HK1191253A - Compositions and methods of delivery of pharmacological agents - Google Patents

Description

Compositions and methods for delivering pharmaceutical agents

This application is a divisional application of International application PCT/US2003/038941 (national application number 200380109606.9, title of the invention: compositions and methods of delivering pharmaceutical agents) filed on 9/12/2003.

Cross reference to related patent applications

This patent application claims the benefit of U.S. provisional patent application 60/432,317, filed on 9/12/2002, U.S. provisional patent application filed on 3/12/2003 (attorney docket No. 225519), U.S. provisional patent application filed on 4/12/2003 (attorney docket No. 225549), and U.S. provisional patent application filed on 5/12/2003 (attorney docket No. 225585).

Technical Field

The present invention relates to pharmaceutical compositions for parenteral or other internal use comprising pharmaceutically active agents which have the effect of reducing certain undesirable side effects upon administration when compared to similar pharmaceutical formulations available.

Background

It is well recognized that many drugs for parenteral use, particularly those administered intravenously, cause undesirable side effects such as venous irritation, phlebitis, burning and pain upon injection, venous thrombosis, extravasation and other administration related side effects. Many of these drugs are insoluble in water and are therefore formulated with solubilizing agents, surfactants, solvents and/or emulsifiers that are irritating, allergic or toxic when administered to a patient (see, e.g., Briggs et al, anesis 37,1099(1982), and Waugh et al, am.j.hosp.pharmacists,48,1520 (1991)). Often, the free drug present in the formulation induces pain or irritation upon administration. For example, phlebitis is observed in 50% of patients who received peripheral intravenous administration of ifosfamide and vinorelbine as first-line chemotherapy for advanced non-small cell lung cancer. (see, e.g., Vallejo et al, am.J.Clin.Oncol.,19 (6)), 584-8 (1996)). In addition, vancomycin has been shown to induce side effects such as phlebitis (see, e.g., Lopes Rocha et al, braz.j.infection.dis., 6 (4)), 196-200 (2002)). The use of cisplatin, gemcitabine and SU5416 in solid tumor patients has resulted in adverse events such as deep vein thrombosis and phlebitis (see, e.g., Kuenen et al, j.clin. oncol.,20(6),1657-67 (2002)). In addition, propofol, an anesthetic, can induce pain, burning and venous irritation when injected, particularly when administered as a lecithin-stabilized fat emulsion (see, e.g., Tan et al, antathenia, 53,468-76, (1998)). Other drugs that show side effects associated with administration include, for example, taxol (see, e.g., taxol's package insert i.v.), Codarone (amiodarone hydrochloride) (see, e.g., Codarone's package insert), thyroid hormone T3 or liothyronine (commercially available as compound liothyronine), thiotepa, bleomycin, and diagnostic radiographic contrast agents.

Another problem associated with the preparation of injectable drugs, particularly water-insoluble drugs, is to ensure sterility. Sterile preparation of the pharmaceutical emulsions/dispersions can be accomplished by absolute sterilization of all components prior to preparation, followed by absolute aseptic techniques in all stages of preparation. However, these methods are time consuming and expensive. In addition, oxidation of the pharmaceutical formulation by exposure to air during manufacture or storage can result in, for example, reduced pH, drug degradation, and discoloration, thereby destabilizing the pharmaceutical formulation and/or reducing shelf life.

In order to avoid the problems associated with the side effects associated with administration of pharmaceutical formulations, alternative formulations have been attempted. For example, with respect to propofol, methods of reducing propofol-induced pain include increasing the fat content of the solvent (e.g., Long Chain Triglycerides (LCT)), premedication, pretreatment with non-steroidal drugs, local anesthesia, opioids, addition of lidocaine, addition of cyclodextrins, and microfiltration (see, e.g., Mayer et al, Anaesthessist, 45(11),1082-4(1996), Davies, et al, Anaesthesia,57,557-61(2002), Doenicke, et al, Anasth.Analg, 82,472-4(1996), Larsen et al, Anaesthessis, 50,842-5(2001), Lilley et al, Anaesthesia,51,815-8(1996), Bielen et al, Anasth.Analg, 82 (1996), and Kbbe et al, Br.J.Clin.Pharma, 653-60 (1999)). However, these formulations induce other side effects (e.g. cardiovascular complications) or lead to instability of the propofol formulation.

To overcome the problem of bacterial contamination, propofol formulations have been developed with antibacterial agents such as EDTA equivalents (e.g., edetate), pentetate, or sulfite-containing agents, or they have been formulated at lower pH (see, e.g., U.S. Pat. nos. 5,714,520, 5,731,355, 5,731,356, 6,028,108, 6,100,302, 6,147,122, 6,177,477, 6,399,087, 6,469,069, and international patent application No. WO 99/39696). However, because edetate and pentetate are metal ion chelators, they have the dangerous potential to clear essential metal ions from body cells. In addition, the addition of sulfites to pharmaceutical formulations brings about potential adverse effects to those in the pediatric population and in the general population that are sensitive to sulfur.

Thus, there remains a need for compositions and methods that reduce or eliminate the side effects associated with parenteral or in vivo administration of drugs. There is also a need for sterile pharmaceutical compositions and methods of preparing such compositions. In addition, there is a need for pharmaceutical compositions and methods that reduce or eliminate oxidation of pharmaceutical formulations to prevent drug instability.

The present invention provides such compositions and methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

Summary of The Invention

The present invention provides various embodiments of pharmaceutical compositions. One, some, or all of the features of various embodiments may be found in different embodiments of the invention and still fall within the scope of the appended claims.

The present invention provides a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein such as albumin, more preferably human serum albumin, in an amount effective to reduce one or more side effects of administration of the pharmaceutical composition to a human, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit microbial growth in the pharmaceutical composition. The invention also provides a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects of administration of the pharmaceutical composition to a human, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.

The present invention provides a method of reducing one or more side effects associated with administration of a pharmaceutical composition to a human, the method comprising (a) administering to the human a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin and deferoxamine. The invention also provides methods for inhibiting microbial growth, or inhibiting oxidation, or inhibiting microbial growth and oxidation in a pharmaceutical composition. These methods comprise preparing a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit microbial growth in the pharmaceutical composition or in an amount effective to inhibit oxidation of the pharmaceutical composition.

The invention also provides a method for enhancing transport of a pharmaceutical agent to a site of frailty, the method comprising administering to a human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the ratio of albumin to pharmaceutical agent in the pharmaceutical composition is about 18:1 or less. The invention further provides a method for enhancing binding of an agent to a cell in vitro or in vivo, the method comprising administering to the cell in vitro or in vivo a pharmaceutical composition comprising the agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the ratio of albumin to agent in the pharmaceutical composition is about 18:1 or less.

The invention also provides a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin in an amount effective to increase transport of the drug to the site of weakness in the human, and wherein the ratio of albumin to pharmaceutical agent is about 18:1 or less.

The invention further provides a method of increasing transport of an agent to a cell in vitro or in vivo by combining the agent with a protein, wherein the protein binds to a specific cell surface receptor on the cell, wherein binding of the protein-agent combination to the receptor results in transport occurring, and wherein the ratio of albumin to agent is about 18:1 or less.

Detailed Description

The present invention provides a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein such as albumin, preferably human serum albumin, in an amount effective to reduce one or more side effects of administration of the pharmaceutical composition to a human, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit microbial growth in the pharmaceutical composition. The invention also provides a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects of administration of the pharmaceutical composition to a human, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.

Any suitable agent may be used in the pharmaceutical compositions of the present invention. Suitable agents include, but are not limited to, anticancer or antineoplastic agents, antimicrotubule agents, immunosuppressive agents, anesthetics, hormones, agents for cardiovascular disorders, antiarrhythmic agents, antibiotics, antifungal agents, antihypertensive agents, antiasthmatic agents, analgesics, anti-inflammatory agents, antiarthritic agents, and vasoactive agents. The present invention is also effective with many other drug classes. More specifically, suitable agents include, but are not limited to, taxanes, (e.g., paclitaxel)(paclitaxel), and taxotere^TM(docetaxel)), epothilones (epothilones), camptothecins, colchicines, amiodarone, thyroid hormones, vasoactive peptides (e.g. vasoactive intestinal peptide), amphotericin, corticosteroids, propofol, melatonin, cyclosporine, rapamycin (sirolimus), tacrolimus, mycophenolic acid, ifosfamide, vinorelbine, vancomycin, gemcitabine, SU5416, thiotepa, bleomycin, diagnostic radiocontrast agents, and derivatives thereof. Other drugs that may be used in the compositions of the present invention are described, for example, in U.S. Pat. No. 5,916,596 and co-pending U.S. patent application No. 09/446,783, preferably the agent is propofol, paclitaxel, or docetaxel. More preferably, the agent is propofol or paclitaxel. Most preferably, the pharmaceutical agent is propofol.

Tai (traditional Chinese medicine)(paclitaxel) (Bristol-Myers Squibb) is active against ovarian, breast, lung, esophageal, and head and neck cancers. However, taxol has been shown to induce toxicity associated with drug administration, as well as significant acute and cumulative toxicities, such as myelosuppression, neutropenic fever, anaphylaxis, and peripheral neuropathy. Because paclitaxel is poorly soluble in water, cremo (r) castor oil is typically usedphor) is used as a solvent, requiring large infusion volumes and special tubing and filters. Polyoxyethylated castor oil is associated with side effects which can be severe, including allergies and other allergic reactions, which may require pretreatment with corticosteroids, antihistamines, and H2 blockers (see, e.g., Gelderblom et al, eur. j of Cancer,37, 1590-. Teddi^TM(docetaxel) is used to treat anthracycline-resistant breast cancer, but has also been shown to induce side effects of allergy and fluid retention, which may be severe. Epothilones (and their derivatives) are also typically administered in polyoxyethylated castor oil and have been shown to induce severe neutropenia, allergy and neuropathy.

Propofol (2, 6-diisopropylphenol) is a hydrophobic, water-insoluble oil widely used as a venous anesthetic to induce and maintain general anesthesia and sedation in humans and animals. Propofol is typically administered directly into the bloodstream and across the blood brain barrier. Pharmaceutical compositions comprising propofol must be sufficiently lipid soluble to pass this barrier and inhibit the relevant mechanisms of the brain. Propofol has a maximum solubility of 1.0 +/-0.02. mu.M in water at 22.5 ℃ (see, e.g., Tonner et al, Anesthesiology,77,926-931 (1992)). Thus, propofol is typically formulated as an emulsion containing solubilizers, surfactants, solvents, or as an oil-in-water emulsion (see, e.g., U.S. patents 6,150,423, 6,326,406, and 6,362,234). In addition to the active agent, the compositions of the present invention also include a pharmaceutical carrier or excipient. The choice of carrier is not necessarily critical and any carrier known in the art may be used in the composition. The choice of carrier is preferably determined in part by the particular site at which the pharmaceutical composition is to be administered and the particular method used to administer the pharmaceutical composition. Preferably, the pharmaceutically acceptable carrier comprises a protein. Any suitable protein may be used. Examples of suitable proteins include, but are not limited to, albumin, immunoglobulins including IgA, lipoproteins, apolipoprotein B, beta-2-macroglobulin, thyroglobulin, and the like. Most preferably, the pharmaceutically acceptable carrier comprises albumin, most preferably human serum albumin. Proteins suitable for the present invention, including albumin, may be of natural origin or synthetically prepared.

