81, and 15.60°2θ when irradiated with Cu Kα rays. For example, a particular three polymorph of the present invention has major X-ray powder diffraction pattern peaks at 5.11, 8.89, 10.34, 11.76, and 15.60°2θ. For example, a particular three polymorph of the present invention has major X-ray powder diffraction pattern peaks at 5.11 and 15.60°2θ.

[0091] [Table 3]

[0092] The tosylate salt of compound 1 crystallized as very small, irregular particles typically measuring 5–8 microns in size. Figure 1 shows the X-ray powder diffraction (XRPD) of the crystalline solid of the tosylate salt of compound 1. This compound was shown to melt and decompose at 190°C.

[0093] Gravimetric vapor sorption was performed on compound 1 or its tosylate salt. It was determined that there were 2.5 molecules of water per molecule of compound 1. XRPD was performed to compare the starting material (E00285) with the dehydrated material. The data did not show any changes in morphology.

[0094] 2. Synthesis of Compound 1 9-(aminomethyl)-minocycline dihydrochloride (200 mg, 1 equivalent), DMF, and trimethylacetaldehyde (45 μL, 1 equivalent) were mixed and stirred in a 40 mL flask. Next, triethylamine (150 μL, 3 equivalents) was added. After stirring at room temperature for several minutes, NaBH(OAc)3 (175 mg, 2 equivalents) and InCl3 (9 mg, 0.1 equivalent) were added. After 1 hour, the reaction product was clear and red. Liquid chromatography revealed one product of the reaction. The reaction was stopped with methanol, the solvent was removed, and the product was purified by column chromatography.

[0095] purification Compound 1 was purified by chromatography by the steps of injecting a low pH aqueous solution of the compound into HPLC with a polar organic solvent gradient, and mixing the product fraction to purify the compound. Process stability and selectivity were enhanced by selecting a suitable acidic mobile phase. Organic and inorganic acidic mobile phases were effective in separating by-products containing epimer impurities and by-products that eluted nearby, through pH control or acid selection. The acidic mobile phase also prevented oxidative degradation of the compound.

[0096] For example, low pH solutions had a pH of approximately 2–3. Examples of solutions used included a 0.1% aqueous solution of methanesulfonic acid and a 0.1% aqueous solution of trifluoroacetic acid. In certain embodiments, the compound was purified from the epimer byproduct and nearby eluting byproducts using an isocratic gradient of a 94% aqueous solution and 6% acetonitrile or another polar organic solvent.

[0097] The resulting aqueous product fractions may be combined and the pH adjusted to approximately 4.0–4.5 using a base (e.g., NaOH). Hydrophobic impurities and oxidative decomposition products of the compound can be removed by washing the aqueous solution with a nonpolar organic solvent (e.g., CH2Cl2). The organic layer was discarded, and the aqueous layer was mixed and retained.

[0098] It is noteworthy that slow-eluting hydrophobic impurities, such as 4-carbonyl byproducts and other oxidative decomposition products, can be selectively removed from acidic aqueous solutions of compounds using organic solvents such as methylene chloride.

[0099] The pH of the combined aqueous layer may then be adjusted to a neutral pH, for example, about 7.5 to 8.5. The pH may also be adjusted by adding a base such as NaOH. Next, the neutral solution was washed with a nonpolar organic solvent such as methylene chloride. It should be noted that selective pH adjustment to a neutral pH range allows the compound to be extracted into the organic solvent while retaining undesirable β-epimers and byproducts in the aqueous phase.

[0100] In addition, antioxidants may be similarly added to aqueous solutions of the compounds described herein. Antioxidants may be provided to prevent oxidative degradation of the compounds. Antioxidants such as sulfurous acid or ammonium bisulfite can be used.

[0101] 3. Method for preparing polymorphic forms of compound 1 The present invention also relates to a method for preparing polymorphic forms of crystalline compound 1.

[0102] In one embodiment, type 1 of the tosylate salt of compound 1 can be prepared by any method apparent to those skilled in the art, based on the teachings herein. In a particular embodiment, type 1 may be formed from the maturation of the amorphous tosylate salt of compound 1 in isopropanol, acetone, ethyl acetate, methylpentanone, toluene, or acetonitrile solution. Type 1 can also be obtained from the recrystallization of the amorphous tosylate salt that has formed a slurry in isopropanol. Type 1 can also be obtained by dissolving a free base in a suitable solvent or mixture of solvents, such as two alcohols, or an alcohol and an antisolvent such as a ketone, ether, or ester. After adding an acid, the salt can be gradually crystallized in the correct form.

[0103] A solvent system can be selected in which impurities and free bases of compound 1 are soluble, but the stable crystalline salt of compound 1 is insoluble, for example, in which a crystalline slurry can be formed by precipitation.

[0104] In another embodiment, the second form of the tosylate salt of compound 1 may be prepared by any method apparent to those skilled in the art who prepare the second form based on the teachings herein. In a particular embodiment, the second form may be formed from the maturation of the amorphous tosylate salt of compound 1 in dichloromethane.

[0105] In another embodiment, form 3 of the tosylate salt of compound 1 may be prepared by any method apparent to those skilled in the art who prepare form 3 based on the teachings herein. In a particular embodiment, form 3 may be formed from the maturation of the amorphous tosylate salt of compound 1 in methyl ethyl ketone, ethyl acetate, or methyl pentanone. Form 3 may also be obtained from the maturation of form 1 in methyl pentanone.

[0106] In a further embodiment, the polymorphic form of the tosylate salt of compound 1 described above may be produced by a method comprising the steps of miscible compound 1 with a solvent to produce a slurry, and the addition of p-toluenesulfonic acid.

[0107] A slurry may be prepared using any suitable solvent. Solvents that may be used in this embodiment include alcoholic solvents such as isopropanol. A solution from which the salt crystallizes may be prepared using a mixture of any suitable solvent. Mixtures of solvents that may be used in this embodiment include, but are not limited to, methanol and methyl-t-butyl ether or ethanol and isopropanol.

[0108] For example, a slurry of compound 1 in a solvent or solvent mixture may be produced at a temperature of approximately 0°C to approximately 60°C, such as approximately 15°C to approximately 45°C, or approximately 20°C to approximately 25°C. After production, the slurry may optionally be heated and / or maintained at a temperature of approximately 15°C to approximately 60°C, such as approximately 20°C to approximately 50°C, or approximately 45°C.

[0109] After preparing the slurry, p-toluenesulfonic acid may be added in an amount sufficient to produce the p-toluenesulfonate of compound 1. In one embodiment, p-toluenesulfonic acid is provided in an amount of about 25 to about 75% by weight, about 25 to about 50% by weight, about 30 to about 40% by weight, or about 33% by weight relative to the amount of compound 1. The p-toluenesulfonic acid may be added in the form of p-toluenesulfonic acid monohydrate.

