RU2522977C2 - Compositions and methods of delivery of pharmacological agents - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a pharmaceutical composition for the delivery of a pharmaceutical agent to a focus of a disease. The composition contains a water-insoluble pharmaceutical agent which is paclitaxel, a pharmaceutically acceptable carrier which is albumin, preferentially human serum albumin. The relation (wt/wt) of albumin to paclitaxel makes 9:1. The pharmaceutical composition contains nanoparticles containing paclitaxel and albumin wherein the nanoparticles have a size of less than 200 nm.

EFFECT: administering the pharmaceutical composition according to the invention provides enhanced characteristics of the delivery of paclitaxel to the site of the disease and reduced adverse side effects.

24 cl, 5 tbl, 51 ex

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This patent application claims priority on the basis of a provisional application for the grant of US patent No. 60/432317 filed on December 9, 2002, a provisional application for the grant of a US patent (attorney dossier No. 225519), filed on December 3, 2003, a provisional application for the grant of a patent ( dossier of Attorney No. 225549) filed on December 4, 2003 and a provisional application for the grant of a US patent (dossier of Attorney No. 225585) filed on December 5, 2003.

FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions containing pharmaceutically active agents for parenteral or other internal use, which, when administered, have the effect of reducing some undesirable side effects when compared with available drugs of similar drugs.

BACKGROUND

It is well known that many drugs for parenteral use, especially drugs administered intravenously, cause unwanted side effects such as vein irritation, phlebitis, burning and pain during injection, venous thrombosis, hemorrhage and others associated with the administration, side effects. Many of these drugs are insoluble in water, and are thus prepared using solubilizing agents, surfactants, solvents and / or emulsifiers that are irritating, allergenic or toxic when administered to patients (see, for example, Briggs et al., Anesthesis 37, 1099 (1982) and Waugh et al., Am. J. Hosp. Pharmacists , 48, 1520 (1991)). Often, unrelated drugs presented in the preparation cause pain and irritation when administered. For example, phlebitis was observed in 50% of patients who received ifosfamide and vinorelbine as initial chemotherapy for advanced non-small cell lung carcinoma through peripheral vein (see, for example, Vallejo et al., Am. J. Clin. Oncol ., 19 ( 6), 584-8 (1996)). In addition, vancomycin has been shown to cause side effects such as phlebitis (see, for example, Lopes Rocha et al., Braz. J. Infect. Dis ., 6 (4), 196-200 (2002)). The use of cisplatin, gemcitabine and SU5416 in patients with solid tumors led to the appearance of adverse reactions, such as deep vein thrombosis and phlebitis (see, for example, Kuenen et al., J. Clin. Oncol., 20 (6), 1657-67 ( 2002)). In addition, propofol, an anesthetic, can cause soreness when injected, burning and irritating veins, especially when a lecithin-stabilized fat emulsion is administered (see, for example, Tan et al., Anathesia , 53, 468-76, (1998) ) Other drugs that exhibit side effects associated with their administration include, for example, taxol (paclitaxel) (see, for example, the package insert in the package of taxol for intravenous use), cordarone (amiodarone hydrochloride) (see, for example, cordardone package insert for intravenous use), thyroid hormone T3 or lyiotironin (commercially available as a triostat), thiotepa, bleomycin and diagnostic radiopaque agents.

Another problem associated with the industrial production of drugs for injection, especially water-insoluble drugs, is sterility. Sterile production of medicinal emulsions / dispersions can be carried out with full sterilization of all components before production, followed by an absolutely aseptic technique at all stages of industrial production. However, such methods are laborious and expensive. In addition, the oxidation of drugs under the influence of air during industrial production or storage can lead, for example, to a decrease in pH, degradation of the drug and discoloration, thereby violating the stability of the drug and / or shortening the shelf life.

In order to overcome the problems associated with side effects related to the introduction of drugs, they tried to prepare alternative drugs. With respect to propofol, for example, methods for reducing the pain caused by the administration of propofol include increasing the fat content in the solvent (e.g. long chain triglycerides (LCT)), pre-medication preparation, pre-treatment with non-steroid drugs, local anesthetics, opiates, addition of lidocaine, cyclodextrin addition and microfiltration (see, e.g., Mayer et al., Anaesthesist , 45 (11), 1082-4 (1996), Davies, et al. Anaesthesia , 57, 557-61 (2002), Doenicke, et al., Anaesth. Analg ., 82, 472-4 (1996), Larsen et al., Anaesthesist , 50, 842-5 (2001) , Lilley et al., Anaesthesia , 51, 815-8 (1996), Bielen et al., Anaesth. Analg ., 82 (5), 920-4 (1996) and Knibbe et al., Br. J. Clin. Pharmacol ., 47 (6), 653-60 (1999)). These formulations, however, cause other side effects (for example, cardiovascular complications) or cause a violation of the stability of emulsions with propofol.