Human Serum Albumin (HSA) is M_r65K, consisting of 585 amino acids. HSA is the most abundant protein in plasma and accounts for 70-80% of the human plasma colloid osmotic pressure. The amino acid sequence of HSA contains a total of 17 disulfide bridges, one free thiol (Cys34), and a single tryptophan (Trp 214). Intravenous HSA solutions have been shown to be useful in the prevention and treatment of hypovolemic shock (see, e.g., Tullis, JAMA,237,355-360,460-463, (1977)) and Houser et al, Surgery, Gynecology and Obstrics, 150,811-816(1980)) along with hemocatheterism in the treatment of neonatal hypercholesterolemia (see, e.g., Finlayson, Seminars in thrombosis and hemostatis, 6,85-120, (1980)).

Human Serum Albumin (HSA) has multiple hydrophobic binding sites (a total of 8 endogenous ligands for fatty acids, HSA) and binds a wide variety of drugs, particularly neutral or negatively charged hydrophobic compounds (Goodman et al, The pharmaceutical Basis of Therapeutics, 9 th edition, McGraw-Hill New York (1996)). Two high affinity binding sites have been proposed in subdomains IIA and IIIA of HSA, which are highly extended hydrophobic vesicles with charged lysine and arginine residues near the surface to serve as attachment points for polar ligand features (see, e.g., Fehske et al, biochem. Pharmcol.,30,687-92(1981), Vorum, dan. Med. Bull.,46,379-99(1999), Kragh-Hansen, dan. Med. Bull.,1441,131-40(1990), Curry et al, Nat. struct. biol.,5,827-35(1998), Sugio et al, protein. Eng.,12,439-46(1999), He et al, Nature,358,209-15(1992), and Carter et al, Adv. protein. chem.,45,153-203 (1994)). Paclitaxel and propofol have been shown to bind HSA (see, e.g., Paal et al, Eur. J. biochem.,268(7),2187-91(2001), Purcell et al, Biochim. Biophys. acta,1478(1),61-8(2000), Altmayer et al, Arzneimitelforkung, 45,1053-6(1995), and Garrido et al, Rev. Esp. Anestestrol. Reanim.,41,308-12 (1994)). In addition, docetaxel has been shown to bind to human plasma proteins (see, e.g., Urein et al, invest. New drugs,14(2),147-51 (1996)). Thus, while not wishing to be bound to any one particular theory, it is believed that the inclusion of a protein such as albumin in the pharmaceutical compositions of the present invention results in a reduction in the side effects associated with administration of the pharmaceutical compositions due, at least in part, to the binding of human serum albumin to any free drug present in the composition.

The amount of albumin included in the pharmaceutical compositions of the invention will vary depending on the pharmaceutically active agent, other excipients, and the intended route and site of administration. Ideally, the amount of albumin included in the composition is an amount effective to reduce the side effects of one or more active pharmaceutical agents upon administration of the pharmaceutical composition of the invention to a human. Typically, the pharmaceutical composition is prepared in liquid form and then albumin is added to the solution. Preferably, the pharmaceutical composition in liquid form comprises from about 0.1% to about 25% (e.g., about 0.5%, about 5%, about 10%, about 15%, or about 20%) by weight of albumin. Most preferably, the pharmaceutical composition in liquid form comprises from about 0.5% to about 5% by weight albumin. The pharmaceutical composition may be dehydrated, for example, by lyophilization, spray drying, fluid bed drying, wet granulation, and other suitable methods known in the art. When the compositions are prepared in solid form, such as by wet granulation, fluid bed drying, and other methods known to those skilled in the art, albumin is preferably applied as a solution to the active agent and other excipients, if present. The HSA solution preferably has about 0.1 wt% to about 25 wt% (about 0.5 wt%, about 5 wt%, about 10 wt%, about 15 wt%, or about 20 wt%) of albumin.

In addition to albumin, the composition of the invention preferably also comprises deferoxamine. Deferoxamine is a natural product isolated from Streptomyces pilous and is capable of forming iron complexes. Deferoxamine mesylate, for example for injection USP, is approved by the food and drug administration as an iron chelator and is available for intramuscular, subcutaneous and intravenous administration. Deferoxamine mesylate USP is a white to off-white powder. It is readily soluble in water and its molecular weight is 656.79. The chemical name of deferoxamine mesylate is N- [5- [3- [ (5-aminopentyl) -hydroxycarbamoyl]-propanamido]Pentyl radical]-3[ [5- ((N-hydroxyacetamido) pentyl ] group]-carbamoyl radical]Propionyl hydroxamic acid monomethanesulfonate having the formula C₂₅H₄₈N₆O₈.CH₃SO₃H. As described in the examples, deferoxamine or an analogue, derivative or salt thereof (e.g. the mesylate salt) inhibits microbial growth and oxidation in the pharmaceutical composition and is believed to bind free drug in the composition. Deferoxamine has also been shown to bind phenolic compounds (see, e.g., Juven et al, J.Appl.Bacteriol.,76(6),626-31 (1994)). Paclitaxel, docetaxel, propofol, etc. or similar phenols or with phenol or phenyl substituents. Thus, it is believed that deferoxamine may bind to or reduce the amount of free drug in the pharmaceutical composition of the invention, thereby also reducing or alleviating irritation or pain on injection.

The amount of deferoxamine or its preferred salt (i.e. the mesylate salt of deferoxamine) included in the composition will depend on the active agent and other excipients. Desirably, the amount of deferoxamine, salts thereof and analogues thereof in the composition is an amount effective to inhibit microbial growth and/or inhibit oxidation. As noted above, typically the pharmaceutical compositions are prepared in liquid form, and then deferoxamine and its analogues are added in solution. Preferably, the pharmaceutical composition in liquid form comprises about 0.0001% to about 0.5% (e.g., about 0.005%, about 0.1%, or about 0.25%) by weight deferoxamine, its salt, or the like. More preferably, the composition in liquid form comprises a similar amount of the preferred deferoxamine salt, deferoxamine mesylate. More preferably, the pharmaceutical composition in liquid form comprises about 0.1% by weight of deferoxamine mesylate. When the composition is prepared in solid form, as described above, such as by wet granulation, fluid bed drying, and other methods known to those skilled in the art, it is preferred to add deferoxamine mesylate as a solution to the active agent and other excipients, if present. The deferoxamine mesylate solution preferably has from about 0.0001% to about 0.5% by weight (e.g., about 0.005%, about 0.1%, or about 0.25%) deferoxamine.

In accordance with the present invention, the pharmaceutical composition may include other agents, excipients or stabilizers to improve the performance of the composition. For example, to increase stability by increasing the negative zeta potential of the nanoparticle or nanodrop, certain negatively charged components may be added. These negatively charged components include, but are not limited to, bile salts of bile acids consisting of: glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid (glycochenodeoxycholic acid), taurochenodeoxycholic acid (taurochenodeoxycholic acid), lithocholic acid, ursodeoxycholic acid (ursodeoxycholic acid), dehydrocholic acid, and others; phospholipids, including lecithin (egg yolk) based phospholipids, including the following phosphatidylcholines: palmitoyl oleoyl phosphatidylcholine, palmitoyl linoleoyl phosphatidylcholine, stearoyl oleoyl phosphatidylcholine, stearoyl arachidoyl phosphatidylcholine, and dipalmitoyl phosphatidylcholine. Other phospholipids include L- α -dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), distearoyl phosphatidylcholine (DSPC), Hydrogenated Soy Phosphatidylcholine (HSPC), D- α -phosphatidylcholine, β -acetyl- γ -O-hexadecyl, L- α -phosphatidylcholine, β -acetyl- γ -O-hexadecyl, DL- α -phosphatidylcholine, β -acetyl- γ -O-hexadecyl, L- α -phosphatidylcholine, β -acetyl- γ -O-octadecyl, L- α -phosphatidylcholine, β -arachidoyl- γ -O-hexadecyl, L- α -phosphatidylcholine, beta-acetyl-gamma-O- (octadec-9-cis-enyl), D-alpha-phosphatidylcholine, beta-arachidonoyl-gamma-O-palmitoyl, 3-sn-phosphatidylcholine, 2-arachidonoyl-1-stearoyl, L-alpha-phosphatidylcholine, beta-arachidonoyl-gamma-stearoyl, L-alpha-phosphatidylcholine, diarachidonoyl, L-alpha-phosphatidylcholine, di (behenoyl), L-alpha-phosphatidylcholine, beta- (cis-8, 11, 14-eicosatrienoyl) -gamma-O-hexadecyl, L-alpha-phosphatidylcholine, beta-oleoyl-gamma-myristoyl, l- α -phosphatidylcholine, β - (pyrene-1-yl) decanoyl- γ -palmitoyl, 3-sn-phosphatidyl-N, N-dimethylethanolamine, 1, 2-dipalmitoyl, L- α -phosphatidylethanolamine, di (heptadecanoyl), 3-sn-phosphatidylethanolamine, 1, 2-dilauroyl, 3-sn-phosphatidylethanolamine, 1, 2-dimyristoyl, 3-sn-phosphatidylethanolamine, 1, 2-dioleoyl, 3-sn-phosphatidylethanolamine, 1, 2-dipalmitoyl, L- α -phosphatidylethanolamine, dipalmitoyl, N-danoyl, L- α -phosphatidylethanolamine, dipalmitoyl, N, N-dimethyl, L- α -dimyristoyl phosphatidylglycerol (sodium salt) (DMPG), dipalmitoyl phosphatidylglycerol (sodium salt) (DPPG), distearoyl phosphatidylglycerol (sodium salt) (DSPG), N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine sodium (MPEG-DSPE), L- α -phosphatidic acid, didecanoyl sodium salt, L- α -phosphatidic acid, di (heptadecacarbonyl) sodium salt, 3-sn-phosphatidic acid, 1, 2-dimyristoyl sodium salt, L- α -phosphatidic acid, dioctanoyl sodium salt, L- α -phosphatidic acid, dioleoyl sodium salt, L- α -phosphatidic acid, dipalmitoyl sodium salt, l- α -phosphatidyl-DL-glycerol, dimyristoyl sodium salt, L- α -phosphatidyl-DL-glycerol, dioleoyl sodium salt, L- α -phosphatidyl-DL-glycerol, dipalmitoyl ammonium salt, L- α -phosphatidyl-DL-glycerol, distearoyl ammonium salt, L- α -phosphatidyl-DL-glycerol, β -oleoyl- γ -palmitoyl ammonium salt, L- α -phosphatidylinositol sodium salt, L- α -phosphatidyl-L-serine, dioleoyl sodium salt, L- α -phosphatidyl-L-serine, and dipalmitoyl sodium salt. Surfactants of negatively charged emulsifiers are also suitable as additives, for example cholesteryl sodium sulfate and the like.

The pharmaceutical agent (e.g., propofol) can be used alone or dissolved in a water-immiscible solvent. A wide variety of water immiscible solvents may be used such as soybean oil, safflower oil, cottonseed oil, corn oil, sunflower oil, peanut oil, castor oil, olive oil. Preferred oils are vegetable oils, with soybean oil being most preferred. Soybean oil may be used in the range of 1% to 10% by weight of the composition. Preferably, soybean oil is present in the composition in an amount of about 3% by weight.