[0110] The polymorphic forms of the tosylate salt of compound 1 can be formed by a solution method. For example, a solution of compound 1 may be produced at a temperature of about 0°C to about 60°C, such as about 15°C to about 45°C or about 20°C to about 25°C. After production, the solution may optionally be heated and / or maintained at a temperature of about 15°C to about 60°C, such as about 20°C to about 50°C or about 45°C.

[0111] After preparing the solution, p-toluenesulfonic acid may be added in an amount sufficient to produce the p-toluenesulfonate of compound 1. In one embodiment, p-toluenesulfonic acid is provided in an amount of about 25 to about 75% by weight, about 30 to about 50% by weight, about 35 to about 45% by weight, or about 40% by weight relative to the amount of compound 1. The p-toluenesulfonic acid may be added in the form of p-toluenesulfonic acid monohydrate.

[0112] In one embodiment, a type 1 polymorph used to introduce seed crystals into the solution may be added. A p-toluenesulfonic acid solution may be formed using any suitable solvent. Suitable solvents include alcohol solvents such as isopropanol, or solvent mixtures such as methanol and methyl-t-butyl ether. In a preferred embodiment, the volume / volume ratio of methanol to methyl-t-butyl ether is 1:1.2. Suitable solvents include mixtures of two or more alcohol solvents, such as a mixture of ethanol and isopropanol. In a preferred embodiment, the volume / volume ratio of ethanol to isopropanol is 2:1. In a particular embodiment, the p-toluenesulfonic acid solution contains the same solvent used to prepare the slurry or solution of compound 1.

[0113] After adding p-toluenesulfonic acid to a suitable solvent, a slurry of the type 1 polymorph of the tosylate salt of the compound is formed. After adding p-toluenesulfonic acid, the water content of the slurry supernatant may be adjusted to a suitable level. Typically, the water content of the slurry supernatant may be in the range of about 0.2 to about 1.0 mg / mL, such as about 0.4 to about 0.8 mg / mL, for example, about 0.6 mg / mL, about 0.54 mg / mL, etc.

[0114] After adding p-toluenesulfonic acid, the slurry or solution may be stirred to produce a crystalline slurry. Stirring may be carried out for longer than 48 hours. However, stirring is typically performed for a period of about 5 to 36 hours, such as about 10 to 24 hours or about 18 hours.

[0115] Stirring may be carried out at any temperature suitable for producing a crystalline slurry. For example, the slurry may be stirred at a temperature of about 0°C to about 60°C, such as about 15°C to about 45°C, or about 20°C to about 25°C.

[0116] After crystal formation, the crystal slurry may be filtered to remove the supernatant, or the crystals may be washed with any suitable solvent. In some embodiments, the crystals may be washed 1 to 4 times, and the solvent may be any solvent suitable for preparing the crystal slurry. In particular, the solvent used to wash the crystals may be the same one or more solvents used to form the initial slurry or solution, or the p-toluenesulfonic acid solution.

[0117] Next, the produced crystals may be dried to remove excess solvent by any suitable method. For example, drying may be achieved by one or more methods, including but not limited to, raised temperatures in the range of about 0°C to about 60°C, such as about 15°C to about 45°C; blowing dry nitrogen onto the crystals; and blowing wet nitrogen onto the crystals.

[0118] A maturation test was performed in which samples of the tosylate salt of compound 1 were prepared as slurries in different solvents, filtered, and the wet solids were analyzed by XRPD. Three polymorphic forms of the tosylate salt of compound 1 were observed. Figure 2 shows the XRPD spectra of the starting material (E00285), the type 1 tosylate salt of compound 1, the type 2 tosylate salt, the type 3 tosylate salt, and the amorphous form.

[0119] Table 4 lists the solvents used in the aging experiment.

[0120] [Table 4]

[0121] The amorphous material of compound 1 was recrystallized in various solvents. Only recrystallization in 2-propanol (isopropyl alcohol, IPA) yielded the type 1 tosylate salt, as shown in Table 5. Figure 3 compares the XRPD spectra of the reference compound 1 (E00285) and the type 1 tosylate salt recrystallized from IPA.

[0122] [Table 5]

[0123] Scheme 1 shows an overview of the polymorphic tosylate salt of compound 1. TIFF2026121464000015.tif68129

[0124] After recrystallization, samples of type 1, type 2, and type 3 tosylate salts were dried overnight under vacuum as shown in Figure 4 and analyzed by XRPD. No changes were observed in the morphology after drying.

[0125] XRPD at various temperatures was performed on the tosylate salts of compound 1, both form 2 and form 3. See Figures 5 and 6, respectively.

[0126] The relative stability of the polymorphic forms of compound 1 was analyzed. For example, the type 1 tosylate salt was subjected to a 24-hour aging experiment in either IPA or methylpentanone, using either the type 2 or type 3 tosylate salt as a seed crystal. During this experiment, no change from type 1 to type 2 or type 3 occurred. A 50:50 mixture slurry of type 1 and type 3 was analyzed in IPA at 0°C, 25°C, 40°C, and 60°C for 18 hours, as shown in Figure 7. No change from type 1 to type 3 occurred.

[0127] Recrystallization experiments demonstrated that type 1 tosylate salts can be reproducibly obtained from amorphous tosylate salts by slurry formation in IPA. Similarly, type 1 of compound 1 can be reproducibly obtained by adding tosylic acid. A high-resolution XRPD scan of type 1 is shown in Figure 8, and the characteristics of the diffraction pattern are shown in Table 1.

[0128] 4. Pharmaceutical compositions comprising the compound, salt, crystalline form, or polymorph of the present invention. In further embodiments, the present invention relates to a pharmaceutical composition comprising a tetracycline compound of the present invention (for example, synthesized or purified by the methods of the present invention) or a pharmaceutically acceptable salt, prodrug, or ester thereof. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0129] As used herein, the term “composition” is intended to encompass not only products containing the specified components (and the specified amounts, if indicated) but also any products directly or indirectly resulting from admixtures of the specified components in specified amounts. “Pharmacologically acceptable” means that diluents, excipients, or carriers must be compatible with the other components of the formulation and must not be harmful to its recipient.

[0130] As previously stated, certain embodiments of the compounds of the present invention may contain basic functional groups such as amino or alkylamino, and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term “pharmaceutically acceptable salt” is recognized in the art and includes relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or by reacting the purified compounds of the present invention in their free base form with suitable organic or inorganic acids individually and isolating the salts thus formed. Typical salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naptylate, mesylate, glucoheptate, lactobionate, and lauryl sulfonate (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Farm. SCI. 66:1-19).

[0131] In other cases, the compounds of the present invention may contain one or more acidic functional groups, which can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salt” in these examples includes relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ during the final isolation and purification of the compounds, or by individually reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Typical alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Typical organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine.