In order to overcome the problem of bacterial contamination, propofol preparations with antibacterial agents such as an EDTA analogue (e.g., edetate), pentetate or sulfite-containing agents were developed, or they were prepared at a lower pH value (see, for example, US Pat. 5714520, 5731355, 5731356, 6028108, 6100302, 6147122, 6177477, 6399087, 6469069 and provisional patent application No. WO 99/39696). Despite the fact that edetate and pentetate are chelators of metal ions, they, however, can be dangerous by removing important metal ions from the body system. Moreover, the addition of sulfites to the composition of drugs is the cause of possible side effects in children and in those people in the general population who are allergic to sulfur.

Thus, there remains a need for a composition and method that reduce or remove side effects associated with parenteral or in vivo administration of drugs. There is also a need for a pharmaceutical composition that is sterile and in methods for preparing such a composition. Additionally, there is a need for a pharmaceutical composition and method that reduces or eliminates the oxidation of pharmaceuticals to prevent drug resistance.

The present invention relates to such compositions and methods. Other advantages of the invention exist, as well as additional features of the invention will be apparent from the description of the invention presented here.

SUMMARY OF THE INVENTION

The invention relates to various embodiments of pharmaceutical compositions. One, some or all of the properties of different variants can be found in different implementations and yet they are within the scope of the attached claims.

The invention relates to a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which 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 when the pharmaceutical composition is administered to a human, and in which the pharmaceutically acceptable carrier contains deferoxamine in an amount effective to inhibit the growth of microorganisms in the pharmaceutical tion of the composition. The invention also relates to a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects when the pharmaceutical composition is administered to a human, and in which a pharmaceutically acceptable carrier includes deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.

The invention relates to a method of reducing one or more side effects associated with the administration of a pharmaceutical composition to a person, comprising (a) administering to the person a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises albumin and deferoxamine. The invention also relates to methods for inhibiting the growth of microorganisms or inhibiting oxidation, or inhibiting the growth of microorganisms and oxidation in a pharmaceutical composition. These methods encompass the preparation of a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier in which the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit the growth of microorganisms, or in an amount effective to inhibit oxidation in the pharmaceutical composition.

The invention also relates to a method for increasing the transport of a pharmaceutical agent to a disease focus, which comprises administering to a human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises albumin and in which the ratio of albumin to pharmaceutical agent in the pharmaceutical composition is approximately 18: 1 or less. The invention further relates to a method for increasing the binding of a pharmaceutical agent to an in vitro or in vivo cell, wherein the method comprises applying to said cells an in vitro or in vivo pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises albumin and in which albumin and a pharmaceutical agent in the pharmaceutical composition is approximately 18: 1 or less.

The invention also relates to a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises albumin in an amount effective to increase the transport of the drug to the focus of the disease in humans, and in which the ratio of albumin to pharmaceutical agent is approximately 18: 1 or less.

The invention also relates to a method for increasing the transport of a pharmaceutical agent to a cell.in vitro orin vivoby combining the specified agent with a protein, where the specified protein binds to a specific receptor on the cell surface of the specified cell, where the specified binding of a combination of a protein and a pharmaceutical agent to the specified receptor causes transport, and where the ratio of protein and pharmaceutical agent is approximately 18: 1 or less.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to 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 when the pharmaceutical composition is administered to a human, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition. The invention also relates to a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects when the pharmaceutical composition is administered to a human, and in which a pharmaceutically acceptable carrier includes deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.

Any suitable pharmaceutical agent may be used in the pharmaceutical composition of the invention. Suitable pharmaceutical agents include, but are not limited to, anticancer agents or antitumor agents, microtubule agents, immunosuppressive agents, anesthetics, hormones, agents for use in cardiovascular disorders, antiarrhythmic agents, antibiotics, antifungal agents, antihypertensive agents, anti-asthma drugs, analgesics, anti-inflammatory agents, anti-arthritic agents and vasoactive agents. The invention is also applicable to many other classes of drugs. More specifically, suitable pharmaceutical agents include, but are not limited to, taxanes (e.g., Taxol ^® (paclitaxel) and Taxotere ^™ (docetaxel)), epothilones, campothecin, colchicine, amiodarone, thyroid hormones, vasoactive peptides (e.g., vasoactive intestinal peptide) , amphotericin, corticosteroids, propofol, melatonin, cyclosporin, rapamycin (sirolimus), tacrolimus, mycophenolic acids, ifosfamide, vinorelbine, vancomycin, gemcitabine, SU5416, thiotepu, bleomycin, diagnostic radiocontrast agents and their derivatives. Other drugs that are applicable in the composition of the invention are described, for example, in US Pat. No. 5,916,596 and in U.S. Patent Application Serial No. 09/446783. Preferably, the pharmaceutical agent is propofol, paclitaxel or docetaxel. More preferably, the pharmaceutical agent is propofol or paclitaxel. Most preferably, the pharmaceutical agent is propofol.