The pharmaceutical compositions of the present invention may be stabilized with a pharmaceutically acceptable surfactant. The term "surfactant" as used herein refers to the surface active groups of an amphiphilic molecule. The surfactant may be anionic, cationic, nonionic and zwitterionic. Any suitable surfactant may be included in the pharmaceutical compositions of the present invention. Suitable surfactants include non-ionic surfactants such as phospholipids, polyoxyethylene sorbitan esters, and tocopheryl polyoxyethylene succinates. Preferred surfactants are lecithin, tween 80 and vitamin E-t d-alpha-tocopheryl polyoxyethylene-1000 succinate (TPGS). For formulations containing soybean oil, lecithin is preferred and for formulations containing 3% soybean oil no more than 1.2%, preferably 1.1% by weight of the composition. For formulations without soybean oil, tween 80 or vitamin E-TPGS are preferred surfactants. Typically, 0.1-1.5 wt% tween 80 or 0.5-4 wt% vitamin E-TPGS is suitable. Preferably, 1.5 wt% tween 80 or 1 wt% vitamin E-TPGS is used. Examples of other suitable surfactants are described, for example, in Becher, Emulsions: principles and Practice, Robert E.Krieger Publishing, Malabar, Fla. (1965).

There is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention (see, e.g., U.S. patent 5,916,596). The following formulations and methods are merely exemplary and are in no way limiting. Formulations suitable for oral administration may consist of: (a) a liquid solution, such as an effective amount of the compound dissolved in a diluent, such as water, saline or orange juice, (b) a capsule, sachet or tablet, each containing a predetermined amount of the active ingredient as a solid or particulate, (c) a suspension in a suitable liquid, and (d) a suitable emulsion. Tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms may comprise the active ingredient in a flavoring agent, typically sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, excipients such as are known in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Preferably an injectable formulation.

Formulations suitable for aerosol administration include pharmaceutical compositions of the invention, including aqueous and non-aqueous isotonic sterile solutions, which may contain antioxidants, buffers, bacteriostats and solutes, and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives, which may be formulated, alone or in combination with other suitable ingredients, into aerosol formulations for administration by inhalation. These aerosol formulations may be placed into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as medicaments for non-pressurized formulations, such as in a nebulizer or atomizer.

Other suitable formulations are possible, for example suppositories may be prepared by using various bases (bases) such as emulsifying bases or water-insoluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

In a preferred embodiment of the invention, the pharmaceutical composition is formulated to have a pH of 4.5-9.0, more preferably a pH of 5.0-8.0. The pharmaceutical composition may also be made isotonic with blood by the addition of suitable osmotic adjusting agents such as glycerol. In addition, the pharmaceutically acceptable carrier preferably also contains pyrogen-free water or water for injection, USP. Preferably, the pharmaceutical composition of the present invention is prepared as a sterile aqueous formulation, nanoparticle, oil-in-water emulsion, or water-in-oil emulsion. Most preferably, the pharmaceutical composition is an oil-in-water emulsion.

In accordance with the present invention, for pharmaceutical compositions comprising propofol, an oil-in-water emulsion can be prepared by dissolving propofol alone in a water-immiscible solvent and preparing an aqueous phase containing albumin, deferoxamine, surfactant and other water-soluble ingredients, and mixing the oil with the aqueous phase. The crude emulsion is homogenized under high pressure at pressures of 10,000 to 25,000psi and recycled for 5-20 cycles to form the desired emulsion. Preferred pressures are 15,000 to 20,000psi, more preferably 10,000 psi. The crude emulsion may be recycled for 7-15 cycles and preferably for 15 cycles. Alternatively, a separate passage through the homogenizer may be used.

Preferably, the pharmaceutical compositions of the present invention may contain particles or droplets having a size of less than about 200 nanometers (nm). For example, in the case of paclitaxel, docetaxel, rapamycin, cyclosporine, propofol, and others, the mean size of these dispersions is less than 200 nm.

The invention further provides methods of reducing one or more side effects associated with administration of a pharmaceutical composition to a human. The method comprises administering to a human a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin and deferoxamine. The descriptions of the pharmaceutical compositions, agents and pharmaceutically acceptable carriers, and components thereof, set forth above in connection with the pharmaceutical compositions of the invention also apply to those same aspects of the methods of the invention.

In the context of the present invention, the dosage of a pharmaceutical composition of the present invention administered to a human will vary with the particular pharmaceutical composition, the method of administration, and the particular site being treated. The dose should be sufficient to achieve a desired response, such as a therapeutic or prophylactic response to a particular disease, or an anesthetic response when the agent is an anesthetic, such as propofol, over a desired time frame.

Although any suitable means of administering a pharmaceutical composition to a human may be used in the context of the present invention, it is preferred that the pharmaceutical composition of the present invention is administered to a human by intravenous administration, intraarterial administration, intrapulmonary administration, oral administration, inhalation, intravesical administration, intramuscular administration, intratracheal administration, subcutaneous administration, intraocular administration, intrathecal administration or transdermal administration. For example, the pharmaceutical compositions of the present invention may be administered by inhalation to treat respiratory disorders. Side effects associated with inhalation of the pharmaceutical compositions of the present invention are minimal because albumin is a natural component of the lining and secretions of the respiratory tract. The compositions of the present invention may be used to treat respiratory disorders such as pulmonary fibrosis, bronchiolitis obliterans, lung cancer, bronchoalveolar carcinoma, and the like.

The methods of the invention result in a reduction in one or more side effects associated with administration of the pharmaceutical composition to a human. Such side effects include, for example, myelosuppression, neurotoxicity, allergy, inflammation, venous irritation, phlebitis, pain, skin irritation, and combinations thereof. These side effects are, however, merely exemplary and other side effects or combinations of side effects associated with various agents may be reduced or avoided by using the novel compositions and methods of the present invention.

The invention further provides methods of inhibiting microbial growth in a pharmaceutical composition. "inhibiting microbial growth" refers to the complete elimination of a microorganism from a pharmaceutical composition, or the reduction of the amount or rate of microbial growth in a pharmaceutical composition. The method comprises preparing a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises deferoxamine, salts thereof, analogs thereof, and combinations thereof, in an amount effective to inhibit microbial growth in the pharmaceutical composition. In addition, the present invention provides methods of inhibiting oxidation of a pharmaceutical composition. The method comprises preparing a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises deferoxamine, salts thereof, analogs thereof, and combinations thereof, in an amount effective to inhibit oxidation of the pharmaceutical composition. The descriptions of the pharmaceutical compositions, agents, and pharmaceutically acceptable carriers, and components thereof, set forth above in connection with the pharmaceutical compositions of the invention, also apply to those same aspects of the methods of the invention.

The amount of deferoxamine, or its preferred salt (deferoxamine mesylate), included in the composition will depend on the active agent and other excipients. Desirably, the amount of deferoxamine, salts thereof, or analogues thereof in the composition is an amount effective to inhibit microbial growth and/or inhibit oxidation. As described above, typically, the pharmaceutical composition is prepared in liquid form, and then deferoxamine, its salts, and the like are added to the solution. Preferably, the pharmaceutical composition in liquid form comprises about 0.0001% to about 0.5% by weight (e.g., about 0.005%, about 0.1%, or about 0.25%) deferoxamine, a salt thereof, or the like. More preferably, the composition in liquid form comprises a similar amount of the preferred deferoxamine salt, deferoxamine mesylate. Most preferably, the pharmaceutical composition in liquid form comprises about 0.5% by weight of deferoxamine mesylate. When the composition is prepared in solid form, as described above, it is preferred to apply the deferoxamine mesylate as a solution to the active agent, and other excipients, if present, as prepared by wet granulation, fluid bed drying and other methods known to those skilled in the art. The deferoxamine mesylate solution preferably has from about 0.0001 wt.% to about 0.5 wt.% (e.g., about 0.005 wt.%, about 0.1 wt.%, or about 0.25 wt.%) deferoxamine.

The invention also provides a method for enhancing transport of a pharmaceutical agent to a site of frailty, the method comprising administering to a human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the ratio of albumin to pharmaceutical agent in the pharmaceutical composition is about 18:1 or less. The invention further provides a method for enhancing binding of an agent to a cell in vitro or in vivo, the method comprising administering to the cell in vitro or in vivo a pharmaceutical composition comprising the agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the ratio of albumin to the agent in the pharmaceutical composition is about 18:1 or less. The descriptions of the pharmaceutical compositions, agents, pharmaceutical carriers, routes of administration, and components thereof set forth above in connection with the pharmaceutical compositions of the invention and the methods of the invention also apply to those same aspects of the transport and binding methods.

In the method for transporting the agent to the site of weakness or for enhancing binding of the agent to cells, the pharmaceutically acceptable carrier preferably comprises albumin, most preferably human serum albumin. Without being bound to any one particular theory, it is believed that examples of proteinsSuch as human serum albumin, to the agent in the pharmaceutical composition affects the ability of the agent to bind to the cell and the transport of the agent to the cell. In this regard, a higher ratio of protein to agent is often associated with poor cellular binding and transport of the agent, which may be the result of receptor competition on the cell surface. The ratio of protein, e.g., albumin, to active agent must be such that a sufficient amount of the agent binds to or is transported to the cell. An exemplary range of protein-drug formulations is a protein to drug ratio (w/w) of 0.01:1 to about 100: 1. More preferably, the ratio is from 0.02:1 to about 40: 1. Although the ratio of protein to agent must be optimized for different combinations of protein and agent, typically the ratio of protein, e.g., albumin, to agent is about 18:1 or less (e.g., about 15:1, about 10:1, about 5:1, or about 3: 1). More preferably, the ratio is from about 0.2:1 to about 12: 1. Most preferably, the ratio is from about 1:1 to about 9: 1. Preferably, the formulation is substantially free of polyoxyethylated castor oil, more preferably free of polyoxyethylated castor oil(BASF). Polyoxyethylene Castor oilNonionic emulsifiers are polyethers which are castor oil and polyoxyethylene. As noted above, polyoxyethylated castor oils are typically used as solvents for paclitaxel and are associated with side effects that may be serious (see, e.g., Gelderblom et al, supra).

The agent can be any suitable agent described herein (e.g., propofol, paclitaxel, or docetaxel). In addition, the agent may be a nucleic acid sequence, most preferably a DNA sequence. In this regard, the pharmaceutical compositions of the present invention can deliver genes to cells via receptor-mediated/cavity (caveolar)/vesicle transport. Pharmaceutical compositions comprising albumin-bound genetic material can be prepared for the purpose of transporting DNA sequences, such as genes or other genetic material, including but not limited to plasmids or c-DNA, into cells (e.g., endothelial cells or tumor cells). Because tumor cells and other cells at the site of inflammation have a high uptake of proteins, it is preferred that genetic material be inhaled into these cell types and can be integrated into the genetic material of the cells to achieve an effective therapeutic effect. The use of proteins such as human serum albumin serves as a non-viral vector for the delivery of genetic material without the risk of viral-related diseases or side effects. For example, a pharmaceutical composition comprising a nucleic acid sequence encoding β -galactosidase or Green Fluorescent Protein (GFP) and albumin can be prepared and contacted with an endothelial cell derived from a human umbilical vein or human pulmonary microvasculature to facilitate integration of the nucleic acid sequence into the endothelial cell. Integration of a nucleic acid sequence can be detected using methods known in the art, such as, for example, fluorescence or staining.

