HK1089972A - Drug formulations having long and medium chain triglycerides - Google Patents

Description

Pharmaceutical formulations containing long and medium chain triglycerides

RELATED APPLICATIONS

The following priority claims are hereby claimed and incorporated by reference herein in their entirety: U.S. provisional patent application No. 60/491,050 entitled "Ansamycin formulations and methods of making and using the same", filed on 29.7.2003 by Ulm et al; U.S. provisional patent application No. 60/478,430 entitled "phospholipid-based formulations and methods of making and using the same", filed on 12.6.2003 by Ulm et al; U.S. provisional patent application No. 60/454,812 entitled "HSP 90 inhibitor formulation and data", filed 3/13/2003 by Ulm et al; and U lm et al, filed 4/2003, PCT application No. PCT/US03/10533 entitled "novel ansamycin formulations and methods for making and using same", claiming priority from the similarly-titled U.S. provisional patent application 60/371,668 filed 10/4/2002.

Technical Field

The present invention relates generally to pharmaceutical formulations and methods, and in more particular embodiments to emulsified formulations of ansamycins, such as 17-AAG.

Background

The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

17-allylamino-geldanamycin (17-AAG) is a synthetic analog of Geldanamycin (GDM). Both of these molecules belong to a large class of antibiotic molecules known as ansamycins. GDMs were first isolated from the microorganism streptomyces hygroscopicus, were initially identified as potent inhibitors of certain kinases, and later shown to act by promoting kinase degradation, particularly targeting "molecular chaperones", such as heat shock protein 90(HSP 90). Later, various other ansamycins also showed more or less this activity, of which 17-AAG is one of the most promising, and it has been the subject of intensive clinical studies currently being conducted by the National Cancer Institute (NCI). See, e.g., Federal Register, 66 (129): 35443-; erichmann et al, proc.aacr (2001), 42, abstrate 4474.

HSP90 is a ubiquitous chaperone protein that is involved in the folding, activation and assembly of many proteins, including key proteins involved in signal transduction, cell cycle control and transcriptional regulation. It has been reported by researchers that HSP90 chaperone proteins are involved in important signaling proteins, such as steroid hormone receptors and protein kinases, including, for example, Raf-1, EGFR, V-Src family kinases, Cdk4 and ErbB-2(Buchner J., 1999, TIBS, 24: 136-141; Stepanova, L.et al., 1996, Genes Dev.10: 1491-502; Dai, K.et al., 1996, J.biol.Chem.271: 22030-4). Studies have further shown that certain co-chaperone molecules, such as Hsp70, p 60/Hop/sil, Hip, Bagl, Hsp40/Hdj2/Hsj1, immunophilin, p23, and p50, can contribute to the performance of Hsp90 function (see, e.g., Caplan, a., Trends in Cell biol., 9: 262-68 (1999)).

Ansamycins, such as Herbimycin A (HA), Geldanamycin (GM) and 17-AAG, are believed to exert their anti-cancer effects by tightly binding to the N-terminal ATP-binding pocket of HSP90 (Stebbins, C.et al, 1997, Cell, 89: 239-. This pocket is highly conserved and has weak homology to the ATP binding site of DNA gyrase (Stebbins, C.et al, supra; Grenert, J.P.et al, 1997, J.biol.chem., 272: 23843-50). In addition, it has been shown that both ATP and ADP bind to the pocket with low affinity and have weak ATPase activity (Proromou, C.et al, 1997, Cell, 90: 65-75; Panaretou, B.et al, 1998, EMBO J., 17: 4829-36). In vitro and in vivo studies have shown that occupancy of the N-terminal pocket by ansamycins and other HSP90 inhibitors alters HSP90 function and inhibits protein folding. At high concentrations, ansamycins and other HSP90 inhibitors can prevent binding of protein substrates to HSP90 (Scheibel, T.H.et al, 1999, Proc.Natl.Acad.Sci.USA 96: 1297-. Ansamycins have also been shown to inhibit ATP-dependent release of chaperone-associated protein substrates (Schneider, C.L.et al, 1996, Proc.Natl.Acad.Sci.USA, 93: 14536-41; Sepp-Lorenzino et al, 1995, J.biol.chem.270: 16580-. In either case, the substrate is degraded by a ubiquitin-dependent process present in the proteasome (Schneider, C., L., supra; Sepp-Lorenzino, L., et al, 1995, J.biol.chem.270: 16580-l 6587; Whitesell, L.et al, 1994, Proc.Natl.Acad.Sci.USA, 91: 8324-.

Destabilization of this substrate occurs in tumor and non-transformed cells and has been shown to be particularly effective against a subset of signal modulators, such as Raf (Schulte, T.W.et al., 1997, biochem. Biophys.Res.Commun.239: 655-9; Schulte, T.W.et al., 1995, J.biol.Chem.270: 2458-8), nuclear steroid receptors (Segnitz, B.and U.Gehring.1997, J.biol.Chem.272: 18694-18701; Smith, D.F.et al., 1995, mol.cell.biol.15: 6804-12), Whv-src (Seitecell, L.biol.15: 6804-12), and certain transmembrane tyrosine kinases (Sep.Acad.USA 8391: 24; 1994, Proc.Natl.Acad.Acad.Sci.S.91: 24, J.S.J.J.S.S. 31, J.J.S.J.S.S.27, J.S.J.S.31, J.S.S.31, J.S.S.S.20, EGF. J.S.D.D.D.D.15: 2, J.S.S.20, J.S.S. 31, S.32, J.S.S.S.S.23, S.S.S.D.D.S.S.S.S.S.S.S.S.S.S.23, S.S.S.23, S.S.S.S.S.S.S.D.S.S.S.S.S.S.S.S.D.D.S.S.D.S.S.S.D.S.D.15, S.S.15, S, CDK4 and mutant p 53. Erichman et al, proc.aacr (2001), 42, abstrat 4474. Ansamycin-induced loss of these proteins results in selective disruption of certain regulatory pathways and growth arrest at specific stages of the cell cycle (Muise-Heimericks, R.C.et al, 1998, J.biol.chem.273: 29864-72), as well as apoptosis and/or differentiation of treated cells (Vasilevskaya, A.et al, 1999, Cancer Res., 59: 3935-40).

Recently, WO01/72779 to Nicchitta et al (PCT/US01/09512) showed that HSP90 could be assumed to have a different conformation after heat shock and/or binding by the fluorophore bis-ANS. In particular, Nicchitta et al demonstrated that this induced conformation showed higher affinity for certain HSP90 ligands than the different morphology of HSP90 that predominates in normal cells. The commonly owned application PCT/US02/39993 further supports this finding by demonstrating the use and application of cancer cell lysates as an excellent source of high affinity HSP 90.