Taxol ^® (paclitaxel) (Bristol-Myers Squibb) is active against carcinomas of the ovary, breast, lung, esophagus and head, and neck. However, taxol has been shown to cause toxicity associated with its administration, as well as significant acute and cumulative toxicity, such as myelosuppression, neutropenic fever, anaphylactic reaction and peripheral neuropathy. Since paclitaxel is slightly soluble in water, cremophor is typically used as a solvent requiring large volumes of infusions and special tubes and filters. Cremophor is associated with side effects that can be serious, including anaphylaxis and other allergic reactions that may require pretreatment with corticosteroids, antihistamines, and H2 blockers (see, for example, Gelderblom et al., Eur. J. Of Cancer , 37 , 1590-1598, (2001)). Taxotere ^TM (docetaxel) is used in the treatment of anthracycline-resistant breast cancer, but also, as previously described, causes side effects in the form of allergies and swelling, which can be severe. Epothilone (and its derivatives) is also typically administered with cremophor, and has been shown to cause severe neutropenia, allergies, and neuropathy.

Propofol (2,6-diisopropylphenol) is a hydrophobic water-insoluble oil that is widely used as an intravenous anesthetic to cause and maintain general anesthesia and sedation in humans and animals. Propofol is typically injected directly into the bloodstream, and it passes through the blood-brain barrier. Pharmaceutical compositions containing propofol must be sufficiently fat soluble in order to pass through this barrier and have an inhibitory effect on the corresponding mechanisms of the brain. The maximum solubility of propofol in water is 1.0 ± 0.02 μM at a temperature of 22.5 ° C (see, for example, Tonner et al., Anesthesiology , 77, 926-931 (1992)). As such, propofol is generally prepared as an emulsion containing solubilizing agents, surfactants, solvents, or as an oil-in-water emulsion (see, for example, US Pat. Nos. 6,150,423, 6,326,406 and 6,362,234). The compositions of the present invention include, in addition to the active pharmaceutical agent, pharmaceutical carriers or excipients. The choice of carrier is not necessarily decisive, and any of the carriers known in the art can be used in the composition. The choice of carrier is preferably partially determined by the particular site into which the pharmaceutical composition is to be administered and the specific method used to administer the pharmaceutical composition. Preferably, the pharmaceutically acceptable carrier includes proteins. 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 including albumin suitable for the invention may be naturally occurring or synthetically prepared.

Human Serum Albumin (HSA) is a highly soluble globular protein M _r 65K and consists of 585 amino acids. HSA is present in the largest amount in plasma and accounts for 70-80% of the colloidal osmotic pressure of human plasma. The amino acid sequence of HSA contains a total of 17 disulfide bridges, one free thiol (Cys 34) and one tryptophan (Trp 214). Intravenous use of HSA solutions is prescribed for the prevention and treatment of hypovolemic shock (see, for example, Tullis, JAMA , 237, 355-360, 460-463, (1977) and Houser et al., Surgery, Gynecology and Obstetrics , 150, 811- 816 (1980)) and in conjunction with blood transfusion for the treatment of neonatal hyperbilirubinemia (see, for example, Finlayson, Seminars in Thrombosis and Hemostasis , 6, 85-120, (1980)).

Human serum albumin (HSA) contains many hydrophobic binding sites (a total of eight for fatty acids, the endogenous HSA ligand) and binds a diverse range of drugs, especially neutral and negatively charged hydrophobic compounds (Goodman et al., The Pharmacological Basis of Therapeutics , 9 ^{th ed, McGraw-Hill New York} ( 1996)). The presence of two high affinity binding sites was suggested in the IIA and IIIA HSA subdomains, which are highly elongated hydrophobic pockets with charged lysine and arginine residues on the surface, which function as attachment points of the polar structural elements of the ligand (see, for example, 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 to HSA (see, for example, 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., Arzneimittelforschung , 45, 1053-6 (1995) and Garrido et al., Rev. Esp. Anestestiol. Reanim ., 41, 308-12 ( 1994)). Additionally, it was shown that docetaxel binds to human plasma proteins (see, for example, Urien et al., Invest. New Drugs, 14 (2), 147-51 (1996)). Thus, although not wanting to be attached to any particular theory, it is believed that the inclusion of proteins, such as albumin, in the pharmaceutical compositions of the invention reduces the side effects associated with the introduction of the pharmaceutical composition, which is a consequence of at least in part, the binding of human serum albumin to any drug present in the composition in free form.