In the methods of the invention for enhancing the transport of an agent to a site of weakness, the weakness may be any suitable disease or condition. Preferably, the frailty is cancer, cardiovascular disease, or arthritis.

In the methods of the invention for enhancing binding of an agent to a cell in vitro or in vivo, a pharmaceutical composition is administered to a cell in vitro or in vivo. Ideally, the cell is an animal cell. More preferably, the cell is a mammalian cell, most preferably, the cell is a human cell. Preferably, the pharmaceutical composition is administered to cells in vivo. The cell may be any suitable cell that is the desired target for administration of the pharmaceutical composition. For example, the cells may be located in or derived from digestive system tissues including, for example, esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Cells may also be located in or derived from respiratory tissues including, for example, the larynx, lungs, and bronchi. The cells may be located in or derived from, for example, the cervix, uterus, ovaries, vagina, prostate, testis, and penis that make up the male or female reproductive system, and the bladder, kidney, renal pelvis, and ureter that make up the urinary system. The cells may be located in or derived from tissues of the cardiovascular system, including, for example, endothelial cells and cardiomyocytes. The cells may also be located in or derived from tissues of the lymphatic system (e.g., lymphocytes), the nervous system (e.g., neurons or glial cells), and the endocrine system (e.g., thyroid cells). Preferably, the cells are located in or derived from tissue of the cardiovascular system. Most preferably, the cells are endothelial cells. In the context of the methods of the present invention for enhancing transport and binding of an agent to a cell, the pharmaceutical composition desirably contacts more than one cell.

In another aspect of the invention, the methods of the invention for enhancing transport and enhancing binding of agents to cells can be used to treat tumor cells. Tumor cells exhibit enhanced protein uptake compared to normal cells, including albumin and transferrin. Because tumor cells divide rapidly, they require more sources of nutrients than normal cells. The tumor research of the pharmaceutical composition containing the paclitaxel and the human serum albumin shows that the albumin-paclitaxel is high in uptake by the tumor. This has been found to be due to a previously unrecognized phenomenon of albumin-drug transport through the glycoprotein 60 ("gp 60") receptor, which is specific for albumin.

Thus, according to another aspect of the invention, albumin-specific gp60 receptors and other protein transport receptors present on tumor cells can be used as targets for inhibiting tumor growth. By blocking the gp60 receptor and blocking other protein transport receptors on tumor cells or tumor endothelial cells using antibodies against gp60 receptor or other large or small molecule compounds that bind, block or inactivate gp60, the transport of proteins to these cells can be blocked, thereby reducing their growth rate and leading to cell death. Thus, blockade of this mechanism leads to treatment of patients (e.g., humans) with cancer or another disease. Identification of specific protein receptor blockade/binding is accomplished by screening any number of compounds against isolated gp60 or other receptors such as gp16 or gp30 or by using whole cell preparations. In addition, suitable animal models can also be used for this purpose, such as, for example, mice containing a "knock-out" mutation of the gene encoding gp60 or caveolin-1 or other proteins specific for transport. Accordingly, methods of identifying compounds that block or bind gp60, gp16, gp30 or other protein receptors are within the scope of the invention.

In addition, compounds that block or bind the gp60 receptor or other protein receptors may be useful in the treatment of several diseases, including cancer. With respect to the treatment of cancer, blocking or binding compounds may be used as single agents or in combination with other standard or multiple chemotherapies. For example, it is used in the treatment of cancer with conventional chemotherapy or with the albumin-pharmaceutical composition of the invention, which shows high accumulation in tumors, followed by compounds that block the transport of proteins to tumor cells. The blocking compound may be administered prior to or in combination with other chemotherapeutic or anti-cancer agents. Thus, any compound that blocks or binds the gp60 receptor or other protein receptor is within the scope of the invention.

The albumin-pharmaceutical compositions of the invention, such as, for example, albumin-paclitaxel, albumin-docetaxel, albumin-epothilone, albumin-camptothecin, or albumin-rapamycin, among others, can be used to treat diseases. These pharmaceutical compositions are believed to be effective due to increased receptor-mediated transport of the protein-pharmaceutical composition to the desired site (e.g., tumor). Without wishing to be bound by any particular theory, transport of a protein-pharmaceutical composition through receptor-mediated transport resulting in a therapeutic effect is believed to be, for example, a transport mechanism for albumin-paclitaxel compositions to tumors, and for albumin-paclitaxel and albumin-rapamycin transport across the lung. Transport is affected by the presence of gp60, gp16 or gp30 in these tissues. Thus, drugs and protein-drug combinations that are associated with gp60, gp16 or gp30 receptors and that lead to therapeutic effects for transport to disease sites such as inflammation (e.g., arthritis) or tumors are contemplated as compositions of the invention.

According to another aspect of the invention, endothelial cells may be co-cultured with cells having a specific function. Incubation of endothelial cells with other cell types such as pancreatic islet cells, hepatocytes, neuroendocrine cells, and other cells allows transfer of desired components such as proteins and other beneficial components to these cells. Endothelial cells provide for the transfer of these components to cultured cell types in order to mimic in vivo conditions, i.e., where these cell types are typically in close proximity to endothelial cells and will rely on endothelial cells to transport nutrients, growth factors, hormone signals, etc., as required for their normal function. When endothelial cells are not present, it has not previously been possible to adequately culture these different cell types and obtain physiological properties. The presence of endothelial cells in culture with the desired cell type allows differentiation and proper function of pancreatic islets, hepatocytes, or neuroendocrine tissue in vitro or ex vivo. It was thus found that co-culturing of endothelial cells and islets results in islets with improved physiological properties (e.g. the ability to secrete insulin) compared to those cultured in the absence of endothelial cells. The tissue can then be used in vitro or transplanted into the body to treat diseases resulting from the lack of proper cellular function (e.g., diabetes in the case of islet cells, liver dysfunction in the case of hepatocytes, and neuroendocrine disorders or pain relief in the case of neuroendocrine cells). Cells derived from other tissues and organs (as described above) can also be co-cultured with endothelial cells to provide the same benefits. In addition, co-culture can be used to integrate genetic material into a target cell type. The presence of albumin in these cultures was found to be very beneficial.

The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

Example 1

This example illustrates the preparation of a pharmaceutical composition containing paclitaxel and albumin. The preparation of paclitaxel-albumin compositions is described in U.S. Pat. Nos. 5,439,686 and 5,916,596, which are incorporated herein by reference in their entirety. Specifically, 30mg of paclitaxel was dissolved in 3.0ml of dichloromethane. This solution was added to 27.0ml of human serum albumin solution (2% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (vitaris homogenizer, model tempest i.q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The obtained dispersion was translucent and the obtained paclitaxel particles had a typical average diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.

Example 2

This example illustrates the preparation of a pharmaceutical composition containing amiodarone and albumin. 30mg of amiodarone were dissolved in 3.0ml of dichloromethane. The solution was added to 27.0ml of human serum albumin solution (1% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The obtained dispersion was translucent and the obtained amiodarone particles had a typical average diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the invention shows significantly lower toxicity when compared to the toxicity of amiodarone dissolved in tween formulation.

Example 3

This example illustrates the preparation of a pharmaceutical composition containing liothyronine and albumin composition. Liothyronine (or a suitable salt) is dissolved in an aqueous or alkaline solution at a concentration of 0.5-50 mg/ml. The alcoholic (or alkaline) solution was added to the albumin solution (0.1-25% w/v) and stirred. The agitation is low shear using a stirrer or high shear using a sonicator or homogenizer. At low concentrations of liothyronine, clear solutions (5-1000. mu.g/ml) were obtained. As the concentration increases, a stable milky suspension is obtained. These solutions or suspensions are filtered through sterile filters. The organic solvent is removed by evaporation or other suitable means.

Example 4

This example illustrates the preparation of a pharmaceutical composition containing rapamycin and albumin. Rapamycin (30 mg) was dissolved in 2ml chloroform/ethanol. The solution was added to 27.0ml of human serum albumin solution (3% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.

Example 5

This example illustrates the preparation of a pharmaceutical composition containing epothilone B and albumin. 30mg of epothilone B was dissolved in 2ml chloroform/ethanol. The solution was added to 27.0ml of human serum albumin solution (3% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the invention shows significantly lower toxicity when compared to the toxicity of epothilone B dissolved in the polyoxyethylene castor oil formulation.

Example 6

This example illustrates the preparation of a pharmaceutical composition containing colchicine dimer and albumin. 30mg of colchicine-dimer are dissolved in 2ml of chloroform/ethanol. The solution was added to 27.0ml of human serum albumin solution (3% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical compositions of the present invention containing albumin show significantly lower toxicity when compared to the toxicity of colchicine dimer dissolved in emetic warms.

Example 7

This example illustrates the preparation of a pharmaceutical composition containing docetaxel and albumin. 30mg of docetaxel was dissolved in 2ml of chloroform/ethanol. The solution was added to 27.0ml of human serum albumin solution (3% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of docetaxel dissolved in tween/ethanol, a standard solvent for the drug.

Example 8

This example illustrates the preparation of a pharmaceutical composition containing docetaxel and albumin. 150mg of docetaxel was dissolved in 1ml of ethyl acetate/butyl acetate and 0.5ml of an oil such as soybean oil or vitamin E oil. These compositions are also contemplated as part of the present invention using other ratios of solvents and oils. Optionally, a small amount of a negatively charged component, such as benzoic acid (0.001% -0.5%) is also added. The solution was then added to 27.0ml of human serum albumin solution (5% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of docetaxel dissolved in tween/ethanol, a standard solvent for the drug.

Example 9

This example illustrates the preparation of a pharmaceutical composition containing taxane IDN5390 and albumin. 150mg of IDN5390 was dissolved in 1ml ethyl acetate/butyl acetate and 0.5ml oil such as soybean oil or vitamin E oil. These compositions are also contemplated as part of the present invention using other ratios of solvents and oils. Optionally, a small amount of a negatively charged component, such as benzoic acid (0.001% -0.5%) is also added. The solution was then added to 27.0ml of human serum albumin solution (5% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition of the invention containing albumin shows significantly lower toxicity when compared to the toxicity of IDN5390 dissolved in tween.

Example 10

This example illustrates the preparation of a pharmaceutical composition containing taxane IDN5109 and albumin. 150mg of IDN5109 was dissolved in 2ml of chloroform/ethanol. These compositions are also contemplated as part of the present invention using other ratios of solvents and oils. Optionally, a small amount of a negatively charged component, such as benzoic acid (0.001% -0.5%) is also added. The solution was then added to 27.0ml of human serum albumin solution (5% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the invention showed significantly lower toxicity when compared to the toxicity of IDN5109 dissolved in emetic.

Example 11

This example illustrates the preparation of a pharmaceutical composition containing 10-hydroxycamptothecin (10HC) and albumin. 30mg of 10HC was dissolved in 2.0ml of DMF/dichloromethane/soybean oil. The solution was then added to 27.0ml of human serum albumin solution (3% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.

Example 12

This example illustrates the preparation of a pharmaceutical composition containing cyclosporin and albumin. 30mg of cyclosporin were dissolved in 3.0ml of dichloromethane. The solution was then added to 27.0ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the particles obtained typically have an average diameter of from 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

Example 13

This example illustrates the preparation of a pharmaceutical composition containing oil and comprising cyclosporin and albumin. 30mg of cyclosporin was dissolved in 3.0ml of an appropriate oil (sesame oil containing 10% orange oil). The solution was then added to 27.0ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The particles obtained typically have an average diameter of 50 to 220nm (Z-average, Malvern Zetasizer). The dispersion was used directly or lyophilized for 48 hours by optionally adding a suitable cryoprotectant. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.