In addition to anti-cancer and anti-tumorigenic activity, HSP90 inhibitors have a variety of other applications, including as anti-inflammatory agents, anti-infective agents, agents for treating autoimmunity, agents for treating stroke, ischemia, cardiac disorders, and agents that help promote nerve regeneration (see, e.g., Rosen et al, WO02/09696(PCT/US 01/23640); Degranco et al, WO99/51223(PCT/US 99/07242); Gold, U.S. patent 6,210,974B 1; DeFranco et al, U.S. patent 6,174,875). Overlapping with some of the above, the literature also reports that fibrogenic disorders may also be treated, including but not limited to scleroderma, polymyositis, systemic lupus erythematosus, rheumatoid arthritis, cirrhosis, keloid formation, interstitial nephritis, and pulmonary fibrosis. (Strehlow, WO 02/02123; PCT/US 01/20578). In addition, the modulation, modulators and uses of HSP90 are also reported in the following documents: PCT/US03/04283, PCT/US02/35938, PCT/US02/16287, PCT/US02/06518, PCT/US98/09805, PCT/US00/09512, PCT/US01/09512, PCT/US01/23640, PCT/US01/46303, PCT/US01/46304, PCT/US02/06518, PCT/US02/29715, PCT/US02/35069, PCT/US02/35938, PCT/US02/39993, 60/293,246, 60/371,668, 60/331,893, 60/335,391, 06/128,593, 60/337,919, 60/340,762 and 60/359,484.

At present, ansamycins, like many other lipophilic drugs, are difficult to formulate into pharmaceutical formulations, especially intravenous formulations. Attempts have been made to prepare lipid vesicles and oil-in-water emulsions to date, but these require complicated processing steps, harsh or clinically unacceptable solvents and/or cause formulation instability. See generally Vemuri, S.and Rhodes, C.T., Preparation and characterization of lipids for therapeutic delivery systems: a review, pharmaceutical Acta Helvetiae 70, pp.95-111 (1995); see also PCT/US99/30631, published as WO00/37050, 6/29/2000. Commonly owned application PCT/US03/10533, to which this application claims priority, describes emulsions containing medium chain triglycerides. However, the medium chain fatty acids and triglycerides carrying them may lead to a metabolic production of caprylate, leading to undesired central nervous system effects, such as, for example, drowsiness, nausea, drowsiness and changes in the EEG. See Cotter et al, am.j.clin.nutr.50: 794-800 (1989); miles et al, Journal of scientific and Enteral Nutrition 15: 37-41 (1991); traul et al, Food chem.toxicol.38: 79-98(2000). To date, such side effects have been partially ameliorated by the use of long chain fatty acids in the form of nutritional supplements, the mechanism of which is that long chain fatty acids compete for the critical caprylate pathway enzymes with higher binding efficiency. However, to the applicant's knowledge so far, they have not been used in combination with medium chain triglycerides and ansamycins.

Thus, there is a need for additional formulations and methods of making the same that are relatively simple to manufacture and that ameliorate one or more of the above-mentioned disadvantages typically associated with medium chain triglycerides.

Summary of The Invention

By formulating long chain triglycerides as a component of a formulation with a compound, applicants have found that the formulations claimed herein can be used to better tolerate intravenous administration of lipophilic compounds such as ansamycin.

In a first aspect, the invention features a pharmaceutical composition that includes a pharmaceutically active compound, e.g., an ansamycin, such as 17-AAG, in combination with an emulsifier (e.g., a phospholipid such as that found in lecithin) and an oil. The oil may and preferably comprises long chain triglycerides. The composition may also comprise medium chain triglycerides. The emulsifier and oil together form a lipid phase.

In certain embodiments, the lipid phase comprises 5-30% w/w, preferably 5-20% w/w of the total formulation.

In certain embodiments, the total w/w percentage of long chain triglycerides is no more than 10%, more preferably in the range of 7% or less, and more preferably in the range of 6% or less, to accommodate viscosity limitations.

In certain embodiments, the w/w ratio of medium chain triglycerides to long chain triglycerides is from 10: 0.0001 to 0.0001: 10, more preferably from 10: 1 to 1: 10.

In certain embodiments, the phospholipid comprises 3-10% w/w of the total weight.

In certain embodiments, the triglyceride is 5-20% w/w of the total weight.

In certain embodiments, the triglyceride is, or is at least partially, in the form of a naturally occurring oil, for example, a vegetable oil, such as soybean oil, sesame oil, safflower oil, and corn oil.

In certain embodiments, the composition further comprises one or more of water, a preservative (e.g., sodium edetate), an anti-freeze agent, a buffer, a chelating agent, and a tonicity modifier (tonicifier).

In certain embodiments, the drug is 17-AAG (17-allylaminogeldanamycin, 17-allylamino-17-demethoxy-geldanamycin) and is present in an amount of 0.5mg/ml to 4mg/ml or 0.05% w/w to 0.4% w/w of the total formulation weight.

In one embodiment, the composition contains the following ingredients: 2mg/ml 17-AAG, 3.3% soybean oil, 6.6% lecithin, 9.9% Miglyol 812N, 7.15% sucrose, and water.

In another embodiment, the composition comprises the following ingredients: 2mg/ml 17-AAG, 7.5% lecithin, 15% Miglyol 812N, 10% sucrose, and water.

17-AAG is 17-allylaminogeldanamycin having the following structure:

in certain embodiments, the compositions of the present invention also comprise short chain triglycerides.

In a particularly preferred embodiment, the composition comprises a sufficient amount of long chain triglycerides to reduce or avoid the occurrence of medium chain triglyceride-mediated Central Nervous System (CNS) effects, particularly where medium chain triglycerides are also included in the composition. CNS effects are generally negative, undesirable effects, including but not limited to one or more of lethargy, nausea, drowsiness, and EEG changes. However, in certain embodiments, such effect(s) may be desirable in a given context, such that the amount of medium chain triglycerides relative to long chain triglycerides is increased.

In certain embodiments, the composition is frozen and/or lyophilized, as outlined in PCT/US 03/10533. In the lyophilized embodiment, the weight percentages of each of the triglyceride, phospholipid, drug and non-volatile components must be increased and exceed the above ranges to account for the increase in their relative fractions after the loss of the water and other volatile components that were originally present but lost after lyophilization.

In certain embodiments, the composition may also be formulated and/or stored under inert gas conditions, for example in dark and/or light-tight bottles, vials or ampoules.

Combinations of any of the above embodiments are also contemplated where appropriate.

In another aspect, the invention features a method of reducing the occurrence of central nervous system effects mediated by medium chain triglycerides in a patient, comprising: a pharmaceutical formulation is provided comprising a pharmaceutically active agent and long chain triglycerides in an amount sufficient to reduce or prevent the occurrence of central nervous system effects mediated by medium chain fatty acids, and administering the product to a patient. Embodiments of this aspect may be made with reference to any of the compositions of the above aspects

Embodiments and combinations thereof.

In another aspect, the invention features methods of using the pharmaceutical compositions, formulations, and methods and products described above for treating or preventing a disease in an organism, such as a mammal, by administering a pharmaceutically effective amount of the product to the organism. The disease, at least in the case of treatment of a mammal, is preferably selected from the group consisting of ischemia, proliferative diseases and nerve injury, and comprises an HSP90 inhibitor, such as one or more ansamycins, as a pharmaceutically active agent. Proliferative diseases include, but are not limited to, tumors and cancers, inflammatory diseases, fungal infections, yeast infections, and viral infections. In certain preferred embodiments, the mammal is a human. In certain preferred embodiments, the mode of administration is intravenous, although other modes of administration are contemplated as described in more detail below.