[0132] The term "pharmaceutically acceptable esters" refers to relatively non-toxic esterification products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by reacting the purified compound or hydroxyl in its free acid form separately with a suitable esterifying agent. Carboxylic acids can be converted to esters by treatment with alcohol in the presence of a catalyst. Hydroxyls can be converted to esters by treatment with esterifying agents such as halogenated alkanoyls. This term also includes lower hydrocarbon groups that can be solvated under physiological conditions, such as alkyl esters, methyl, ethyl, and propyl esters (see, e.g., Berge et al., above).

[0133] The present invention also relates to tetracycline compounds synthesized and / or purified by the methods of the present invention, and pharmaceutically acceptable salts thereof.

[0134] The phrase “pharmaceutically acceptable carrier” is recognized in the art and includes pharmaceutically acceptable materials, compositions, or vehicles suitable for administering the compounds of the present invention to mammals. A carrier includes liquid or solid extenders, diluents, excipients, solvents, or encapsulating materials involved in carrying or transporting the test substance from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; water free of pyrogens; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0135] The composition may contain not only wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, but also colorants, release agents, coating agents, sweeteners, flavorings, as well as fragrances, preservatives, and antioxidants.

[0136] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bicarbonate, sodium metabisulfate, and sodium sulfite; lipid-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0137] The formulations of the present invention include formulations suitable for oral, intranasal, topical, transdermal, oral cavity, sublingual, rectal, vaginal, and / or parenteral administration. The formulations may be conveniently expressed in unit dosage forms and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be mixed with a carrier material to produce one dosage form will generally be the amount of compound that produces the therapeutic effect. Generally, out of 100 percent, this amount will be in the range of about 1 percent to about 99 percent of the active ingredient, preferably in the range of about 5 percent to about 70 percent, and most preferably in the range of about 10 percent to about 30 percent.

[0138] Methods for preparing these formulations or compositions include the step of associating the compound of the present invention with a carrier and optionally one or more auxiliary components. Generally, formulations are prepared by the steps of uniformly and completely associating the compound of the present invention with a liquid carrier, a fine powder solid carrier, or both, and, if necessary, shaping the product.

[0139] Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavoring base, usually sucrose and acacia or tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or flavored tablets (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or mouthwash, etc., each containing a specified amount of the compound of the present invention as an active ingredient. The compound of the present invention may also be administered as a bolus, lick, or paste.

[0140] In the solid dosage forms of the present invention for oral administration (capsules, tablets, pills, sugar-coated tablets, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate and / or any of the following: fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol and / or silicic acid; binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; dissolution retarders such as paraffin; absorption accelerators such as quaternary ammonium compounds; wetting agents such as cetyl alcohol and glycerol monostearate; absorbents such as kaolin and bentonite soil; lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and colorants. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules, using not only excipients such as lactose or milk sugar, but also high molecular weight polyethylene glycol, etc.

[0141] Tablets may be prepared by compression or molding with one or more auxiliary components as optional. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycol or cross-linked sodium carboxymethylcellulose), a surface-active agent, or a dispersant. Molded tablets may be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent in suitable equipment.

[0142] Tablets, as well as other solid dosage forms of the pharmaceutical compositions of the present invention, such as sugar-coated tablets, capsules, pills, and granules, may optionally be etched or prepared with coatings and casings, such as enteric coatings and other coatings well known in the pharmaceutical art. They may be formulated to provide a slow or controlled release of the active ingredient therein, for example, using various ratios of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide a desired release profile. They may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injection medium immediately before use. These compositions may also optionally contain opacifying agents and may optionally release the active ingredient in a delayed manner, either to specific parts of the gastrointestinal tract or preferentially to specific parts. Examples of embedding compositions that can be used include polymer materials and waxes. The active ingredient may, where appropriate, be in a microencapsulated form containing one or more of the aforementioned excipients.

[0143] Liquid dosage forms for oral administration of the compounds of the present invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain water or other solvents, solvents, and emulsifiers commonly used in the art, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), fatty acid esters of glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan, as well as mixtures thereof.

[0144] In addition to inert diluents, oral compositions may also contain adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavorings, colorants, fragrances, and preservatives.

[0145] In addition to the active compound, the suspension agent may contain other suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0146] Formulations of the pharmaceutical compositions of the present invention for rectal or vaginal administration may be represented as suppositories, which may be prepared by mixing one or more compounds of the present invention with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylate, which are solid at room temperature but liquid at body temperature and therefore melt in the rectal or vaginal cavity to release the active compound.

[0147] Formulations of the present invention suitable for intravaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing carriers known to be suitable in the art.

[0148] Dosage forms for topical or transdermal administration of the compounds of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, liquids, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, any preservatives, buffers, or propellants.

[0149] The ointments, pastes, creams, and gels may contain, in addition to the active compounds of the present invention, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0150] The powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof, in addition to the compounds of the present invention. The sprays may further contain conventional propellants such as chlorofluorohydrocarbons, as well as volatile unsubstituted hydrocarbons such as butane and propane.

[0151] Transdermal patches offer the further advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be prepared by dissolving or dispersing the compounds in a suitable culture medium. Absorption enhancers can also be used to increase the influx of the compounds through the skin. Such influx rates can be controlled either by providing a rate-controlled membrane or by dispersing the active compounds in a polymer matrix or gel.

[0152] It is intended that ophthalmic preparations, ophthalmic ointments, powders, liquids, etc., are also within the scope of the present invention.

[0153] A pharmaceutically acceptable composition of the present invention suitable for parenteral administration comprises one or more compounds of the present invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous aqueous solutions, dispersants, suspensions, or emulsions, or sterile powders which may be reconstituted immediately before use in a sterile injectable solution or dispersion that may contain antioxidants, buffers, bacteriostatic agents, solutes, suspensions, or concentrators to make the formulation isotonic with respect to the blood of the intended recipient.

[0154] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersants, and by using surfactants.

[0155] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action may be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Similarly, it may be desirable to include isotonic substances such as sugars and sodium chloride in the composition. In addition, sustained absorption of injectable pharmaceutical dosage forms may be achieved by including absorption-delaying substances such as aluminum monostearate and gelatin.

[0156] In some cases, it is desirable to delay the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effect. This may be achieved by using a liquid suspension of a crystalline or amorphous material with low water solubility. The rate of drug absorption may depend on its dissolution rate, and then on the size and morphology of the crystals. Alternatively, delayed absorption of a parenterally administered drug form may be achieved by dissolving or suspending the drug in an oily vehicle.

[0157] Injectable depot formulations are prepared by forming a microencapsulation matrix of the compound of the present invention in a biodegradable polymer such as polylactic acid-polyglycolate ester. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydride esters). Injectable depot formulations are also prepared by capturing the drug in liposomes or microemulsions that are compatible with body tissues.

[0158] The preparations of the present invention may be administered orally, parenterally, topically, or rectally. They are provided in a dosage form suitable for each route of administration. For example, they may be administered in the form of tablets or capsules. For example, they may be administered by injection, infusion, inhalation, lotion, ointment, suppository, etc. Oral administration is preferred.