Example 14

This example illustrates the preparation of a pharmaceutical composition containing amphotericin and albumin. 30mg of amphotericin were dissolved in 3.0ml of methylpyrrolidone/dichloromethane. The solution was added to 27.0ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the solvent was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The resulting dispersion was translucent and the resulting amphotericin particles had a typical mean diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The addition of other components such as lipids, bile salts, etc. also produces suitable formulations.

Example 15

This example illustrates the preclinical pharmacokinetics and pharmacodynamics of a pharmaceutical composition containing albumin and paclitaxel.

Several preclinical pharmacokinetic studies in mice and rats were performed to evaluate the possible advantages of albumin-paclitaxel pharmaceutical compositions over polyoxyethylated castor oil-paclitaxel (taxol) pharmaceutical compositions. These studies demonstrate that: (1) the pharmacokinetics of albumin-paclitaxel in rats are linear, while the pharmacokinetics of taxol are non-linear with respect to dose, (2) albumin-containingThe pharmaceutical composition with paclitaxel showed lower plasma AUC and C_maxSuggesting a faster distribution of albumin-paclitaxel composition to the tissue compared to taxol (excretion is similar), (3) pharmaceutical composition comprising albumin and paclitaxel showed lower C_maxThis may explain the reduced toxicity associated with peak blood levels compared to taxol, (4) pharmaceutical compositions comprising albumin and paclitaxel showed half-lives that were about 2-fold higher in rats and 4-fold higher in tumor-bearing mice compared to taxol, and (5) the metabolism of paclitaxel in pharmaceutical compositions comprising albumin and paclitaxel was lower than that of paclitaxel in taxol pharmaceutical compositions. At 24 hours after injection in rats, for the pharmaceutical composition comprising albumin and paclitaxel, 44% of the total radioactivity is still associated with paclitaxel compared to only 22% for taxol. The final effect of the pharmacokinetics described above, i.e. enhanced intracellular uptake, prolonged half-life and lower metabolism, shown for the pharmaceutical composition containing albumin and paclitaxel, resulted in a 1.7-fold higher tumor AUC, tumor C, in tumor-bearing mice compared to taxol_max1.2 times higher and 1.7 times longer tumor half-life.

Example 16

This example illustrates the reduced side effects and reduced toxicity associated with pharmaceutical compositions comprising paclitaxel and albumin.

Due to the unique properties of the pharmaceutical composition comprising paclitaxel and albumin in the absence of polyoxyethylated castor oil, the pharmaceutical composition comprising paclitaxel and albumin is significantly less toxic than taxol. In preclinical studies in mice and rats, single dose acute toxicity studies in mice have shown LD for pharmaceutical compositions containing paclitaxel and albumin₅₀The dosage is about 59 times that of taxol. In multiple dose toxicity studies in mice, for pharmaceutical compositions containing paclitaxel and albumin, the LD thereof₅₀The dosage is about 10 times that of taxol. Further studies evaluated the degree of bone marrow suppression in rats treated with pharmaceutical compositions containing paclitaxel and albumin and taxol. Results are shown at equal doses, packageThe pharmaceutical composition containing paclitaxel and albumin produced significantly less myelosuppression than taxol in rats. In acute toxicity studies in rats, cortical necrosis or severe neurotoxicity was observed in animals receiving 9mg/kg taxol, but not in animals receiving up to 120mg/kg doses of the pharmaceutical composition comprising paclitaxel and albumin. Thus, the presence of albumin in the pharmaceutical composition comprising paclitaxel results in a significant reduction in side effects and toxicity compared to conventional pharmaceutical compositions comprising paclitaxel.

Example 17

This example illustrates the clinical effect of a pharmaceutical composition comprising paclitaxel and albumin in humans.

Clinical studies to date in more than 500 human patients provide evidence for pharmaceutical compositions comprising paclitaxel and albumin ("albumin-paclitaxel") that support reduced toxicity and side effects compared to polyoxyethylated castor oil-paclitaxel compositions (taxol). In a phase I study of 19 patients, the maximum tolerated dose of albumin-paclitaxel was provided every 3 weeks at 300mg/m². This is significantly higher than the commonly administered polyoxyethylated castor oil-paclitaxel dose, i.e. 175mg/m is provided once every 3 weeks². In these patients, the blood toxicity is mild, no allergy, mild neuropathy, and no administration-related side effects such as venous irritation, etc.

In another phase I study of 27 patients, the maximum tolerated dose of albumin-paclitaxel on a weekly schedule was 125-150mg/m². This is significantly higher than the commonly administered polyoxyethylated castor oil-paclitaxel dose, i.e. 80mg/m provided on a weekly schedule². In these patients, the blood toxicity is mild, no allergy, mild neuropathy, and no administration-related side effects such as venous irritation, etc.

175 or 300mg/m were provided every 3 weeks in 43 and 63 patients, respectively²In two phase II studies of albumin-paclitaxel, the blood toxicity was low at 175mg/m²And 300mg/m²Only 7% and 24% of patients ANC, respectively<500/mm³. At 175mg/m²And 300mg/m²In the lower, 0% and 14% of patients, respectively, developed severe neuropathy. No serious allergy occurred, no administration-related side effects such as venous irritation, pain upon injection etc. occurred, which were significantly lower than those experienced with taxol.

In phase III trials comparing albumin-paclitaxel composition ABI-007 with taxol (with cremophor oil-paclitaxel), the dose of ABI-007 was significantly higher (260 mg/m)²Comparison of 175mg/m for taxol²) Indicating that it is better tolerated. The albumin-paclitaxel composition also shows significantly reduced neutropenia when compared to the polyoxyethylated castor oil-paclitaxel.

Example 18

This example illustrates enhanced preclinical efficacy using a pharmaceutical composition containing albumin and paclitaxel.

An in vitro cytotoxicity study comparing the effects of albumin-paclitaxel and taxol on cervical squamous cell carcinoma a431 showed that: the cytotoxic activity of albumin-taxol is increased by about 4 times, and the IC of albumin-taxol and taxol are increased₅₀0.0038 and 0.012. mu.g/ml, respectively.

In five different human xenograft tumor models (MX-1 breast, NCI-H522 lung, SK-OV-3 ovary, PC-3 prostate, and HT-29 colon) in athymic mice, the MTD or isotoxic dose of ABI-007 was 1.5-3.4 times higher than that of taxol, resulting in a statistically significant improvement in tumor growth delay (p <0.05) in all tumors except lung tumor (p = 0.15).

In the MX1 mammary model, one hundred percent (100%) of albumin-paclitaxel treated animals survived for 103 days, compared to 20-40% survival in the group treated with equivalent doses of taxol.

Example 19

This example illustrates the enhanced clinical efficacy using an intra-arterial administration of a pharmaceutical composition comprising albumin and paclitaxel.

In a phase I/II study with intra-arterial administration of a pharmaceutical composition comprising albumin and paclitaxel, patients with head and neck cancer (N =31) and patients with anal canal cancer (N =12) were recruited as described herein. By percutaneous super-selective intra-arterial infusion, the dose administered within 30 minutes is from 120mg/m²Gradually increased to 300mg/m²Q3-4 weeks. Patients with head and neck cancer showed a response rate of 76% (N =29), while patients with anal canal cancer showed a response rate of 64% (N = 11).

Example 20

This example illustrates the preparation of a pharmaceutical composition containing 3% oil and comprising propofol and albumin.

An oil-in-water emulsion containing 1% by weight propofol was prepared as follows. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2um filter). The oil phase was prepared by dissolving lecithin (0.4 wt%) and propofol (1 wt%) in soybean oil (3 wt%) at about 50-60 ℃ and stirring until dissolved. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The crude emulsion was homogenized at 20,000psi under high pressure and recycled for 15 cycles at 5 ℃. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.5-3% of human serum albumin, 0.5-3.0% of soybean oil, 0.12-1.2% of lecithin, 2.25% of glycerol, a proper amount of water for injection to 100% and a pH value of 5-8. Optionally, a suitable chelating agent, such as deferoxamine (0.001-0.1%) is added.

Example 21

This example illustrates the preparation of a pharmaceutical composition containing 5% oil and comprising propofol and albumin.

An oil-in-water emulsion containing 1% by weight propofol was prepared as follows. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2um filter). The oil phase was prepared by dissolving lecithin (0.8 wt%) and propofol (1 wt%) in soybean oil (5 wt%) at about 50-60 ℃ and stirring until dissolved. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The crude emulsion was homogenized at 20,000psi under high pressure and recycled for 15 cycles at 5 ℃. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.5-3% of human serum albumin, 0.5-10.0% of soybean oil, 0.12-1.2% of lecithin, 2.25% of glycerol, a proper amount of water for injection to 100% and a pH value of 5-8. Optionally, a suitable chelating agent, such as deferoxamine (0.001-0.1%) is added.

Example 22

This example illustrates the preparation of a propofol and albumin, oil-free pharmaceutical composition.

A propofol composition containing albumin and tween 80 was prepared using a method analogous to that described in example 18. The aqueous phase was prepared by adding glycerol (2.25 wt%), human serum albumin (0.5 wt%), tween 80(1.5 wt%) and deferoxamine mesylate (0.1 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). Propofol (1 wt%) was added to the aqueous phase and homogenized for 5 minutes at 10,000 RPM. The crude emulsion was homogenized at 20,000psi under high pressure and recycled for 15 cycles at 5 ℃. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.5-3% of human serum albumin and 800.1-1.5% of tween; 0.0001-0.1% of deferoxamine mesylate, 2.25% of glycerol, a proper amount of water for injection to 100%, and the pH value of 5-8.

Example 23

This example illustrates the preparation of an oil-free pharmaceutical composition containing propofol, albumin and vitamin E-TPGS.

Propofol compositions containing albumin and vitamin E-TPGS were prepared using a method analogous to that described in example 19. The aqueous phase was prepared by adding glycerol (2.25 wt%), human serum albumin (0.5 wt%), vitamin E-TPGS (1 wt%) and deferoxamine mesylate (0.1 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). Propofol (1 wt%) was added to the aqueous phase and homogenized for 5 minutes at 10,000 RPM. The crude emulsion was homogenized at 20,000psi under high pressure and recycled for 15 cycles at 5 ℃. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5 to 5 percent of propofol, 0.5 to 3 percent of human serum albumin, 0.5 to 4.0 percent of vitamin E-TPGS; optionally 0.0001-0.1% of deferoxamine mesylate, 2.25% of glycerol, a proper amount of water for injection to 100%, and a pH of 5-8.

Example 24

This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS and 1% oil.

An emulsion containing 1% by weight propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) was added to the aqueous phase. The oil phase consisted of propofol (1% by weight) and 1% soybean oil. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000psi and recycled at 5 ℃ for a maximum of 15 cycles. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.

The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.01-3% of human serum albumin, 0.1-2% of vitamin E-TPGS; soybean oil or other oils (0.1% -5%); 2.25 percent of glycerin, proper amount of water for injection to 100 percent and pH 5-8. Desferrioxamine (0.001 wt% to 0.1 wt%) is optionally added.

Example 25

This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil, and negatively charged components.