According to particular embodiments, advantages of the present invention include one or more of the following: ease of preparation, use of clinically acceptable agents (e.g., to reduce toxicity to the environment and/or patient), improved stability of the formulation, ease of shipment and storage, ease of formulation and bedside handling, intravenous and systemic tolerance for administration, and avoidance of certain undesirable side effects often caused by medium chain fatty acids and triglycerides in the body. Other advantages, aspects, and embodiments will become apparent from the following drawings, detailed description, and claims.

Brief Description of Drawings

Figure 1 shows that lethargy in rats is reduced due to the addition of long chain fatty acids to a formulation comprising medium chain triglycerides.

Description of the preferred embodiments

The formulations of the present invention have particular advantages over water-soluble drugs which are suitable for intravenous and other administration to patients. The formulation methods are relatively simple, generally employ clinically acceptable agents, and the products prepared have advantages over existing methods and products in terms of storage, stability, and biological tolerance.

Definition of

The following terms have the following meanings, and the following terms not particularly specified also have the meanings commonly used in the art:

the term "pharmaceutically active compound" has the same meaning as "drug" and refers to any compound that exerts a biological effect, directly or indirectly, when administered to a cultured cell or organism in vivo or in vitro. The drug is preferably capable of being encapsulated in liposomes and/or emulsified, and although not required, the drug is generally lipophilic.

The terms "evaporation" and "lyophilization" do not imply that 100% removal of solvent and solution is necessary, and may be only a minor proportion. However, in lyophilized embodiments, substantial removal is preferred, with removal of about 95% or more being preferred.

"inert gas conditions" refers to gas conditions that have relatively low reactivity compared to the gas in standard atmospheric conditions. An example of such inert gas conditions is the use of pure or substantially pure nitrogen gas during the formulation process. Other situations are well known to those skilled in the art.

The term "hydration" or "rehydration" refers to the addition of an aqueous solution, such as water or a physiologically compatible buffer, e.g., hanks 'solution, ringer's solution, or physiological saline buffer.

The term "about" is meant to encompass a range of deviation of 20% around the stated value. The terms "comprising" and "between" or "between about" when used in conjunction with "are meant to include the endpoints of the recited ranges.

The term "ansamycin" is a broad term characterizing a compound having a "loop" structure comprising one of a benzoquinone, benzohydroquinone, naphthoquinone, or naphthohydroquinone moiety bridged by a long chain. Examples of naphthoquinone or naphthohydroquinone type compounds are the clinically important drugs rifampin and rifamycin, respectively. Examples of benzoquinone like compounds are geldanamycin (including its synthetic derivatives, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-N, N-dimethylamino-ethylamino-17-Demethoxygeldanamycin (DMAG)), dihydrogeldanamycin and herbimycin. An example of a benzohydroquinone is macbecin. Ansamycins and benzoquinone ansamycins according to the present invention may be synthetic, naturally occurring, or a combination of the two, i.e., "semi-synthetic", and may include dimer and conjugated variants and prodrugs. Some exemplary benzoquinone ansamycins useful in the present invention and methods for their preparation include, but are not limited to, those described in U.S. Pat. Nos. 3,595,955 (describing the preparation of ansamycins), 4,261,989, 5,387,584, and 5,932,566. Geldanamycin is also commercially available, for example from CN Biosciences, an affiliate of Merck KGaA, Darmstadt, Germany, headquartered in San Diego, California, USA (cat. No. 345805). Biochemical purification of the geldanamycin derivative 4, 5-dihydrogeldanamycin and its hydroquinone from cultures of Streptomyces hygroscopicus (ATCC55256) is described in an application filed by Pfizer Inc, filed by Cullen et al, published on 7.22.1993, published under WO93/14215, international application No. PCT/US 92/10189; an alternative process for the synthesis of 4, 5-dihydrogeldanamycin by catalytic hydrogenation of geldanamycin is also known. See, for example, Progress in the Chemistry of organic Natural Products, Chemistry of the antibiotic Antibiotics, 33: 278(1976). Other ansamycins useful in various embodiments of the invention are described in the references cited in the "background" section above.

"oils" include fatty acids and fatty acid-containing glycerides, such as mono-, di-, and tri-glycerides as known in the art. The fatty acids and glycerides used in the present invention may be saturated and/or unsaturated, natural and/or synthetic, charged or neutral. "synthetic" may be fully synthetic or semi-synthetic, and these terms are all known in the art. The oil may also be uniform or heterogeneous in composition and/or source.

Herein, a "medium chain triglyceride" is a triglyceride composition containing fatty acids of 8 to 12 straight chain carbon atoms in length, more preferably 8 to 10 carbon atoms in length. Various embodiments of the present invention include the use of Miglyol * 812 supplied by CONDEA (Cranford, NJ, USA) 812. Miglyol * 812 contains roughly 50-65% octanoic acid (8 carbon atoms) and 30-45% decanoic acid (10 carbon atoms). Caproic acid (6 carbon atoms) may also be present, up to about 2%, as may lauric acid (12 carbon atoms). Myristic acid (14 carbon atoms) is present in a smaller amount (up to 1%). Condea also provides Miglyol * 810, 818, 829 and 840, and it is envisioned that one or more of these other Miglyol * solutions and other medium chain triglyceride solutions may also be used with more or less success in various aspects and embodiments of the present invention. As to the latter, those skilled in the art are familiar with their nature, source and/or method of preparation and can obtain or prepare them without undue research or experimentation. Miglyol 812N has monograph papers as medium chain triglycerides in the european pharmacopoeia, fractionated coconut oil in the british pharmacopoeia, and caprylic/capric triglyceride in the japanese pharmacopoeia. Other sources of medium chain triglycerides include coconut oil, palm kernel oil, and butter.

"short chain triglycerides" are triglyceride compositions containing fatty acids less than 8 linear carbon atoms in length.

A "long chain triglyceride" is a triglyceride composition containing fatty acids greater than 12 linear carbon atoms in length. Common sources for them are vegetable oils, such as soybean oil, which typically contain 55-60% linoleic acid (9, 12-octadecadienoic acid), 22% oleic acid (cis-9-octadecenoic acid), and small amounts of palmitic and stearic acids.

The terms "short", "medium" and "long" may also be used for individual fatty acids, in which case the definition includes less than 8 straight chain carbon atoms, 8-12 straight chain carbon atoms and more than 12 straight chain carbon atoms, respectively.