[0159] As used herein, the phrases “parenteral administration” and “administered parenterally” mean administration by conventional injection other than intestinal and topical surface administration, including, but not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intravertebral, and intrasternal injections and infusions.

[0160] As used herein, the phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” mean the administration of a compound, drug, or other material other than direct administration to the central nervous system, such as subcutaneous administration, in which the compound enters the patient’s system and thus undergoes metabolism and other similar processes.

[0161] These compounds may be administered to humans and other animals for therapeutic purposes by any suitable route of administration, including orally, intranasally (e.g., by spray), rectally, intravaginally, parenterally, intracisionally, and topically (e.g., powders, ointments, or drops, including oral and sublingual applications).

[0162] Regardless of the selected route of administration, the compounds of the present invention, which may be used in suitable hydrated forms and / or pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0163] The term "therapeutic effective dose" refers to an amount of the salt or polymorph of the present invention that is sufficient to induce a biological or medical response in a tissue, system, animal, or human being, as sought by researchers, veterinarians, physicians, or other clinicians, or to prevent or partially alleviate the onset of one or more symptoms of a disease being treated.

[0164] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient that is effective in achieving a desired therapeutic response with respect to a particular patient, composition, and mode of administration, without causing toxicity to the patient.

[0165] The selected dose level will depend on a variety of factors, including the activity of the specific compound of the present invention used, or its ester, salt, or amide; the route of administration; the time of administration; the excretion rate of the specific compound used; the duration of treatment; other drugs, compounds, and / or materials used in combination with the specific compound used; the age, sex, weight, condition, overall health, and prior medical history of the patient being treated; and similar factors well known in the art of medicine.

[0166] A physician or veterinarian skilled in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may start the dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the dose required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0167] Generally, the suitable daily dose of the compound of the present invention will be the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above. Generally, when used in relation to the analgesic effect described, the intravenous and subcutaneous doses of the compound of the present invention per patient will be in the range of about 0.0001 to about 100 mg / kg body weight / day, more preferably about 0.01 to about 50 mg / kg / day, and even more preferably about 0.1 to about 10 mg / kg / day. For example, in some embodiments, the dose is 0.5 to about 4.0 mg / kg / day. If desired, the effective daily dose of the active compound may be administered in unit dose form at appropriate intervals of one day, one week, or other suitable period, as one, two, three, four, five, six or more lower doses administered individually.

[0168] While the compound of the present invention can be administered alone, it is preferable to administer the compound as a pharmaceutical composition.

[0169] 5. Method using the tetracycline compound of the present invention The present invention also relates to a method for treating a tetracycline response state in a subject by administering to the subject an effective amount of a composition comprising compound 1 or a pharmaceutically acceptable salt thereof according to the present invention, so that the state is treated.

[0170] As used herein, the terms “to treat,” “treating,” or “treatment” refer to a method of alleviating or eliminating a disease or disorder (e.g., a tetracycline compound response state) and / or its associated symptoms. As used herein, the terms “to prevent,” “preventing,” or “prevention” refer to a method of preventing a subject from acquiring a disease or disorder. As used herein, “subject” includes mammals. A mammal may be any mammal, for example, a human, a primate, a mouse, a rat, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. Preferably, the mammal is a human.

[0171] The terms “tetracycline compound-responsive condition” or “tetracycline-responsive condition” include conditions that can be treated, prevented, or otherwise improved by administration of the tetracycline compounds of the present invention. Tetracycline compound-responsive conditions include bacterial, viral, and fungal infections (including infections that are resistant to other tetracycline compounds), cancer (for example, prostate cancer, breast cancer, colon cancer, lung cancer, melanoma, lymphoma, and other disorders characterized by undesirable cell proliferation, including but not limited to the disorders described in US 6,100,248), arthritis, osteoporosis, diabetes, and other conditions in which tetracycline compounds have been found to be active (see, for example, U.S. Patents 5,789,395; 5,834,450; 6,277,061, and 5,532,227, each of which is expressly incorporated herein by reference). The compounds of the present invention can be used to prevent or control important mammalian and animal diseases such as diarrhea, urinary tract infections, infections of the skin and skin structures, infections of the ear, nose, and throat, wound infections, mastitis, and the like. In addition, methods for treating neoplasms using the tetracycline compounds of the present invention are also included (van der Bozert et al., Cancer Res., 1998, 48:6686-6690). In one embodiment, the tetracycline response state is not a bacterial infection. In another embodiment, the tetracycline compounds of the present invention are inherently non-antibacterial. For example, the non-antibacterial tetracycline compounds of the present invention may have an MIC greater than about 4 μg / ml when measured by assays known in the art.

[0172] Tetracycline compound response states also include inflammation-related states (IPAS). The term “inflammation-related states” includes states in which inflammation or inflammatory factors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins, plasma proteins, cellular defense systems, cytokines, lipid metabolism, proteases, toxic radicals, adhesion molecules, etc.) are involved or present in abnormal amounts in a region. Inflammatory processes are the response of living tissue to damage. The causes of inflammation can be physical damage, chemicals, microorganisms, tissue necrosis, cancer, or other substances. Acute inflammation is short-lived, lasting only a few days. However, if it lasts longer, it may be called chronic inflammation.

[0173] IPAS includes inflammatory disorders. Inflammatory disorders are generally characterized by fever, redness, swelling, pain, or loss of function. Examples of causes of inflammatory disorders include, but are not limited to, microbial infections (e.g., bacterial and fungal infections), physical agents (e.g., burns, radiation, and trauma), chemical agents (e.g., toxins and caustic substances), tissue necrosis, and various types of immune responses. In a further embodiment, IPAS includes disorders described in U.S. Patents 5,929,055 and 5,532,227, the entire contents of which are incorporated herein by reference.

[0174] Examples of inflammatory disorders include, but are not limited to, osteoarthritis, rheumatoid arthritis, acute and chronic infections (bacterial and fungal, including diphtheria and pertussis), acute and chronic bronchitis, sinusitis, upper respiratory infections (such as the common cold), acute and chronic gastroenteritis and colitis, acute and chronic cystitis and urethritis, acute and chronic dermatitis, acute and chronic conjunctivitis, acute and chronic serositis (pericarditis, peritonitis, synovitis, pleurisy and tendinitis), uremic pericarditis, acute and chronic cholecystitis, acute and chronic vaginitis, acute and chronic uveitis, drug reactions, insect bites, burns (thermal, chemical and electrical), and sunburn.

[0175] The term “inflammatory process-related conditions” includes, in one embodiment, NO-related conditions. The term “NO-related conditions” includes conditions involving or related to nitric oxide (NO) or inducible nitric oxide synthase (iNOS). NO-related conditions include conditions characterized by abnormal amounts of NO and / or iNOS. Preferably, NO-related conditions can be treated by administering the tetracycline compounds of the present invention. The disorders, diseases, and conditions described in U.S. Patents 6,231,894; 6,015,804; 5,919,774; and 5,789,395 are also included as NO-related conditions. The entire contents of each of these patents are incorporated herein by reference.