An emulsion containing 1% by weight propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) was added to the aqueous phase. The oil phase consisted of propofol (1% by weight) and 1% soybean oil. A small amount of a negatively charged component (0.001% -1%) such as a phospholipid or bile salt is added. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000psi and recycled at 5 ℃ for a maximum of 15 cycles. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.

The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.01-3% of human serum albumin, 0.1-2% of vitamin E-TPGS; soybean oil or other oils (0.1% -5%); 2.25 percent of glycerin, proper amount of water for injection to 100 percent and pH 5-8. Desferrioxamine (0.001 wt% to 0.1 wt%) is optionally added.

Example 26

This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil and a negatively charged component (sodium deoxycholate).

An emulsion containing 1% by weight propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) was added to the aqueous phase. The oil phase consisted of propofol (1% by weight) and 1% soybean oil. A small amount of a negatively charged component (0.001% -1%) such as sodium deoxycholate is added. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000psi and recycled at 5 ℃ for a maximum of 15 cycles. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.

The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.01-3% of human serum albumin, 0.1-2% of vitamin E-TPGS; soybean oil or other oils (0.1% -5%); 2.25 percent of glycerin, proper amount of water for injection to 100 percent and pH 5-8. Desferrioxamine (0.001 wt% to 0.1 wt%) is optionally added.

Example 27

This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil and negatively charged components (phospholipids, bile salts, polyamino acids, etc.).

An emulsion containing 1% by weight propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25 wt%) and human serum albumin (0.5 wt%) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) was added to the aqueous phase. The oil phase consisted of propofol (1% by weight) and 1% soybean oil. A small amount of a negatively charged component (0.001% -1%), such as phosphatidylcholine, is added. The oil phase was added to the water phase and homogenized at 10,000RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000psi and recycled at 5 ℃ for a maximum of 15 cycles. Alternatively, a separate channel through the homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.

The resulting pharmaceutical composition comprises the following components (in weight%) in the usual ranges: 0.5-5% of propofol, 0.01-3% of human serum albumin, 0.1-2% of vitamin E-TPGS; soybean oil or other oils (0.1% -5%); 2.25 percent of glycerin, proper amount of water for injection to 100 percent and pH 5-8. Desferrioxamine (0.001 wt% to 0.1 wt%) is optionally added.

Example 28

This example illustrates the binding of propofol to albumin.

Binding of propofol to albumin was determined as follows. Propofol solubility was tested in water and in solutions containing albumin. 250 μ L of propofol was added to 10mL of water or albumin solution and stirred in scintillation vials for 2 hours. The solution was then transferred to a 15mL polyethylene centrifuge tube and held at 40 ℃ for about 16 hours. Propofol was measured in samples of water and albumin solution. The solubility of propofol in water was determined to be 0.12 mg/ml. The solubility of propofol in albumin solutions depends on albumin concentration, increasing to 0.44mg/ml when the albumin concentration is 2% (20 mg/ml). The solution was ultrafiltered through a 30kD MWCO filter and the filtrate was assayed for propofol. It was found that 61% of propofol could be recovered in the filtrate for the propofol/water solution, while only 14% was recovered in the filtrate for the propofol/albumin solution, showing considerable binding of propofol to albumin. Based on these results, the addition of albumin to a pharmaceutical composition comprising propofol results in a reduction in the amount of free propofol due to binding of albumin to propofol.

Example 29

This example illustrates the reduction of free propofol in a pharmaceutical composition by filtration/membrane contact.

As observed in the experiment described in example 28, filtration or ultrafiltration of a pharmaceutical composition comprising propofol resulted in a reduction in the amount of free propofol. Bipropofol and the albumin-containing pharmaceutical composition prepared according to the invention, each containing 1% propofol (10mg/ml), were ultrafiltered using a 30kD membrane. The amount of free propofol in the filtrate was measured using HPLC. The concentration of free propofol in the filtrate for bipropofol is about 17 μ g/ml, whereas the concentration of free propofol in the filtrate for the pharmaceutical compositions of the present invention is about 7 μ g/ml. The results correspond to an effective reduction of free propofol of more than 2-fold for a pharmaceutical composition comprising propofol and albumin.

Example 30

This example illustrates the administration of a pharmaceutical composition comprising propofol and albumin to a human.

A randomized, double-blind clinical trial was conducted to compare the adverse skin feel of a pharmaceutical composition comprising propofol and albumin with the commercial propofol formulation, bipropofol. The test was performed according to Good Clinical practice (Good Clinical Practices) and informed consent was obtained from the subjects. Adult subjects of any gender may participate, provided they have unbroken, apparently normal dorsal skin of their hands.

The formulation originally stored in the refrigerator was brought to a room temperature environment, and then 10. mu.L of the formulation was slowly placed on the backs of both hands of the subject at the same time. The overall reaction and feel of their hands to the formulation was recorded. The results of this study are listed in table 1.

TABLE 1

Example 31

This example illustrates the use of deferoxamine as an antioxidant in a pharmaceutical composition comprising propofol.

Pharmaceutical compositions comprising propofol and deferoxamine mesylate, and containing tween or TPGS were stored at 4 ℃, 25 ℃ or 40 ℃ to examine the effect of deferoxamine mesylate in preventing propofol oxidation. The propofol concentration was measured over time for these formulations to determine the antioxidant activity of deferoxamine. This data is reported in tables 2 and 3 below as% efficacy relative to time zero.

TABLE 2 Albumin/Tween formulations

TABLE 3 Albumin/TPGS formulations

Under these conditions, deferoxamine is sufficient to reduce the oxidation level of propofol. This effect is more pronounced at higher temperatures. No significant oxidation occurred at 4 ℃. The study was conducted using a plug that was not coated with an inert material or polytetrafluoroethylene.

Example 32

This example illustrates intrapulmonary delivery of a pharmaceutical composition containing paclitaxel and albumin (ABI-007).

The object of this study was to determine³H]Time course of ABI-007 in blood and selection of tissues after intratracheal instillation into Sprague Dawley rats.

The target volume of the intratracheal dosage formulation administered to the animal was calculated based on a dose volume of 1.5mL per kg body weight. The dosing device consisted of a Penn-Century micro-nebulizer (model No. 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from De Long diagnostics, Long Branch, N.J.) connected to a 1-mL air-tight luer-lock syringe. The appropriate volume of the dosage formulation is inhaled into the dosing device, the filled device is weighed and the weight recorded. The catheter was placed into the trachea of the anesthetized animal and the micro-nebulizer portion of the dosing device was placed through the catheter into the trachea to administer the dose. After dosing, the empty dosing device was reweighed and the dose administered was calculated as the difference in weight of the dosing device before and after dosing. The average dose was 4.7738. + -. 0.0060(CV1.5059) mg paclitaxel/kg body weight for all animals.

Approximately 250 μ L of blood samples were collected from the indwelling jugular vein cannulas of JVC rats at a time post-administration of the following pre-determined dose: 1,5,10,15,30, and 45 minutes (min), and 1, 4, 8, and 24 hours (h). Blood samples were collected at 24 hours, and at 10 minutes, 45 minutes and 2 hours from sacrificed animals by cardiac puncture from anesthetized rats at sacrifice. All blood samples used for total radioactivity analysis were dispensed into pre-weighed sample tubes, the sample tubes were re-weighed, and the weight of each sample was calculated by subtraction. Blood samples collected from the jugular vein and 250- μ L aliquots of blood collected from each animal at sacrifice were assayed for total tritium content.

For all rats, the maximum concentration of tritium in the blood was observed at 5 minutes (0.0833 hours) after dose administration. The elimination half-life of tritium, measured in the time interval from 4h to 24h, is from 19.73h to 43.02 h. It should be noted that the interval includes only three data points, which may account for the variability of the parameter. The apparent clearance of tritium from blood was about 0.04L/h. The results of these experiments are set forth in table 4 below.

TABLE 4 instillation in trachea³H]Non-compartmental analysis of blood tritium concentration (mg-eq/L) versus time curves in rats after ABI-007

Analysis of the source of [ alpha ], [ alpha ] form of a peptide, which is derived from a polypeptide³H]Average blood concentration of radioactivity of ABI-007 to evaluate that derived from intratracheal administration³H]Bioavailability of tritium of ABI-007. This analysis resulted in a 24-hour AUC (AUC terminal) of 6.1354mg-eq hr/L. Based on these data, the data are derived from intratracheal administration³H]The radioactivity of ABI-007 is highly bioavailable. These assays are based on total radioactivity.

Derived from [ 2 ]³H]Tritium of ABI-007 is rapidly absorbed after intratracheal instillation. Administration in trachea³H]The mean absorption and elimination half-lives of tritium in blood after ABI-007 (k 01 half-life and k10 half-life, respectively) were 0.0155+/-0.0058 hours and 4.738+/-0.366 hours, respectively. The mean apparent clearance of tritium from blood was 0.1235 +/-0.0180L/hr (see Table 4 above).

Derived from [ 2 ]³H]Tritium of ABI-007 is absorbed and distributed after intratracheal administration. The time course of tritium in blood is well described by a two-compartment model, with average absorption and elimination half-lives of 0.0155 and 4.738 hours, respectively. Approximately 28% of the administered dose was recovered in the lungs 10 minutes after intratracheal administration. Except for the gastrointestinal tract, a maximum dose of less than 1% was recovered in other tissues at all times examined.

Based on the value from the previous use³H]Capxol^TMThe intravenous dosing study performed resulted in a bioavailability of tritium derived from intratracheal administration of 1.229 ± 0.268 (mean ± SD) for three animals of the dose group. It should be noted, however, that this estimate of bioavailability is based on total radioactivity. Surprisingly, paclitaxel delivered by the pulmonary route using the albumin-containing compositions of the invention is rapidly bioavailable, showing excellent transport across the lung endothelium. Toxicity was not noted in animals, which was surprising, since pulmonary delivery of cytotoxic species is known to cause pulmonary toxicity.

A considerable amount of radioactivity is present in the gastrointestinal tract (including the contents) 24 hours after dosing (27% intratracheal administration). The presence of tritium in the gastrointestinal tract may be due to biliary excretion or clearance of tritium from the respiratory tract through mucociliary clearance and subsequent swallowing.

Example 33

This example illustrates Aerotech II and Pari nebulizers for pulmonary delivery of pharmaceutical compositions comprising paclitaxel and albumin.

The study was performed using paclitaxel-albumin pharmaceutical composition ABI-007 under the following conditions: room temperature (20-23 ℃), relative humidity (48-54%), ambient pressure (629mmHg), nebulizer flow rate (10L/min for Aerotech II; 7L/min for Pari), total flow rate (28.3L/min), nebulizer pressure drop (23 lb/in for Aerotech II)²32lb/in for Pari²) Run time (15-60 seconds), sample volume (1.5mL), ABI-007 VioletPaclitaxel concentrations (5,10,15 and 20 mg/mL).

Both Aerotech II and Pari nebulizers provided acceptable overall efficiencies (30% -60%) when ABI-007 was reconstituted at concentrations of 5-15 mg/mL. The Pari nebulizer efficiency was higher than the Aerotech II nebulizer efficiency. The efficiency of the Pari nebulizer decreased slightly with increasing concentrations of ABI-007. An excellent fine fraction (74% -96%) was observed. Aerotech II sprayers have a higher fine fraction than Pari sprayers. The fine fraction is independent of concentration.