"emulsifier" is synonymous with "surfactant" and includes, but is not limited to, phospholipids, such as lecithin. "lecithin" is a naturally occurring mixture of the diglycerides of stearic, palmitic and oleic acids linked to choline ester of phosphoric acid. The term surfactant or emulsifier also includes phosphatidylcholine, and such compounds are well known. A preferred emulsifier for use in the present invention is soy Lecithin, such as Phospholipon 90G available from American Lecithin Company (Oxford, CT, USA). Phospholipon 90G has been used earlier in parenteral nutrition products, such as at a concentration of about 1.2% in Intralipid *, about 1% in Doxil * (doxorubicin), about 2% in Ambisome * (amphotericin B), and about 1.2% in Propofol *. For the latter, see, for example, U.S. patent 6,140,374. The surfactant/emulsifier is typically present in a concentration of about 0.5-25% w/v based on the amount of water and/or other components in which the surfactant is dissolved. The surfactant concentration is preferably from about 0.5 to about 10% w/v, most preferably from about 1 to about 8% w/v.

Examples of the anionic surfactant include sodium lauryl sulfate, triethanolamine lauryl sulfate, sodium polyoxyethylene lauryl ether sulfate, sodium polyoxyethylene nonylphenyl ether sulfate, triethanolamine polyoxyethylene lauryl ether sulfate, sodium cocoyl sarcosinate, sodium N-cocoyl methyltaurate, sodium polyoxyethylene (coconut) alkyl ether sulfate, sodium diether hexyl sulfosuccinate, sodium alpha-olefin sulfonate, sodium lauryl phosphate, sodium polyoxyethylene lauryl ether phosphate, and perfluoro alkyl carboxylate (manufactured by Daikin Industries ltd., under the trade name uni ne dids-101 and 102).

Examples of cationic surfactants include dialkyl (C12-C22) dimethyl ammonium chloride, alkyl (coconut) dimethyl benzyl ammonium chloride, octadecyl amine acetate, tetradecylamine acetate, tallow alkyl propylenediamine imine acetate, octadecyl trimethyl ammonium chloride, alkyl (tallow) trimethyl ammonium chloride, dodecyl trimethyl ammonium chloride, alkyl (coconut) trimethyl ammonium chloride, hexadecyl trimethyl ammonium chloride, biphenyl trimethyl ammonium chloride, alkyl (tallow) -imidazoline quaternary salts, tetradecyl methyl benzyl ammonium chloride, octadecyl dimethyl ammonium chloride, dioleyl dimethyl ammonium chloride, polyoxyethylene dodecyl monomethyl ammonium chloride, polyoxyethylene alkyl (C12-C22) dimethyl ammonium chloride, alkyl (coconut) dimethyl ammonium chloride, octadecyl trimethyl ammonium chloride, dodecyl trimethyl ammonium chloride, and polyoxyethylene alkyl (C) trimethyl ammonium chloride₁₂-C₂₂) Benzyl ammonium chloride, polyoxyethylene lauryl monomethyl ammonium chloride, 1-hydroxyethyl-2-alkyl (tallow) -imidazoline quaternary salt, and silicone cationic surfactant containing siloxane groups as hydrophobic groups, fluorine-containing cationic surfactant containing fluoroalkyl groups as hydrophobic groups (manufactured by Daikin Industries Ltd under the trade name unitine DS-202).

Examples of the nonionic surfactant include polyoxyethylene lauryl ether, polyoxyethylene tridecyl ether, polyoxyethylene cetyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene stearyl ether, polyoxyethylene oleyl ether, polyoxyethylene nonylphenyl ether, polyoxyethylene octylphenyl ether, polyoxyethylene monolaurate, polyoxyethylene monostearate, polyoxyethylene monooleate, sorbitan monolaurate, sorbitan monostearate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene polyoxypropylene block polymer, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyglycerin fatty acid esters, polyether-modified Silicone oils (manufactured by Toray dow corning Silicone co., Ltd., trade names SH3746, SH3748, SH3749, and SH3771), perfluoroalkyl ethylene oxide adducts (manufactured by Daikin Industries Ltd, trade names unitine DS-401 and DS-403), fluoroalkyl ethylene oxide adducts (manufactured by Daikin Industries Ltd, trade names unitine DS-406), and perfluoroalkyl oligomers (manufactured by Daikin Industries Ltd, trade names unitine DS-451).

By "physiologically acceptable carrier" is meant a carrier or diluent that does not cause significant irritation to a living being and does not diminish the biological activity and properties of the administered compound.

"excipient" refers to a substance added to a pharmaceutical composition that can further facilitate administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose and cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. These may also be physiologically acceptable carriers, as described above, for example sucrose. Further within the definition of excipient are fillers. "bulking agents" generally provide mechanical support to lyophilic formulations by allowing the matrix of the dried formulation to retain its conformation. Preferably a sugar. Sugars as used herein include, but are not limited to, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Specific examples include, but are not limited to, fructose, glucose, mannose, trehalose, sorbose, xylose, maltose, lactose, sucrose, dextrose, and dextran. Sugars also include sugar alcohols such as mannitol, sorbitol, inositol, galactitol, xylitol, and arabitol. Mixtures of sugars may also be used according to the invention. Various bulking agents, such as glycerol, sugars, sugar alcohols, and mono-and disaccharides, may also function as isotonicity agents. Preferably, the bulking agent is chemically inert to the drug and has no or very limited deleterious side effects or toxicity under the conditions of use. In addition to the bulking agent carrier, other carriers may or may not fulfill the purpose of the bulking agent, including, for example, adjuvants and diluents as are known and readily available in the art.

One preferred bulking agent of the present invention is sucrose. Without being bound by any theory, it is believed that sucrose forms an amorphous glass upon freezing and subsequent lyophilization, thereby enhancing the possible stability of the formulation by forming dispersed oil droplets containing the active ingredient in the hard glass. Stability may also be enhanced because the sugar replaces the water lost upon lyophilization. Instead of water molecules, sugar molecules are bound to the phospholipid at the interface via hydrogen bonds. Other fillers that have these characteristics and that can be substituted include, but are not limited to, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

The phrase "reducing or avoiding the amount of central nervous system effects mediated by medium chain triglycerides" in the claims can be determined empirically by one of ordinary skill without undue experimentation using the starting points described herein.

Emulsification action

Emulsions comprising an oil phase and an aqueous phase are well known in the art as carriers for therapeutically effective ingredients or as sources of parenteral nutrition. The emulsion may be oil-in-water or water-in-oil. As is the case currently, if the therapeutic ingredient is particularly soluble in the oil phase, the oil-in-water type is a preferred embodiment. Simple emulsions are thermodynamically unstable systems because of the separation of the oil and water phases (coalescence of the oil droplets). The key to reducing the coalescence process to a slight level is the addition of an emulsifier to the emulsion.

To prevent or minimize oxidative degradation or lipid peroxidation, in addition to or as an alternative to deoxygenation (e.g., placing the formulation in an inert gas such as nitrogen and argon, and/or using a light-resistant container), for example, α -tocopherol and butylated benzyl alcohol, and preservatives such as edetate, may be added.