[0176] Other examples of NO-related conditions include malaria, aging, diabetes, vascular stroke, neurodegenerative disorders (Alzheimer's disease, Huntington's disease, and Parkinson's disease), heart disease (post-infarction reperfusion-related injury), juvenile diabetes, inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute, recurrent, and chronic infections (bacterial, viral, and fungal), acute and chronic bronchitis, sinusitis, and respiratory infections (common cold, etc.), and acute and chronic gastrointestinal This includes, but is not limited to, inflammation and colitis, acute and chronic cystitis and urethritis, acute and chronic dermatitis, acute and chronic conjunctivitis, acute and chronic serositis (pericarditis, peritonitis, synovitis, pleurisy, and tendinitis), uremic pericarditis, acute and chronic cholecystitis, cystic fibrosis, acute and chronic vaginitis, acute and chronic uveitis, drug reactions, insect bites, burns (thermal, chemical, and electrical), and sunburn.

[0177] The term “inflammatory process-related states” includes, in one embodiment, matrix metalloproteinase-related states (MMPAS). MMPAS include states characterized by abnormal amounts of MMPs or MMP activity. These also include tetracycline compound-responsive states which may be treated with the compounds of the present invention.

[0178] Examples of matrix metalloproteinase-associated conditions (MMPAS) include atherosclerosis, corneal ulcers, emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann. Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997, 72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronic pulmonary obstructive disease, skin and eye diseases, periodontitis, osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatory disorders, tumor growth and invasion (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907:191-217; Li et al., Mol. Other MMPASs include, but are not limited to, those described in U.S. Patent Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412; 4,704,383; 4,666,897; and RE No. 34,656, which are incorporated herein by reference in their entirety.

[0179] In another embodiment, the tetracycline compound response state is cancer. Examples of cancers for which the tetracycline compounds of the present invention may be useful for treatment include all solid tumors, i.e., carcinomas, such as adenocarcinomas and sarcomas. Adenocarcinomas are carcinomas that originate from glandular tissue or in which tumor cells form recognizable glandular structures. Sarcomas broadly include tumors in which their cells are embedded in fibrils or homogeneous material such as embryonic connective tissue. Examples of carcinomas that may be treated with the compounds of the present invention include, but are not limited to, prostate cancer, breast cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, and breast cancer. The methods of the present invention are not limited to the treatment of these tumor types but extend to any solid tumor originating from any organ system. Examples of treatable cancers include, but are not limited to, colon cancer, bladder cancer, breast cancer, melanoma, ovarian cancer, prostate cancer, lung cancer, and a variety of other cancers. The methods of the present invention also cause inhibition of cancer growth in adenocarcinomas such as prostate cancer, breast cancer, kidney cancer, ovarian cancer, testicular cancer, and colon cancer.

[0180] In one embodiment, the present invention relates to a method for treating subjects who have cancer or are at risk of developing cancer by administering an effective amount of a substituted tetracycline compound such that inhibition of cancer cell growth occurs, i.e., that cell proliferation, invasion, metastasis, or tumor development is reduced, delayed, or stopped. The inhibition may result from inhibition of inflammatory processes, downregulation of inflammatory processes, several other mechanisms, or a combination of mechanisms. Alternatively, tetracycline compounds may be useful to prevent cancer recurrence, for example, to treat residual cancer after surgical resection or radiotherapy. Useful tetracycline compounds according to the present invention are particularly advantageous because they are substantially non-toxic compared to other cancer treatments. In a further embodiment, the compounds of the present invention are administered in combination with standard cancer treatments, but not limited to, chemotherapy.

[0181] Examples of tetracycline response states also include both neuropsychiatric and neurodegenerative disorders, but also include Alzheimer's disease, Alzheimer's disease-related dementias such as Pick's disease, Parkinson's disease and other diffuse Lewy body dementias, senile dementia, Huntington's disease, Gilles de la Tourette syndrome, multiple sclerosis, amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease, as well as autonomic dysfunctions such as hypertension and sleep disorders. This includes neuropsychiatric disorders (such as depression, schizophrenia, schizoaffective disorder, Korsakoff psychosis, mania, anxiety disorders, and phobias), learning or memory impairments (such as amnesia or age-related memory loss, attention deficit disorder, etc.), dysthymia, major depressive disorder, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, and panic disorder, as well as neurological disorders, not limited to bipolar affective disorders (such as severe bipolar affective (mood) disorder (BP-1), bipolar affective neurological disorders, e.g., migraines and obesity). Further neurological disorders include those described in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the full contents of which the most recent edition is incorporated herein by reference.

[0182] In another embodiment, the tetracycline compound response state is diabetes, such as juvenile diabetes, diabetes mellitus, type 1 diabetes, or type 2 diabetes. In a further embodiment, protein glycosylation is not affected by the administration of the tetracycline compound of the present invention. In another embodiment, the tetracycline compound of the present invention is administered in combination with standard diabetes treatment, such as, but not limited to, insulin therapy.

[0183] In another embodiment, the tetracycline compound response state is osteodystrophy. Osteodystrophy includes disorders in which the bone of the subject is impaired and / or conditions in which bone formation, repair, or remodeling is favorable. For example, osteodystrophy includes osteoporosis (e.g., decreased bone strength and density), fracture, bone formation associated with surgical techniques (e.g., facial reconstruction), osteogenesis imperfecta (fragility osteopathy), hypophosphatasia, Paget's disease, fibrous osteodysplasia, osteopetrosis, myeloma, and calcium depletion in the bone, such as in cases associated with primary hyperparathyroidism. Osteodystrophy includes not only all conditions in which bone formation, repair, or remodeling is favorable to the subject, but also all other disorders related to the bone or skeletal system of the subject that can be treated with the tetracycline compounds of the present invention. In a further embodiment, bone density disorders include those described in U.S. Patents No. 5,459,135; No. 5,231,017; No. 5,998,390; No. 5,770,588; RE No. 34,656; No. 5,308,839; No. 4,925,833; No. 3,304,227; and No. 4,666,897, each of which is incorporated herein by reference in its entirety.

[0184] In another embodiment, a tetracycline compound response state is acute lung injury. Acute lung injury includes adult respiratory distress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Trauma includes any injury to living tissue caused by an exogenous substance or event. Examples of trauma include, but are not limited to, bruises, contact with a hard surface, or cuts or other injuries to the lungs.

[0185] The present invention also relates to a method for treating acute lung injury by administering the substituted tetracycline compounds of the present invention.