100mg of paclitaxel was delivered by the Pari nebulizer in less than 30 minutes using a 15mg/mL solution of ABI-007. 100mg of paclitaxel was delivered by Aerotech II nebulizer using 10mg/mL or 15mg/mL ABI-007 solution in about 65 minutes. Performance stability was tested for Aerotech II and Pari nebulizers. Both nebulizers are stable in aerosol concentration and efficiency until the drug is exhausted. At 15mg/mL, the Pari nebulizer consumed twice as much drug as the Aerotech II nebulizer, resulting in a higher aerosol concentration than the Aerotech II nebulizer.

In conclusion, the nanoparticle/albumin formulation of paclitaxel (ABI-007) showed excellent bioavailability in rats when administered by the pulmonary route. At the dose administered, there was no obvious sign of early toxicity. Pulmonary delivery of nanoparticulate paclitaxel (ABI-007) can be achieved using a conventional nebulizer.

Example 34

This example describes the intrapulmonary delivery of a pharmaceutical composition comprising albumin and rapamycin. The objective of this study was to determine the pulmonary absorption of rapamycin in the blood following intratracheal instillation into Sprague Dawley rats and compare with intravenous instillation.

The target volume of the intratracheal dosage formulation administered to the animal was calculated based on a dose volume of 1mL per kg body weight. The dosing device consisted of a Penn-Century micro-nebulizer (model No. 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from De Long diagnostics, Long Branch, N.J.) connected to a 1-mL air-tight luer-lock syringe. The appropriate volume of the dosage formulation is inhaled into the dosing device, the filled device is weighed and the weight recorded. The catheter was placed into the trachea of the anesthetized animal and the micro-nebulizer portion of the dosing device was placed through the catheter into the trachea to administer the dose. After dosing, the empty dosing device was reweighed and the dose administered was calculated as the difference in weight of the dosing device before and after dosing.

Approximately 250 μ L of blood samples were collected from the indwelling jugular vein cannula of the rat at the following scheduled dosing time: 1,5,10,15,30, and 45 minutes (min), and 1, 4, 8, and 24 hours (h). All blood samples analyzed were dispensed into pre-weighed sample tubes, the sample tubes were re-weighed, and the weight of each sample was calculated by subtraction. Total rapamycin concentration was determined using LC/MS/MS on collected blood samples.

Surprisingly, the results show that there is no significant difference between the blood concentration of rapamycin delivered via the pulmonary route and via intravenous delivery. The bioavailability of rapamycin delivered via the pulmonary route was calculated to be 109% using pharmaceutical compositions comprising albumin, showing excellent transport across the pulmonary endothelium.

Example 35

This example illustrates the tissue distribution of albumin-rapamycin after intrapulmonary administration of a pharmaceutical composition comprising rapamycin and albumin prepared in accordance with the present invention. The objective of this study was to determine the pulmonary uptake of rapamycin in tissues following intratracheal instillation into Sprague Dawley rats, and compared to intravenous instillation.

The target volume of the intratracheal dosage formulation administered to the animal was calculated based on a dose volume of 1mL per kg body weight. The dosing device consisted of a Penn-Century micro-nebulizer (model No. 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from De Long diagnostics, Long Branch, N.J.) connected to a 1-mL air-tight luer-lock syringe. The appropriate volume of the dosage formulation is inhaled into the dosing device, the filled device is weighed and the weight recorded. The catheter was placed into the trachea of the anesthetized animal and the micro-nebulizer portion of the dosing device was placed through the catheter into the trachea to administer the dose. After dosing, the empty dosing device was reweighed and the dose administered was calculated as the difference in weight of the dosing device before and after dosing.

Samples were collected from the brain, lung and liver of 3 rats per group at each time point of 10 minutes, 45 minutes, 2 hours and 24 hours. Samples were collected and analyzed for total rapamycin concentration using LC/MS. The results show that rapamycin concentrations are greater in lung tissue when delivered through the lung compared to intravenous delivery. However, the total concentration of rapamycin in the brain is lower when delivered by Intratracheal (IT) compared to Intravenous (IV). In the liver, there appears to be no difference in rapamycin concentration, regardless of IT or IV delivery. Based on these results, intrapulmonary delivery of rapamycin may be useful in treating conditions (i.e., lung transplantation) where high local concentrations of rapamycin would be beneficial.

Example 36

This example illustrates oral delivery of a pharmaceutical composition containing paclitaxel and albumin (ABI-007).

Tritiated ABI-007 was used to determine the oral bioavailability of paclitaxel in rats after oral gavage. After an overnight fast, 5 rats were given 5.5mg/kg paclitaxel in ABI-007 (group A) and another 5 rats (group B) were pre-treated with cyclosporin (5.0mg/kg) followed by 5.6mg/kg paclitaxel in ABI-007. After the radioactivity in the blood samples was determined by combustion, pharmacokinetic analyses of blood samples taken at 0.5,1,2,3,4,5,6,8,12, and 24 hours were performed. Oral bioavailability was determined by comparison with previously obtained intravenous data. The results are set forth in table 5 below.

TABLE 5 after oral administration³Mean AUC0-24, C of H-paclitaxel derived radioactivity_max，T_maxAnd% absorption

AUC0-24IV (6.06 μ g x hr./mL) and the IV dose (5.1mg/kg) were used to calculate percent absorption (based on data for the IV dose of ABI-007).

An oral bioavailability of 44% was observed for ABI-007 alone. This is significantly higher than that observed for other paclitaxel formulations. Bioavailability increased to 121% when animals were treated with cyclosporin (CsA). This is expected because CsA is a known inhibitor of the p-glycoprotein pump, which would normally inhibit the absorption of compounds such as paclitaxel from the GI tract. Bioavailability greater than 100% can be explained by resorption after biliary excretion of paclitaxel to the GI tract. Other known absorption inhibitors or enhancers may also be used for this purpose.

Example 37

This example illustrates the improved penetration of paclitaxel into red blood cells and tumor cells after administration of a pharmaceutical composition comprising paclitaxel and albumin.

Human MX-1 mammary tumor fragments were subcutaneously transplanted into athymic mice. By using³Paclitaxel a pharmaceutical composition comprising paclitaxel and albumin ("paclitaxel-albumin") and paclitaxel as described above were prepared with a specific radioactivity of 25 μ Ci/mg paclitaxel. When the tumor volume reaches about 500mm³In this case, 20mg/kg of radiolabeled paclitaxel-albumin or taxol in saline was administered intravenously. Plasma, blood and tumor tissue were sampled and shown to analyze radioactivity at 5,15, and 30 minutes and at 1,3, 8, and 24 hours post-dose. Tumor pharmacokinetics (AUC and absorption constants) were analyzed using WinNonlin, Pharsight, USA.

Paclitaxel-albumin shows rapid partitioning to Red Blood Cells (RBC) as indicated by the rapid decrease in plasma/blood radioactivity ratio to unity following intravenous drug administration. Complete partitioning into RBCs occurs as early as 1 hour after paclitaxel-albumin administration. In contrast, the partitioning of paclitaxel formulated as taxol to RBCs is much slower and not completed until more than 8 hours.

Paclitaxel-albumin shows a rapid distribution to tumor tissue, absorption constant (K)_a) Is 3.3X greater than taxol. K_aFor paclitaxel-albumin and taxol, respectively, 0.43hr^-1And 0.13hr^-1. Rapid uptake of paclitaxel resulted in a paclitaxel-albumin tumor AUC that was 33% higher than that of taxol. AUC for paclitaxel-albumin and taxol were 3632nCi hr/g and 2739nCi hr/g, respectively.

Example 38

This example illustrates the safety of a pharmaceutical composition containing paclitaxel and albumin administered to mice.

Athymic mice were treated daily with increasing doses of paclitaxel-albumin or taxol for 5 consecutive days. Survival vs dose plots to determine LD₅₀. Paclitaxel-albumin greatly increased survival compared to taxol (p =0.017, ANOVA). LD of paclitaxel-Albumin and taxol for the q1d x5 Schedule₅₀Calculated as 47 mg/kg/day and 30 mg/kg/day, respectively. At a dose level of 13.4 mg/kg/day, both paclitaxel-albumin and taxol were well tolerated with mortality rates of 1% (1 death in 72 mice) and 4% (2 deaths in 47 mice), respectively. At a dose level of 20 mg/kg/day, paclitaxel-albumin mortality was 1% (1 out of 72 mice) and taxol mortality was 17% (8 out of 47 mice) (p = 0.0025). At a dose level of 30 mg/kg/day, paclitaxel-albumin mortality was 4% (3 out of 72 mice) and taxol mortality was 49% (23 out of 47 mice) (p)<0.0001)。

Example 39

This example illustrates the novel paclitaxel transport mechanism across microvascular Endothelial Cells (ECs) for paclitaxel-albumin compositions.

The nanoparticle and albumin-paclitaxel compositions can accumulate in tumor tissues due to the EPR effect caused by "leaky" vessels in the tumor. The albumin-specific gp60 receptor (albondin) transports albumin through the EC by transcytosis of the receptor within a crypt-like invagination of the cell membrane at the cell surface. This transcytosis mechanism allows albumin-paclitaxel to be transported into the underlying interstitial space. In contrast, the polyoxyethylated castor oil in taxol inhibits the binding of taxol to albumin, greatly reducing the transport of taxol to tumors. In addition, gp16 and gp30 receptors are also involved in the intracellular transport of modified albumin containing bound paclitaxel, resulting in increased binding of paclitaxel to endothelial cells with greater anti-angiogenic effect compared to taxol.

Example 40

This example illustrates the increase in endothelial transcytosis of a pharmaceutical composition comprising paclitaxel and albumin compared to paclitaxel.

Human lung microvascular endothelial cells (HLMVEC) were cultured on transwell to confluence. The pharmaceutical composition containing paclitaxel and albumin or taxol of the present invention at a concentration of 20 μ g/mL was added to the upper transwell chamber.

The transport of paclitaxel from the upper chamber into the lower chamber by transcytosis was continuously monitored using a fluorometer. A control containing flutmax alone without albumin was also used. Controls containing flutmax showed no transport, demonstrating the integrity of confluent HLMVEC monolayers. The transport of paclitaxel from the albumin-paclitaxel composition was much faster than that from taxol in the presence of 5% HSA (physiological concentration). Transport rate constant (K)_t) For albumin-paclitaxel composition and taxol, respectively, 1.396hr^-1And 0.03hr^-1. The total amount of paclitaxel transported across the monolayer by the albumin-paclitaxel composition was 3 times higher than the total amount of paclitaxel transported across the monolayer by the taxol.

EXAMPLE 41

This example illustrates the improved Endothelial Cell (EC) binding of a pharmaceutical composition containing paclitaxel and albumin compared to taxol.

Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in 96-well microtiter plates. In one experiment, polyoxyethylated castor oil EL/EtO was used at increasing concentrationsHUVEC were reacted with paclitaxel (Flutax-Oregon Green labeled paclitaxel) in the presence of H (carrier for taxol). In another experiment, pharmaceutical compositions containing albumin and flutmax and the taxol-flutmax composition reacted with HUVEC at various final concentrations. The binding of paclitaxel to cells is inhibited by polyoxyethylated castor oil. 0.02% IC by polyoxyethylated Castor oil EL/EtOH₅₀Inhibition is shown. This concentration of polyoxyethylated castor oil has been shown to last at least 24 hours during taxol chemotherapy. Thus, it is an in vivo relevant process. At all concentrations tested, a significant amount of paclitaxel from the albumin-paclitaxel composition bound to the cells. In contrast, little or no binding was observed for taxol.