Emulsification may be achieved by a variety of techniques well known in the art, such as mechanical mixing, homogenization (e.g., using polytron or Gaulin high energy devices), vortexing, and sonication. Sonication can be carried out using a water bath type or probe type apparatus. Microfluidizers are available, for example, from Microfluidics corp., Newton, mass, as further described in U.S. patent No. 4,533,254, and utilize pressure assistance to pass through a stenotic orifice. Pressure assisted extrusion may also be used to pass through a variety of commercially available polycarbonate membranes. Low voltage devices may also be used. These high and low pressure devices may be used to select and/or adjust the size of the vesicles.

The bacteria were removed by filtration. Filtration may include pre-filtration through a larger diameter filter, such as a 0.45 micron filter, and then re-permeation through a smaller filter, such as a 0.2 micron filter. A preferred filter medium is cellulose acetate (Sartorius-Sartobran)^TM)。

Freeze-drying

Lyophilization is the removal or substantial removal of liquid from a sample, for example by sublimation, as described above under the section entitled "removal of solvent".

Characterization and assessment of physical and chemical stability

Phospholipids and their degradation products can be extracted from the emulsion and assayed. The liquid mixture is then separated using a two-dimensional thin-layer chromatography (TLC) system or a High Performance Liquid Chromatography (HPLC) system. In TLC, spots corresponding to individual components can be taken for determination of the amount of phosphorus. The total amount of phosphorus in the sample can be quantified, as measured by the intensity of the blue color formed relative to water at 825nm with a spectrophotometer. In HPLC, Phosphatidylcholine (PC) and Phosphatidylglycerol (PG) can be separated and quantified accurately. The lipids were detected in the region 203-205 nm. In the 200nm wavelength region of the ultraviolet spectrum, unsaturated fatty acids exhibit high absorbance, while saturated fatty acids exhibit low absorbance. As an example, Vemuri and Rhodes, supra, describe the separation of PC and PG in egg yolk in Licrosorb Diol and Licrosorb S1-60. The mobile phase used for the separation was acetonitrile-methanol containing 1% hexane-water (74: 16: 10 v/v/v). After 8 minutes, the separation of PG from PC was observed. The retention times were approximately 1.1 and 3.2 minutes, respectively.

The appearance, average droplet diameter, and size distribution of the emulsion are important parameters for observation and maintenance. These parameters can be evaluated in a number of ways. For example, dynamic light scattering and electron microscopy techniques may be employed. See, e.g., Szoka and papahahahahadjoulos, annu.rev.biophysis.bioenng, 9: 467-508(1980). In particular, morphological characterization can be performed using freeze fracture electron microscopy. A less powerful optical microscope may also be used.

The emulsion droplet size distribution can be measured, for example, using a particle size distribution analyzer such as CAPA-500 manufactured by Horiba Limited (Ann Arbor, MI, USA), a Coulter sizer (Beckman Coulter Inc., Brea, Calif., USA), or a Zetasizer (Malvern Instruments, Southborough, MA, USA).

Stability testing using HPLC

Similar to the above-described method for the lipid component of an emulsion, the chemical stability of a therapeutically active ingredient, such as 17-AAG, can also be assessed by HPLC after extraction of the emulsion. A special assay can be developed to separate the therapeutically active ansamycin from its degradation products. The extent of degradation can be assessed by a decrease in the HPLC peak signal associated with the therapeutically active ansamycin and/or by an increase in the HPLC peak signal associated with the degradation product. Ansamycin readily and clearly detected a maximum absorbance at 345nm compared to the other components in the emulsion component.

Formulation and mode of administration

Although intravenous administration is preferred in various aspects and embodiments of the present invention, it will be understood by those skilled in the art that the method may be modified or readily adapted for other modes of administration, such as oral, aerosol, parenteral, subcutaneous, intramuscular, intraperitoneal, rectal, vaginal, intratumoral, or peritumoral administration. Much of the following discussion is known to those skilled in the art, but is still provided as background to illustrate other possibilities of the present invention. It is to be understood that the following discussion will have overlapping portions.

The pharmaceutical compositions may be prepared by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutically acceptable compositions may be formulated using conventional methods using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The appropriate formulation method will depend on the route of administration chosen. Some excipients and their use in the preparation of formulations have been described. Other are known in the art, as described in PCT/US99/30631, Remmington's Pharmaceutical Sciences, MeadePlubricing Co., Easton, PA (latest edition) and Goodman and Gilman's the Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y. (latest edition).

For injection, the drug may be formulated in aqueous solution, preferably in a physiologically compatible buffer, such as hanks 'solution, ringer's solution, or physiological saline buffer, as are well known in the art. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The formulations of the invention described above, and their use after hydration of the lyophilized cake, are well suited for immediate or near immediate parenteral administration by injection, such as bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative, e.g., edetate. As discussed above, the pharmaceutical compositions of the present invention may be stored in an inert environment, such as an ampoule or other light-or oxygen-resistant package.

Dosage range

Phase I pharmacological study of 17-AAG in adult patients with advanced solid tumors, administered by 1 hour daily infusion for 5 days every three weeks, established a Maximum Tolerated Dose (MTD) of 40mg/m². Wilson et al, am. Soc. Clin. Oncol., Abstract, Phase IPharmacological Study of 17- (Allylamino) -17-Demethoxgegeramycin (AAG) in Adult Patients with Advanced Solid turbines (2001). In this study, the mean +/-SD values for terminal half-life, clearance and steady state capacity were determined to be 2.5+/-0.5 hours, 41.0 +/-13.5L/hour and 86.6+/-34.6L/m². When the dosage is 40 and 56mg/m²C of plasma_maxLevels were determined 1860+/-660nM and 3170+/-1310nM, respectively. Using these values as a guide, it is contemplated that useful patient dosages of the active ingredient in the formulations of the present invention will range from about 0.40mg/m²To 4000mg/m²。M²Indicating the surface area of the body. Using standard algorithm to convert mg/m²Converted to mg drug/kg body weight.

The following examples are illustrative only, and the components and steps herein are not intended to be limiting unless specifically recited in the claims. Examples 1-5 and 9 refer to commonly owned PCT application PCT/US03/10533 entitled "novel ansamycin formulations and methods for making and using the same", filed on 4/2003, to which this application claims priority.

Examples

Example 1: preparing 17-AAG; alternative 1

To 45.0g (80.4mmol) of geldanamycin dissolved in 1.45L of dry THF placed in a 2L dry flask was added 36.0mL (470mmol) of allylamine dissolved in 50mL of dry THF dropwise over 30 minutes. The reaction mixture was stirred at room temperature under nitrogen for 4 hours until TLC analysis indicated that the reaction had completed [ GDM: bright yellow: rf is 0.40; (5% MeOH-95% CHCl)₃) (ii) a 17-AAG: purple: rf 0.42 (5% MeOH-95% CHCl)₃)]. The solvent was removed by rotary evaporation and the crude product was purified at 25 ℃ in 420mL H₂Slurry in O: EtOH (90: 10) was formed, then filtered and dried at 45 ℃ for 8 hours to give 40.9g (66.4mmol) of violet crystalline 17-AAG (82.6% yield, > 98% purity as monitored by HPLC at 254 nm). The melting point was 206-212 ℃ as determined by Differential Scanning Colorimetry (DSC).¹The results obtained by H NMR and HPLC are consistent with the desired product.