[0186] The tetracycline response states of the present invention also include chronic lung injury. The present invention relates to a method for treating chronic lung injury by administering a tetracycline compound, such as the compounds described herein. The present invention includes the step of administering an effective amount of the substituted tetracycline compound to a target so as to treat chronic lung injury. Examples of chronic lung injury include, but are not limited to, asthma, cystic fibrosis, and emphysema. In further embodiments, the tetracycline compounds of the present invention are used to treat acute and / or chronic lung injury, such as the disorders described in U.S. Patents 5,977,091; 6,043,231; 5,523,297; and 5,773,430, each of which is incorporated herein by reference in its entirety.

[0187] In another embodiment, the tetracycline compound response state is ischemia, stroke, or ischemic stroke. The present invention also relates to a method for treating ischemia, stroke, or ischemic stroke by administering an effective amount of the substituted tetracycline compound of the present invention. In a further embodiment, the tetracycline compound of the present invention is used to treat the disorders described in U.S. Patents 6,231,894; 5,773,430; 5,919,775; and 5,789,395, which are incorporated herein by reference.

[0188] In another embodiment, the tetracycline compound response state is a skin wound. The present invention also relates, at least in part, to a method for improving the healing response of epithelial tissue to acute traumatic injury (e.g., cuts, burns, abrasions, etc.). This method may include a step of using the tetracycline compound of the present invention (which may or may not have antimicrobial activity) to improve the ability of epithelial tissue to heal acute wounds. This method may increase the rate of collagen accumulation in healing tissue. This method may also reduce proteolytic activity in epithelial tissue by reducing the collagen-degrading and / or gelatin-degrading activity of MMPs. In a further embodiment, the tetracycline compound of the present invention is administered to the surface of the skin (e.g., a topical surface). In further embodiments, the tetracycline compounds of the present invention are used to treat skin wounds and other such disorders, as described in, for example, U.S. Patents 5,827,840; 4,704,383; 4,935,412; 5,258,371; 5,308,8391; 5,459,135; 5,532,227; and 6,015,804, each of which is incorporated herein by reference in its entirety.

[0189] In another embodiment, the tetracycline compound response state is an aorta or vascular aneurysm in the vascular tissue of a subject (e.g., a subject having or at risk of having an aortic or vascular aneurysm). The tetracycline compound may be effective in reducing the size of the vascular aneurysm, or may be administered to the subject before the development of a vascular aneurysm to prevent its development. In one embodiment, the vascular tissue is an artery, such as the aorta, such as the abdominal aorta. In a further embodiment, the tetracycline compound of the present invention is used to treat disorders described in U.S. Patents 6,043,225 and 5,834,449, the entire contents of which are incorporated herein by reference.

[0190] Bacterial infections can be caused by a wide variety of Gram-positive and Gram-negative bacteria. The compounds of the present invention are useful as antibiotics against organisms that are resistant to other tetracycline compounds. The antibiotic activity of the tetracycline compounds of the present invention is described in Waitz, JA, National Commission for Clinical Laboratory Standards, Document M7-A2, vol. 10, no. 8, pp. 13-20, 2 nd The standard in vitro broth dilution method described in edition, Villanova, PA (1990) may be used to determine the result.

[0191] Tetracycline compounds may also be used to treat infections conventionally treated with tetracycline compounds, such as rickettsial infections, numerous Gram-positive and Gram-negative bacterial infections, venereal lymphogranuloma, inclusion conjunctivitis, and psittacosis. Tetracycline compounds may also be used to treat infections of, for example, Klebsiella pneumoniae, Salmonella, Streptococcus hirae, Acinetobacter baumanii, B. catarrhalis, Haemophilus influenzae, Pseudomonas aeruginosa, Enterococcus faecium, Escherichia coli, Staphylococcus aureus, or Enterococcus faecalis. In one embodiment, tetracycline compounds are used to treat bacterial infections that are resistant to other tetracycline antibiotic compounds. The tetracycline compounds of the present invention may be administered together with a pharmaceutically acceptable carrier.

[0192] The phrase “in combination with another therapeutic substance or treatment” includes simultaneous administration of a tetracycline compound (e.g., an inhibitor) with another therapeutic substance or treatment, administration of the tetracycline compound first followed by the other therapeutic substance or treatment, and administration of the tetracycline compound first followed by the other therapeutic substance or treatment. The other therapeutic substance may be any substance known in the art for treating, preventing, or reducing the symptoms of a disease or disorder such as IPAS. Furthermore, the other therapeutic substance may be any substance that is beneficial to the patient when administered in combination with the administration of the tetracycline compound. In one embodiment, diseases such as cancer treated by the method of the present invention include the diseases described in U.S. Patents 6,100,248; 5,843,925; 5,837,696; and 5,668,122, the entire contents of which are incorporated herein by reference.

[0193] The term "effective dose" of a compound refers to the amount necessary or sufficient to treat or prevent a tetracycline compound response. The effective dose can vary depending on factors such as the subject's size and weight, the type of disease, or the specific tetracycline compound. For example, the choice of tetracycline compound can affect what constitutes the "effective dose." Those skilled in the art can examine these factors and make a determination regarding the effective dose of a tetracycline compound without excessive experimentation.

[0194] In the therapeutic methods of the present invention, one or more tetracycline compounds of the present invention may be administered to a subject alone, or more typically, the compounds of the present invention may be administered as part of a pharmaceutical composition, mixed with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances that are suitable for parenteral, oral, or other preferred administration, do not react adversely with the active compound, and are not harmful to the recipient.

[0195] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. [Examples]

[0196] 6. Demonstration of the present invention Example 1: Synthesis of 9-alkylaminomethylminocycline Minocycline hydrochloride (compound 2) was dissolved in methylsulfonic acid or hydrofluoric acid along with a similar water scavenger acid such as methylsulfonic anhydride or trifluoromethanesulfonic acid. N-hydroxymethylphthalimide was added to the reaction mixture. The mixture was stirred at 20-35°C until the reaction was complete. When the acid solution was added to the ice / water mixture, the trifurate was readily precipitated and could be filtered and collected. The solid was redissolved in acetone and neutralized with a base to pH. The product was precipitated by adding water. If trifuric acid is present as a scavenger, the product may precipitate without neutralization. The product was isolated as a mixture of bis and trisalkylated products. The isolated material of this reactant was concentrated at the desired bis ratio (90%).

[0197] The solid was suspended in EtOH or MeOH. Aminolysis was performed using methylamine. As the reaction proceeded, a phthalamide byproduct precipitated and was removed by filtration. By adding approximately 1.5 times the amount of t-butyl methyl ether to the reaction mixture, a pale yellow solid product precipitated, which was collected by simple filtration, leaving many small impurities and methylamine reagent in the solution. Further purification of the compound was performed by restrush formation with a lower aliphatic alcohol such as methanol.

[0198] Compound 4, as a free base, was transferred to a hydrogenation vessel filled with methanol and aldehyde. The vessel was then filled with an inactivated Pd / C catalyst and pressurized with hydrogen gas. The reaction mixture was hydrogenated for approximately 24 hours under a hydrogen pressure of approximately 30 psi. After the conversion of compound 4 to compound 1 was complete, the solution was filtered and washed through a Celite pad. At this point, the reaction mixture contained a very low amount of βC-4 epimer, approximately 3-7%.