Example 42

This example illustrates the improved albumin binding of a pharmaceutical composition containing paclitaxel and albumin compared to taxol.

Human Serum Albumin (HSA) was immobilized on plastic ELISA plates. Paclitaxel (Flutax-Oregon Green labeled paclitaxel) was reacted with immobilized HSA in the presence of increasing concentrations of polyoxyethylated castor oil EL/EtOH. In another experiment, albumin-paclitaxel-flutmax and taxol-flutmax compositions were reacted with immobilized HSA at a final concentration of 20 μ g paclitaxel/mL. The binding of paclitaxel to albumin is inhibited by polyoxyethylated castor oil. 0.003% IC by Polyoxyethylene Castor oil EL/EtOH₅₀Inhibition is shown. This concentration of polyoxyethylated castor oil has been shown to last at least 24 hours during taxol chemotherapy. Thus, it is an in vivo relevant process. At the relevant drug paclitaxel concentration (20 μ g/mL), a significant amount of paclitaxel from the albumin-paclitaxel composition binds to immobilized HSA. In contrast, no binding was observed for taxol.

Example 43

This example illustrates the increased transport of paclitaxel to albumin for a pharmaceutical composition containing paclitaxel and albumin compared to paclitaxel.

The taxol-flutmax and albumin-paclitaxel-flutmax compositions were mixed at 20 μ g/mL,40 μ g/mL, and 80 μ g/mL with 5% HSA in Hanks buffer or with serum. The mixture was immediately separated on a native 3-14% polyacrylamide gel and the amount of paclitaxel bound to albumin was determined by scanning fluorimetry. Paclitaxel is transported to HSA faster for albumin-paclitaxel compositions than paclitaxel. When serum or 5% HSA was incubated with either the albumin-paclitaxel-Flutax composition or the taxol-Flutax composition, more paclitaxel was co-electrophoresed with HSA. Compared to the taxol-Flutax composition, 20, 40, and 80 ug/ml of the albumin-paclitaxel-Flutax composition transported 45%,60%, and 33% more paclitaxel, respectively, to HSA after exposure to 5% HSA. For 260mg/m²ABI-007, C of_maxAbout 20. mu.g/mL, and this is therefore an important in vivo process.

Example 44

This example illustrates that the glycoprotein receptor gp60 is responsible for albumin-paclitaxel binding and transcytosis.

The fluorescently labeled paclitaxel (Flutax) albumin composition is contacted with microvascular endothelial cells in culture. Fluorescence staining was observed under the microscope, as evidenced by the spot area, which is presumed to be the gp60 receptor bound to albumin-paclitaxel. This was confirmed by using rhodamine-labeled albumin, which is identical in spot fluorescence position to paclitaxel.

Example 45

This example illustrates that an increased amount of albumin can compete with the binding of paclitaxel.

Albumin was immobilized on a microtiter plate. Fluorescent paclitaxel was added to the wells and paclitaxel binding was measured using a scanning fluorometer. An increasing amount of albumin was added to the wells and the level of inhibition of paclitaxel bound to the immobilized albumin was measured. The data show that as the amount of albumin added increases, a corresponding decrease in binding is observed. A similar effect was observed for binding to endothelial cells. This shows that higher albumin concentrations inhibit paclitaxel binding. Compositions of the invention having lower amounts of albumin are preferred.

Example 46

This example illustrates that lower amounts of albumin in the pharmaceutical compositions of the invention result in stable compositions.

To investigate whether a lower amount of albumin in the composition affects the stability of the pharmaceutical composition of the present invention, an albumin-paclitaxel composition containing a low amount of albumin was prepared. When examining typical parameters of paclitaxel efficacy, impurity formation, particle size, pH and other stability at different temperatures (2-8 ℃, 25 ℃ and 40 ℃), these compositions were found to be as stable as compositions with higher amounts of albumin. Compositions with lower amounts of albumin are therefore preferred as this can greatly reduce costs and allow increased binding and transport to cells.

Example 47

This example illustrates a pharmaceutical composition containing albumin and paclitaxel with a high albumin to paclitaxel ratio.

30mg of paclitaxel was dissolved in 3.0ml of dichloromethane. The solution was added to 27.0ml of human serum albumin solution (3% w/v) (corresponding to a ratio of albumin to paclitaxel of 27). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The obtained dispersion was translucent and the obtained paclitaxel particles had a typical average diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.

Example 48

This example illustrates a pharmaceutical composition containing albumin and paclitaxel with a low albumin to paclitaxel ratio.

Specifically, 300mg of paclitaxel was dissolved in 3.0ml of dichloromethane. The solution was added to 27ml of human serum albumin solution (5% w/v) (corresponding to an albumin to paclitaxel ratio of 4.5). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The obtained dispersion was translucent and the obtained paclitaxel particles had a typical average diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization.

It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.

Example 49

This example illustrates a pharmaceutical composition containing albumin and paclitaxel with an intermediate albumin to paclitaxel ratio.

Specifically, 135mg of paclitaxel was dissolved in 3.0ml of dichloromethane. The solution was added to 27ml of human serum albumin solution (5% w/v). Deferoxamine is added as required. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion, which was then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000psi while the emulsion was recycled for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 ℃ under reduced pressure (30mm Hg) for 20-30 minutes. The obtained dispersion was translucent and the obtained paclitaxel particles had a typical average diameter of 50-220nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted to the original dispersion by addition of sterile water or saline. The particle size after reconstitution was the same as before lyophilization. In the composition of the present invention, the calculated ratio (w/w) of albumin to paclitaxel is about 10.

It should be recognized that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The pharmaceutical composition containing albumin of the present invention shows significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.

Example 50

This example illustrates the treatment of rheumatoid arthritis in an animal model with an albumin-paclitaxel composition.

The collagen-induced arthritis model in Louvain rats was used to examine the therapeutic effect of albumin-paclitaxel compositions on arthritis. Paw size was monitored in experimental animals to assess the severity of arthritis.

After arthritis has fully developed (usually about 9-10 days after collagen injection), the experimental animals are divided into different groups to receive either albumin-paclitaxel 1mg/kg q.o.d, or albumin-paclitaxel 0.5mg/kg + prednisone 0.2mg/kg q.o.d. (combination therapy) intraperitoneally for 6 doses, followed by one dose every week for three weeks. Paw size was measured at the beginning of treatment (day 0) and at each injection of drug. Only one group receiving physiological saline served as a control. At the end of the experiment, a 42% reduction in paw size was achieved in the group receiving albumin-paclitaxel, the combination treatment group showed a 33% reduction in paw size, while the control group increased paw size by about 20% compared to the start of treatment.

In summary, the albumin-paclitaxel composition showed therapeutic effect on arthritis. The albumin-paclitaxel combination may be localized to the site of the arthritic injury through receptor-mediated mechanisms such as gp60 transport.

Example 51

This example illustrates the use of an albumin-paclitaxel composition for the treatment of cardiovascular restenosis.

Paclitaxel eluting stents (Paclitaxel eluting stents) in animals resulted in incomplete healing and, in some cases, a lack of sustained inhibition of neointimal growth in arteries. This study examined the efficacy of the novel systemically delivered albumin-paclitaxel composition of the present invention for reducing in-stent restenosis.

The saline reconstituted albumin-paclitaxel was tested in 38 new zealand white rabbits receiving bilateral iliac artery stents. Administering a dose of albumin-paclitaxel (1.0-5.0mg/kg paclitaxel dose) as a 10-minute intra-arterial infusion; control animals received vehicle (0.9% saline).

In the following chronic experiments, 5.0mg/kg albumin-paclitaxel was delivered to the scaffold and repeated intravenous doses of 3.5-mg/kg albumin-paclitaxel were administered on day 28; these studies were terminated at 3 months. On day 28, mean neointimal thickness (p < =0.02) was reduced by dosing albumin-paclitaxel > =2.5mg/kg, as evidenced by prolonged healing. However, the efficacy of a single dose of 5.0mg/kg albumin-paclitaxel was lost 90 days ago. In contrast, a second repeat administration of 3.5mg/kg albumin-paclitaxel 28 days after stenting resulted in a sustained inhibition of neointimal thickness at 90 days (p < =0.009 compared to a single dose of 5.0mg/kg albumin-paclitaxel and control), with the neointimal nearly completely healing.

Although systemic albumin-paclitaxel reduced neointimal growth at 28 days, a single repeat dose was required for sustained neointimal inhibition. Thus, the compositions of the present invention are useful in the treatment of cardiovascular diseases such as restenosis. The compositions of the invention comprising agents other than paclitaxel (e.g., rapamycin, other taxanes, epothilones, etc.) are all suitable for use in the treatment of restenosis in vascular or artificial vascular grafts, such as those used for arterio-venous access in patients in need of hemodialysis.

Claims (25)

1. A pharmaceutical composition comprising paclitaxel and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, wherein the ratio (weight/weight) of albumin to paclitaxel is from about 1:1 to about 9:1, and wherein the pharmaceutical composition comprises nanoparticles comprising paclitaxel and albumin.

2. The pharmaceutical composition of claim 1, wherein the nanoparticles have a particle size of less than about 200 nm.

3. The pharmaceutical composition of claim 1, wherein said albumin is human serum albumin.

4. The pharmaceutical composition of claim 3, wherein the nanoparticles have a particle size of less than about 200 nm.

5. The pharmaceutical composition of claim 1, wherein the ratio (weight/weight) of albumin to paclitaxel in the pharmaceutical composition is from about 1:1 to about 5: 1.

6. The pharmaceutical composition of claim 1, wherein the ratio (weight/weight) of albumin to paclitaxel in the pharmaceutical composition is about 9: 1.

7. The pharmaceutical composition of claim 6, wherein the nanoparticles have a particle size of less than about 200 nm.

8. The pharmaceutical composition of claim 1, wherein the composition comprises from 0.1% to 25% by weight albumin.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition comprises from 0.5% to 5% by weight albumin.

10. The pharmaceutical composition of claim 1, wherein the paclitaxel is substantially free of polyoxyethylene castor oil。

11. The pharmaceutical composition of any one of claims 1-9, wherein the pharmaceutical composition is dehydrated.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is lyophilized.

13. The pharmaceutical composition of any one of claims 1-9, wherein the pharmaceutical composition is a liquid.

14. The pharmaceutical composition of claim 13, wherein the composition has a pH of 5.0 to 8.0.

15. The pharmaceutical composition of claim 13, wherein the composition comprises saline.

16. The pharmaceutical composition of any one of claims 1-9, wherein the composition is sterile.

17. The pharmaceutical composition of any one of claims 1-9, wherein the composition is a unit dose.

18. The pharmaceutical composition of any one of claims 1-9, wherein the composition is multiple doses.

19. The pharmaceutical composition of any one of claims 1-9, wherein the composition is contained in a sealed container.

20. Use of a pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of cancer.

21. The use of claim 20, wherein the cancer is lung cancer.

22. The use of claim 20, wherein the cancer is breast cancer.

23. The use of claim 20, wherein the cancer is ovarian cancer.

24. The use of claim 20, wherein the cancer is pancreatic cancer.

25. Use of a pharmaceutical composition according to any one of claims 1 to 9 in the manufacture of a medicament for the treatment of cardiovascular disease.