Example 2: preparation of low melting point type 17-AAG

An alternative purification method is to dissolve the crude 17-AAG of example 1 in 800mL of 2-propanol (isopropanol) at 80 ℃ and then cool to room temperature. Filtration and subsequent drying at 45 ℃ for 8 hours gave 44.6g (72.36mmol) of violet crystalline 17-AAG (90% yield, > 99% purity by HPLC at 254 nm). The melting point was determined to be 147-175 ℃ by Differential Scanning Colorimetry (DSC).¹The results obtained by H NMR and HPLC are consistent with the desired product.

Example 3: preparation of the Low melting form of 17-AAG, alternative 1

An alternative purification method is to subject the product 17-AAG of example 2 to 400mL of H₂A slurry of O: EtOH (90: 10) at 25 ℃ was formed, filtered and dried at 45 ℃ for 8 hours to give 42.4g (68.6mmol) of violet crystalline 17-AAG (95% yield, > 99% purity by HPLC at 254 nm). The melting point was 147-175 ℃.¹The results obtained by H NMR and HPLC are consistent with the desired product.

Example 4: preparation of 17-AAG emulsion

The 17-AAG obtained in any of the above examples 1-3 was dissolved in ethanol. The following table shows a 4000gm batch of articles prepared according to one embodiment of the present invention. Those skilled in the art will appreciate that the procedure may be scaled up or down, the amounts of the various components may be varied, etc., and components not listed may also be added.

Components	％w/w	Grams in 4000g batch
			17-AAG	0.2	8
Miglyol 812	15	600
			Phospholipon 90G	7.5	300
Disodium EDTA, dihydrate, USP	0.005	0.2
			Sucrose, NF	15	600
Water for injection, USP	QS	QS
			0.2N NaOH	Adjusting the pH to 6.0 + -0.2	Adding according to the required amount

17-AAG (CNF-101) was weighed into a 5L polypropylene beaker. Ethanol was added at about 50 times the weight of the drug and the solution was sonicated in a water bath to disperse the drug. Miglyol 812(Sasol North America Inc; Houston, TX, USA) and Phospholipon 90G (American Lecithen Co., Oxford, CT, USA) were then added to the dispersion and the mixture was stirred on a stir plate until the solids were approximately completely dissolved. Dissolution of the solid can be aided by a sonicator bath and/or heating to about 45 ℃. The solution can be examined with an optical microscope to ensure the desired level of dissolution.

A stream of dry air or nitrogen (national drug collection) gas is passed over the liquid surface with vigorous agitation to evaporate the ethanol until the ethanol content is reduced, preferably to less than 50% w/w, more preferably less than 10% w/w, most preferably about 5% w/w or less of its original content. The solution is examined under an optical microscope equipped with polarizing filters to ensure the desired level of dissolution, preferably complete dissolution (no crystallization or precipitation).

EDTA (disodium, dihydrate, USP), sucrose and water for injection (WFI) were weighed into a 5L polypropylene beaker and stirred until the solids dissolved. The aqueous phase was then added to the oil phase and thoroughly mixed with a high speed emulsifier with an emulsion head at 5000RPM until the oil adhered to the surface "peeled off". The shear rate was then increased to 10000RPM for 2-5 minutes to obtain a homogeneous primary emulsion. Laser Light Scattering (LLS) can be used to measure the average droplet diameter and the solution can be further examined, such as under an optical microscope to determine the relative presence or absence of crystals and solids.

The pH of the emulsion was adjusted to 6.0. + -. 0.2 with 0.2N NaOH. The primary emulsion was passed through a microfluidizer model 11OS (Mieroflorisics Inc., Newton, Mass., USA) having a 75 micron emulsion action chamber (F20Y) 6-8 times at a static pressure of about 110psi (operating pressure 60-95psi) until the average droplet diameter was ≤ 190 nm. The progress of each pass can be assessed by LLS. The solution can be further examined under a light microscope for the presence of crystals using polarized light.

The emulsion was then passed through a 0.45 micron Gelman micro-capsule filter (Pall Corp., East Hills, NY, USA) followed by a 0.2 micron sterile Sartorius Sartobran P capsule filter (500cm2) (Sartorius AG, Goettingen, Germany) in a laminar flow hood hyperstatic table. Pressures of up to 60PSI were used to maintain smooth and continuous flow. The filtrate was collected into one or more polypropylene bottles and immediately placed in a-20 ℃ freezer. Aliquots of 1ml can be left to allow detection by Laser Light Scattering (LLS) and/or High Performance Liquid Chromatography (HPLC).

Example 5: alternative 17-AAG emulsified formulations and methods of making

When ethanol is used to facilitate the dissolution of 17-AAG into the oil phase of the emulsion, it is most common to first dissolve 17-AAG in ethanol using sonication, followed by the addition of an emulsifier and medium chain triglycerides to the solution. Dissolution of all components was then achieved using sonication and stirring.

Alternatively, 17-AAG can be added to the oil phase of the solution without the use of ethanol, by a process comprising: an emulsifier preformed in a triglyceride solution, such as Phospholipon in Miglyol * 812, is heated, preferably to 65 ℃ or higher, a drug, such as 17-AAG, is added thereto, and mixed by, for example, stirring and/or sonication. It has been found that the lower melting form of 17-AAG, which is easier to prepare in example 2 by crystallizing 17-AAG in isopropanol rather than in ethanol, can be dissolved into Phospholipon in Miglyol solution at room temperature. See commonly owned PCT/US01/29715 entitled "method of making.. -. [17-AAG ] and other ansamycins" filed on 9/18/2002.

The products of examples 5 and 6 were both light purple milky emulsions with oil droplets having an average diameter of about 200nm or less. The oil droplets are stable in size when stored at 20 deg.C, 2-8 deg.C or room temperature for more than 2 months. Concentrations of up to 3mg/ml are achieved overall, whereas in the oil phase only, concentrations of up to 20-30mg/ml are achieved. If stored at 40 ℃, degradation of 17-AAG can be observed after about two weeks.

The mixed solvent solution of the drug was subjected to vacuum evaporation of the ethanol component to form a Miglyol solution of 17-AAG. Emulsification may be achieved by mechanical mixing, ultrasonic irradiation treatment, and finally by microfluidizer, however it will be appreciated that the terms "emulsification" and "emulsification" are not limited to these treatments and that other emulsification techniques exist and may be used alternatively or in combination with one or more of the above techniques.