[0199] Product (1) was prepared as follows to selectively isolate the product from its impurities: The pH of the solution was adjusted to approximately 4.5 with concentrated HCl, and the solution was extracted with dichloromethane. The aqueous layer was extracted with dichloromethane to selectively recover the preferred epimer product (e.g., α). The dichloromethane layers were combined and concentrated, and 2 L of n-heptane was added to precipitate the product. Further purification was obtained by repeating preparation techniques with or without t-butyl methyl ether to dissolve the crude product.

[0200] Example 2: Purification of Compound 1 Free base (40 g) of crude 9-(2',2'-dimethylpropylaminomethyl)minocycline was dissolved in 150 mL of buffer A (0.1% methanesulfonic acid aqueous solution - MSA), and the pH was adjusted to 2-3 with MSA.

[0201] The solution was filtered and injected into an HPLC, where the product was eluted by an isocratic gradient of 94% buffer A and 6% acetonitrile. Once the product peak was detected, fractional collection of the product was initiated. Each fraction was analyzed, and an acceptable criterion greater than the 80% AUC of the main peak was used for the initial product fraction. When combining fractions, the levels of impurities and relative concentrations in the pooled fractions were taken into consideration as selection criteria to meet the specifications of the final product. A 10% aqueous sodium sulfite solution equal to 10% of the original volume of the collected fraction was added to the product fraction.

[0202] The following example shows the output result of a single injection. The output results of multiple injections may be similarly combined. A fractional volume of 3.5 L of the product (containing sodium sulfite) was collected, and its pH was adjusted to 4.0-4.5 using a sodium hydroxide solution. The aqueous solution was washed with 2 L of dichloromethane, and the organic layer was separated and discarded.

[0203] The pH of the aqueous layer was adjusted to 7.5–8.5 using sodium hydroxide, and the product was extracted four times with 2.4 L of dichloromethane. Before each extraction, the pH was readjusted to 7.5–8.5 using sodium hydroxide or MSA.

[0204] The four dichloromethane layers were combined and concentrated to approximately 200 mL, which was gradually added to vigorously stirred n-heptane (2.5 L) over approximately 10 minutes. The suspension was stirred at room temperature for approximately 10 minutes and then gradually diluted with 1.5 L of n-heptane over 5 minutes. The slurry was cooled to 0–5°C and stirred for 1–2 hours. The suspended solid was filtered and washed with 3 × 150 mL of n-heptane. The product was dried under vacuum at 40°C for at least 24 hours until a certain weight was achieved and all residual solvent levels were within specifications. Approximately 13.6 g of the free base of 9-(2',2'-dimethylpropylaminomethyl)minocycline was isolated as a yellow solid.

[0205] Example 3: Preparation of the crystalline HCl salt of compound 1 Compound 1 (13 g) was dissolved in acetone (300 mL), filtered, and the filter paper was further washed with acetone. The combined filtrate and washing solution were cooled to 5°C. A concentrated HCl (3.9 mL) acetone solution (79 mL) was gradually added to the combined filtrate and washing solution while vigorously stirring. The resulting slurry was stirred in an ice bath for 15 minutes and then filtered.

[0206] The initial solid harvest was washed with cold acetone and pentane, and dried under vacuum for 48 hours to obtain 13.7 g of a yellow amorphous solid. The flask containing the saturated filtrate was covered with aluminum foil and left for two weeks, during which time the growth of single crystals was observed. The crystals were collected by filtration and washed with hexane.

[0207] Example 4: Preparation of crystalline methanesulfonate of compound 1 A 25 mL three-necked flask was packed with 3 mL of isopropanol (IPA) under an inert nitrogen atmosphere. A slurry was prepared by adding 225 mg of amorphous free base of compound 1 to the flask. The slurry was heated to a temperature of 45°C. Next, methanesulfonic acid hydrate (98.0 mg) was added to the slurry. After stirring the slurry at 45°C for 1 hour, it was cooled to 22°C to produce a concentrated crystalline slurry. The slurry was filtered and washed with IPA (2 × 1 mL). To achieve a constant weight, excess IPA was removed from the crystal cake by drying at 55°C for longer than 2 hours. The crystalline mesylate (methanesulfonic acid) salt of compound 1 (180 mg) was isolated. The crystalline mesylate salt was determined to be unstable at 5°C.

[0208] Example 5: Preparation of crystalline tosylate salt of compound 1 (using slurry method) A 5 L three-necked flask was packed with 2.0 L of isopropanol (IPA) under an inert nitrogen atmosphere. A slurry was prepared by adding 289 g of amorphous free base of compound 1 to the flask. A solution of p-toluenesulfonic acid hydrate (97.0 g) in IPA (400 mL) was added to the slurry. The water content of the slurry supernatant was adjusted to 0.6 g / L by adding water (9 mL), and the slurry was stirred at 20-25°C for 18 hours to produce a concentrated crystalline slurry. The slurry was filtered and washed with IPA (2 × 500 mL). Excess IPA was removed from the crystalline cake by spraying dry nitrogen into the cake for 24 hours. For solids containing 3 wt% (wt%) IPA, the cake was further dried by spraying humid nitrogen into the cake at a relative humidity of 70-75% for 24 hours. The cake retained 0.9 wt% IPA, which was not further reduced by this method. Excess water was removed from the cake by blowing dry nitrogen into it for 24 hours. The tosylate salt of compound 1 was isolated as an orange powder (337 g). The isolated tosylate salt of compound 1 was crystalline in the only observed form: a non-stoichiometric hemihydrate, as determined by X-ray powder diffraction (XPRD) and thermogravimetric (TG) analysis.

[0209] Example 6: Preparation of crystalline tosylate salt of compound 1 (using solution method) A 5 L three-necked flask was packed with 1.7 L of methanol and 1.7 L of methyl-t-butyl ether under an inert nitrogen atmosphere. A clear solution was obtained by adding 209 g of p-toluenesulfonic acid monohydrate and 556 g of amorphous free base of compound 1 to the flask with stirring. Crystallization was initiated by adding a seed crystal amount (3 g) of the monotosylate salt of compound 1, and further amounts of methanol (0.1 L) and methyl-t-butyl ether (0.5 L) were added. The resulting slurry was stirred at approximately 20°C for 22 hours to produce a concentrated crystalline slurry. The slurry was filtered and washed with a mixture of 1.1 L of methanol and 1.3 L of methyl-t-butyl ether, and then washed with methyl-t-butyl ether (2 × 2.4 L). The tosylate salt of compound 1 was isolated as an orange powder. Excess solvent was removed from the crystal cake by blowing dry nitrogen into the cake for 24 hours. Next, the cake was dried under vacuum at approximately 30°C until the solid contained approximately 6 weight percent (wt%) of solvent. The cake was further dried under vacuum at approximately 45°C until the solid contained less than 3 weight percent (wt%) of solvent. The isolated tosylate salt of compound 1 was a crystal having type 1 as observed when determined by X-ray powder diffraction (XPRD).