Example 6: adding long chain triglycerides

Variations of the above method include the addition of long chain triglycerides, such as in the form of soybean oil. As shown in FIG. 1 for 17-AGG, a source of long chain triglycerides (soybean oil) was mixed with Miglyol 812N (a source of medium chain triglycerides) and an emulsifier (Phospholipon 90G (PL90G)) in a w/w ratio of 16.7% to 50.0% to 33.3%. Homogenization was performed until PL90G was completely dissolved (about 20,000rpm for about 20 minutes). Then 1% w/w 17-AAG is added, which homogenizes/dissolves for about 5 minutes at about 20,000 rpm. The above materials constitute the "oil phase". The oil phase (1 part) was then slowly added to 3.6 parts of an aqueous phase (9.375% w/w sucrose, 0.0063% w/w EDTA in sterile water for injection) homogenized at about 12,000-15,000 rpm. The resulting mixture is adjusted to pH 6.0. + -. 0.2 with sodium hydroxide and/or hydrochloric acid if necessary. The "primary" emulsion was then microfluidised by passage through an F12Y chamber and filter sterilised with a 0.2 μm Polyethersulphone (PES) filter.

By adopting the method, the following two preparations are prepared:

component (w/w)	Preparation 1	Preparation 2
			17-AAG Soybean oil Phospholipon 90G (Soybean lecithin) Miglyol 812N sucrose ethylenediaminetetraacetic acid sodium salt sterile water	2mg/ml3.3％6.6％9.9％7.5％0.005％72.5％	2mg/ml---7.5％15％10％0.005％67.3％

Example 7: freeze-drying

Lyophilization of the emulsions of examples 5 and 6 can be accomplished according to protocols similar to those in the following table.

Initial temperature (. degree. C.)	End temperature (. degree. C.)	Pressure (mTorr)	Operation of
				25	-40	Ambient pressure	Changing the temperature at 1 deg.C/min
-40	-40	Ambient pressure	Keeping for 60 minutes
				-40	-40	50	Condensing at-60 deg.C to-80 deg.C
-40	-28	50	Changing the temperature at 1 deg.C/min
				-28	-28	50	Keeping for 7200 min
-28	30	50	Changing the temperature at 1 deg.C/min
				30	30	50	Keeping for 300 minutes
Complete the process
				N at about 0.9atm2Bottle with lower stopper

The stability profile of the lyophilized 17-AAG emulsion after storage at 2-8 ℃ and reconstitution is as follows.

Detection of	Time (week)
					0	2	4	10
	Determination (% initial)	100	98.5	97.7					97.0
Purity (area%)					98.8	97.2	97.8	97.8
	Droplet diameter (μm)	0.187	0.200	0.197					0.190

Example 8: long chain triglycerides to inhibit lethargy

Miglyol 812N produces sedation upon rapid administration due to metabolic release of caprylate. Sedation was observed during intravenous infusion of 17-AAG emulsion (Miglyol 812N oil) into rats at infusion rates greater than 1.1gm total lipid/Kg/hr. See figure 2. Sedation was also observed in dogs infused with the 17-AAG emulsion formulation intravenously at a rate greater than 1.13gm total lipid/Kg/hr. To counter this effect, soybean oil was added as described above to compete with the in vivo metabolism of Miglyol 812N in order to reduce the fatty acid octanoates produced during intravenous infusion. For the soybean oil/Miglyol 812NCF237 emulsion, no significant sedation was observed in rats at infusion rates up to about 40gm total lipid/kg/hr. Thus, the combination of soybean oil and Miglyol 812N greatly improved the tolerance of the CF237 emulsified formulation to sedation. Similarly, no sedation nor intravenous irritation was observed in monkeys administered a six-fold dose of the CF237 emulsified formulation, i.e., 12mL of formulation per kg/hr of intravenous infusion.

Example 9: preparation of other ansamycins for similar formulations

Ansamycins other than 17-AAG

Essentially all ansamycins can be substituted for 17-AAG and formulated as described in the examples above. A variety of such ansamycins and their preparation are described in detail in PCT/US 03/04283. The preparation of two of these is described below.

Compound 563: 17- (benzoyl) -aminogeldanamycin. A solution of 17-aminogeldanamycin (1mmol) in EtOAc was taken over Na₂S₂O₄(0.1M, 300ml) was treated at room temperature. After 2 hours, the aqueous layer was extracted twice with EtOAc and the combined organic layers were in Na₂SO₄Dried above and concentrated under reduced pressure to obtain 18, 21-dihydro-17-aminogeldanamycin as a yellow solid. The latter was dissolved in anhydrous THF and transferred via cannula to a mixture of benzoyl chloride (1.1mmol) and MS4 * (1.2 g). After 2 hours, EtN (i-Pr)₂(2.5mmol) was added to the reaction mixture. After stirring overnight, the reaction mixture was filtered and concentrated under reduced pressure. Then water was added to the residue, extracted three times with EtOAc and the combined organic layers were in Na₂SO₄Drying and concentration under reduced pressure gave crude product which was purified by flash chromatography to give 17- (benzoyl) -aminogeldanamycin. At CH of 80: 15: 5₂Cl₂EtOAc: MeOH, Rf 0.50. Mp 218-. 1H NMR (CDCl)₃)0.94(t，6H)，1.70(br s，2H)，1.79(br s，4H)，2.03(s，3H)，2.56(dd，1H)，2.64(dd，1H)，2.76-2.79(m，1H)，3.33(br s，7H)，3.44-3.46(m，1H)，4.325(d，1H)，5.16(s，1H)，5.77(d，1H)，5.91(t，1H)，6.57(t，1H)，6.94(d，1H)，7.48(s，1H)，7.52(t，2H)，7.62(t，1H)，7.91(d，2H)，8.47(s，1H)，8.77(s，1H)。

Compound 237: a dimer is provided. In N₂3, 3-diamino-dipropylamine (1.32g, 9.1mmol) was added dropwise to a solution of geldanamycin (10g, 17.83mmol) in DESO (200ml) placed in a flame-dried flask and stirred at room temperature. After 12 hours the reaction mixture was diluted with water. The reaction formed a precipitate and was filtered to obtain the crude product. The crude product was chromatographed on silica (5% CH)₃OH/CH₂Cl₂) Chromatography gave the dimer as a purple solid. Purification by flash chromatography (silica gel) gives the pure product in purple; yield: 93 percent; mp 165 ℃; 1H NMR (CDCl)₃)0.97(d，J＝6.6Hz，6H，2CH3)，1.0(d，J＝6.6Hz，6H，2CH3)，1.72(m，4H，2CH2)，1.78(m，4H，2CH2)，1.80(s，6H，2CH3)，1.85(m，2H，2CH)，2.0(s，6H，2CH3)，2.4(dd，J＝11Hz，2H，2CH)，2.67(d，J＝15Hz，2H，2CH)，2.63(t，J＝10Hz，2H，2CH)，2.78(t，J＝6.5Hz，4H，2CH2)，3.26(s，6H，2OCH3)，3.38(s，6H，2OCH3)，3.40(m，2H，2CH)，3.60(m，4H，2CH2)，3.75(m，2H，2CH)，4.60(d，J＝10Hz，2H，2CH)，4.65(Bs，2H，2OH)，4.80(Bs，4H，2NH2)，5.19(s，2H，2CH)，5.83(t，J＝15Hz，2H，2CH＝)，5.89(d，J＝10Hz，2H，2CH＝)，6.58(t，J＝15Hz，2H，2CH＝)，6.94(d，J＝10Hz，2H，2CH＝)，7.17(m，2H，2NH)，7.24(s，2H，2CH＝)，9.20(s，2H，2NH)；MS(m/z)1189(M+H)。

The corresponding hydrochloride salt was prepared as follows: a solution of HCl in EtOH (5ml, 0.123N) was added to a solution of Compound #237 (1 gm prepared above) in THF (15ml) and EtOH (50ml) at room temperature. The reaction mixture was stirred for 10 minutes. The salt precipitated out, was filtered off and washed with copious amounts of EtOH and dried in vacuo.

* * *

The skilled person will appreciate that the parameters in the above examples and tables can be adjusted according to the conditions employed, and according to whether the formulation method and the amounts of materials used are scaled up or down or changed relative to each other and to what extent.

The above examples are not intended to be limiting and merely represent various aspects and embodiments of the present invention. All documents cited herein are indicative of the level of skill in the art to which this invention pertains. Although none of the cited documents is admitted to be prior art, the contents of each of the cited documents are incorporated by reference herein to the same extent as if each document were incorporated by reference in its entirety and individually. In the event that a clear definition herein conflicts with a definition that exists in the prior art or in the priority text incorporated herein by reference, the clear definition in this application controls.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions are intended to illustrate preferred embodiments, to be exemplary, and not to limit the scope of the invention. Certain modifications and other uses will occur to those skilled in the art and are encompassed within the scope of the invention as defined by the scope of the claims.

The reagents are either commercially available, e.g., from Sigma-Aldrich, or can be readily prepared by conventional methods well known to those skilled in the art without undue experimentation.

It will be apparent to those skilled in the art that various substitutions and modifications may be made to the present invention without departing from the spirit and scope of the invention. Accordingly, such other embodiments are within the scope of the invention and the following claims.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. For example, in all cases herein, the terms "comprising," "consisting essentially of," and "consisting of" may each be substituted for one of the other two terms, and each term has a different meaning in patent law. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. While those skilled in the art will recognize that many variations are possible within the scope of the invention claimed. It will thus be appreciated that while the present invention has been particularly disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the claims.

In addition, where features or aspects of the invention are described in terms of Markush groups or alternatively other groups, those skilled in the art will recognize that the invention can also be described in terms of each member or subgroup of members of the Markush group or other group, if desired to be excluded by the definition of each member.

Other embodiments are within the scope of the following claims.

Claims (32)

1. A pharmaceutical composition comprising:

an HSP90 inhibitor, optionally ansamycin;

an emulsifier; and

an oil comprising both medium chain triglycerides and long chain triglycerides.

2. The pharmaceutical composition of claim 1, wherein said HSP90 inhibitor is ansamycin.

3. The pharmaceutical composition of claim 2, wherein said ansamycin is geldanamycin or a geldanamycin derivative.

4. The pharmaceutical composition of claim 3, wherein said geldanamycin derivative is selected from the group consisting of 17-AAG and DMAG.

5. The pharmaceutical composition of claim 1, wherein the w/w ratio of the medium chain triglycerides to the long chain triglycerides is from 10: 1 to 0.01: 10.

6. A pharmaceutical composition according to claim 1 which is an oil-in-water emulsion having a lipid phase and an aqueous phase, wherein the lipid phase comprises from 5 to 30% w/w of the total weight.

7. The pharmaceutical composition of claim 1, wherein the phospholipid is present in the form of lecithin.

8. The pharmaceutical composition of claim 4, wherein the amount of lecithin is 3-10% w/w of the oil-in-water emulsion.

9. The pharmaceutical composition of claim 1, comprising 3-10% w/w phospholipid and 5-20% w/w oil.

10. The pharmaceutical composition of claim 1, comprising no more than 10% w/w long chain triglycerides.

11. The pharmaceutical composition of claim 10, further comprising less than or equal to 7% w/w long chain triglycerides.

12. The pharmaceutical composition of claim 10, wherein the long chain triglyceride comprises a fatty acid ester consisting of 16-18 linear carbon units, the triglyceride optionally being selected from one or more of linoleic acid, oleic acid, palmitic acid and stearic acid.

13. The pharmaceutical composition of claim 1, wherein the phospholipid is in the form of lecithin, optionally soy or egg lecithin.

14. The pharmaceutical composition of claim 4, wherein said ansamycin is a 17-AAG low melting isomer having a melting point no greater than 200 ℃.

15. The pharmaceutical composition of claim 14 wherein the lower melting isomer is 17-AAG having a melting point of 147 ℃, -175 ℃.

16. The pharmaceutical composition of claim 1, wherein said oil comprises one or more natural oils selected from the group consisting of soybean oil, sesame oil, safflower oil, and corn oil.

17. The pharmaceutical composition of claim 1, which is an emulsion, optionally lyophilized.

18. The pharmaceutical composition of claim 1, comprising one or more of water, a preservative, an anti-freezing agent, a buffer, a chelating agent, and a tonicity adjusting agent.

19. The pharmaceutical composition of claim 1 comprising Miglyol 812N.

20. The pharmaceutical composition of any one of claims 1-19, comprising 0.5mg/ml to 4mg/ml of 17-AAG.

21. The pharmaceutical composition of any one of claims 1-19, comprising 0.05% w/w to 0.4% w/w of 17-AAG.

22. The pharmaceutical composition of claim 1, comprising the following components: 2mg/ml 17-AAG, 3.3% soybean oil, 6.6% lecithin, 9.9% Miglyol 812N, 7.5% sucrose, and water.

23. The pharmaceutical composition of claim 1, comprising the following components: 2mg/ml 17-AAG, 7.5% lecithin, 15% Miglyol 812N, 10% sucrose, and water.

24. The pharmaceutical composition of claim 22 or 23, further comprising sodium edetate.

25. The pharmaceutical composition of claim 24, wherein said sodium edetate is 0.005% w/w.

26. The pharmaceutical composition of claim 1, wherein the long chain triglycerides are present in an amount sufficient to reduce or prevent the occurrence of medium chain triglyceride-mediated central nervous system effects.

27. The pharmaceutical composition of claim 22, wherein the central nervous system effect is selected from one or more of lethargy, nausea, drowsiness, and EEG changes.

28. The pharmaceutical composition of claim 1, further comprising a short chain triglyceride.

29. A method of reducing the occurrence of central nervous system effects mediated by medium chain triglycerides in a patient, wherein the patient receives a pharmaceutical formulation comprising medium chain triglycerides as a component of the formulation, the method comprising:

a) providing a pharmaceutical formulation comprising ansamycin and medium and long chain triglycerides in an amount sufficient to reduce or prevent the occurrence of central nervous system effects mediated by medium chain fatty acids; and are

b) Administering the product of step a) to a patient.

30. The method of claim 29, wherein the central nervous system effect is selected from one or more of lethargy, nausea, drowsiness, and EEG changes.

31. The pharmaceutical composition of claim 1, which is in one or more of lyophilized, frozen, thawed, or reconstituted form.

32. The pharmaceutical composition of claim 1, which is stored in an inert environment.