[0210] Example 7: Characterization of the tosylate salt of compound 1 The XRPD patterns of the isolated tosylate salt of compound 1 included 2θ values ​​of 5.6, 8.0, 8.6, 11.4, 13.0, 15.5, 18.8, 20.4, and 24.5°.

[0211] The crystalline tosylate salt was subjected to thermogravimetric (TG) analysis under a nitrogen stream at a heating rate of 10°C / min. A weight loss of 3.9% was observed by 81.3°C, which was attributed to water loss.

[0212] Upon drying, the moisture content of the crystalline tosylate salt of compound 1 was calculated to be 0.5%. After being left at room temperature for 24 hours, this value increased to 5%. In contrast to amorphous compound 1, the crystalline tosylate salt of compound 1 remained stable at room temperature for several weeks and months, maintaining a moisture content of approximately 5%.

[0213] The hygroscopicity of the target crystalline tosylate salt was determined using a symmetric vapor sorption analyzer and reported as a function of percentage relative humidity (%RH) in weight percentages, ranging from 5% to 95% to 1% at 25.0°C, and in 5% increments at 25°C. A maximum weight increase of 12 wt% at 95%RH was observed, with slight hysteresis during desorption, and a loss of 2 wt% due to IPA was observed.

[0214] The crystalline tosylate salt was determined to have the solubility summarized in Table 6 below. Solubility was determined by mixing an excess amount of solid with a solvent at ambient temperature for 2 hours, followed by filtering of the supernatant. The concentration of the supernatant was determined by high-performance liquid chromatography (HPLC). Clear solutions were obtained when 56.8 mg of the target crystalline tosylate salt was brought to equilibrium in 0.5 mL of water, and when 122.1 mg of the target crystalline tosylate salt was brought to equilibrium in 1.0 mL of water. The pH of 122.1 mg of the crystalline tosylate salt of the present invention in 1.0 mL of water was determined to be 5.70.

[0215] [Table 6]

[0216] Example 8: XRPD of Compound 1 X-ray powder diffraction patterns were collected using a Siemens D5000 diffractometer with Cu Kα irradiation (40 kV, 40 mA) and a θ-θ goniometer, divergence V20 and photodetector slit, graphite secondary monochromator, and scintillation counter. Instrument performance was checked using the accredited Corundum standard (NIST 1976). The software used for data acquisition was Diffrac Plus XRD Commander v2.3.1, and the data were analyzed and displayed using Diffrac Plus EVA v 11,0.0.2 or v 13.0.0.2.

[0217] Powder samples were prepared using flat plate specimens. Approximately 35 mg of the sample was gently packed into a cavity cut into a polished zero-background (510) silicon wafer. The sample was rotated on its own plane during analysis. Details of data acquisition are as follows: Angle range: 2~42°2θ Step size: 0.05°2θ Collection time: 4 seconds / step High-resolution X-ray powder diffraction patterns were collected using a Bruker AXS C2 GADDS diffractometer with Cu Kα irradiation (40 kV, 40 mA), an automated XYZ stage, a laser video microscope for automated sample placement, and a HiStar 2D region detector. The X-ray optics consisted of a single Gobel multilayer mirror coupled to a 0.3 mm pinhole collimator.

[0218] The divergence of the light beam, i.e., the effective size of the X-ray beam in the sample, was approximately 4 mm. Using the θ-θ continuous scan mode with a sample-detector distance of 20 cm, this gives an effective 2θ range of 3.2° to 29.7°. Typically, the sample would be exposed to X-ray beams for 120 seconds. The software used for data acquisition was GADDS for WNT 4.1.16, and the data were analyzed and displayed using Diffrac Plus EVA v 9.0.0.2 or v 13.0.0.2.

[0219] Example 9: Temperature and moisture stability test The tosylate and mesylate salts of compound 1 were tested for stability by monitoring changes in the high-performance liquid chromatography (HPLC) impurity profiles of each salt when exposed to various temperature and / or humidity conditions. Each sample was placed in a sealed container and exposed to three controlled environments: refrigeration (5°C), 20°C at 60% relative humidity, and 40°C at 75% relative humidity. After two weeks, the crystalline tosylate salt was determined to be the most stable crystalline form of compound 1. Stability testing of the crystalline tosylate salt was continued.

[0220] The samples were analyzed by the following reversed-phase HPLC impurity profiling method at 0, 1, 2, and 4 weeks (Table 7) and 3 months (Table 8) for the tosylate salt, and at 0, 1, and 2 weeks (Table 9) for the mesylate salt.

[0221] Reverse-phase HPLC analysis was performed using a SYMMETRY SHIELD RP 18 column (4.6 × 250 mm, particle size 5 pm). Similar results are expected from analyses using similar or equivalent columns. The mobile phase components were 0.01 M aqueous ammonium acetate (A) and acetonitrile (B) at pH 3.3. The mobile phase component was increased from 6% to 15% B over 5 minutes, maintained at 15% B for 15 minutes, increased from 15% to 60% B over 10 minutes, then increased from 60% to 90% B over 2 minutes, and then re-equilibriumated at 6% B for 4 minutes. The flow rate was 1.0 mL / min and the injection volume was 10.0 μL. The column temperature was maintained at 30°C. Detection was performed by ultraviolet (UV) light at 280 nm. The retention time for compound 1 was approximately 15.7 minutes. The sample solution was prepared in mobile phase A to a final concentration of 2.0 mg / mL.

[0222] [Table 7] * RH refers to relative humidity. **Total impurities include all impurities in the specified batch.** *** RRT 1.26 refers to the β-epimer.

[0223] [Table 8] * RH refers to relative humidity. **Total impurities include all impurities in the specified batch.**

[0224] [Table 9]

[0225] The above data demonstrates that the tosylate salt of compound 1 is stable when stored under refrigeration and / or 20°C & 60%RH conditions for at least 4 weeks. The tosylate salt of compound 1 is stable at temperatures in the range of 0°C to 70°C, or 5°C to 50°C, or 20°C to 30°C.

[0226] Example 10: Photostability Test Photostability tests were performed on the tosylate salt of compound 1. Two samples were prepared in clear glass petri dishes. One sample was covered with aluminum foil and used as a control sample. Both samples were placed in an ES 2000 Environmental Light 10 chamber and exposed to 12 kilolux of cool white fluorescence for a total of 47 hours. The samples were analyzed by HPLC impurity profiling, and the results are summarized in Table 10.

[0227] [Table 10]

[0228] equivalent Those skilled in the art will be able to recognize or confirm, through routine experiments, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be covered by the following claims.

[0229] All patents, patent applications, and references cited herein are incorporated herein by express reference.

Claims

[Claim 1] The invention described in the specification of this application.

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