The amount of albumin in the pharmaceutical composition of the present invention will vary depending on the active pharmaceutical agent, other excipients, method of application and place of intended administration. Preferably, the amount of albumin in the composition is an amount effective to reduce one or more side effects of the active pharmaceutical agent due to the administration of the pharmaceutical composition of the invention to humans. Typically, the pharmaceutical composition is prepared in liquid form, and then albumin is added to the solution. Preferably, the pharmaceutical composition in liquid form contains from about 0.1 to about 25 wt.% (For example, about 0.5 wt.%, About 5 wt.%, About 10 wt.%, About 15 wt.% Or about 20 wt.%) albumin. Most preferably, the pharmaceutical composition in liquid form contains from about 0.5 to about 5 wt.% Albumin. The pharmaceutical composition can be dehydrated, for example, by lyophilization, by spray drying, by fluidized bed drying, wet granulation, and other suitable methods known in the art. When the composition is prepared in solid form, such as by wet granulation, by drying in a fluidized bed and by other methods known to those skilled in the art, albumin is preferably used with an active pharmaceutical agent and other excipients, if present, as a solution. The HSA solution preferably contains from about 0.1 to about 25 wt.% (About 0.5 wt.%, About 5 wt.%, About 10 wt.%, About 15 wt.% Or about 20 wt.%) Albumin.

In addition to albumin, the compositions of the present invention preferably contain deferoxamine. Deferoxamine is a natural product isolated from Streptomyces pilous , and it is able to form complexes with iron. Deferoxamine injection mesylate according to USP requirements, for example, is approved by the Food and Drug Administration (USA) as a chelating agent for iron, and is available for intramuscular, subcutaneous and intravenous administration. Deferoxamine mesylate, according to USP requirements, is a powder from white to almost white. It is freely soluble in water, and its molecular weight is 656.79. The chemical name of deferoxamine mesylate is N- [5- [3 - [(5-aminopentyl) hydroxycarbamoyl] propionamido] pentyl] -3 - [[5 - ((N-hydroxyacetamido) pentyl] carbamoyl] propionhydroxamic acid monomethanesulfonate, and its structural formula is C ₂₅ H ₄₈ N ₆ O ₈ .CH ₃ SO ₃ H. As described in the examples, deferoxamine or its analogs, derivatives or salts (eg, mesylate salts) inhibit the growth of microorganisms and inhibit oxidation in the pharmaceutical composition. it was shown that deferoxamine binds to phenolic compounds (see. for example, Juven et al., J. Appl . Bacteriol ., 76 (6), 626-31 (1994)). Paclitaxel, docetaxel, propofol, and the like, are either phenolic-like or contain phenolic or phenyl substituents Therefore, it is believed that deferoxamine can contact the drug in free form or reduce its amount in the pharmaceutical composition of the invention, thus also reducing or weakening the irritation or pain during injection.

The amount of deferoxamine and its preferred salt, that is, the salt of deferoxamine mesylate included in the composition, depends on the active pharmaceutical agent and other excipients. Preferably, the amount of deferoxamine, its salt and its analogues in the composition is an amount effective to inhibit the growth of microorganisms and / or inhibit oxidation. As described above, the pharmaceutical composition is typically prepared in liquid form, and deferoxamine, its salts and its analogs are then added to the solution. Preferably, the pharmaceutical composition in liquid form contains from about 0.0001 to about 0.5 wt.% (For example, about 0.005 wt.%, About 0.1 or about 0.25 wt.%) Of deferoxamine, its salts or its analogues . More preferably, the composition in liquid form contains similar amounts of a preferred salt of deferoxamine, deferoxamine mesylate. Most preferably, the pharmaceutical composition in liquid form contains about 0.1 wt.% Deferoxamine mesylate. When the composition is prepared in solid form, as described above, such as by wet granulation, by drying in a fluidized bed, and by other methods known to those skilled in the art, the deferoxamine mesylate is preferably used with an active pharmaceutical agent and other excipients, if present, as a solution. The deferoxamine mesylate solution preferably contains from about 0.0001 to about 0.5 wt.% (For example, about 0.005 wt.%, About 0.1 or about 0.25 wt.%) Deferoxamine.

In accordance with the invention, the pharmaceutical composition may contain other agents, excipients or stabilizers in order to improve the properties of the composition. For example, to increase stability by increasing the negative zeta potential of nanoparticles or nanodroplets, certain negatively charged components can be added. Such 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, taurochenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, dehydrocholic acid; phospholipids, including lecithin-based phospholipids (egg yolk), which include the following phosphatidylcholines: palmitoyloleoleoylphosphatidylcholine, palmitoylilinoleoylphosphatidylcholine, stearoyllineoleoloylphospholidoliphosphonylphosphonylphosphonylphosphonidylphosphonylphosphonidylphosphonylphosphonidylphosphonylphosphonidylphosphonidylphosphonylphosphonidylphosphonidyl Other phospholipids include L-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), D-α-phosphatidylcholine-γ-β-γ-β-β-γ-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-β-Compound-Compound Compounds of the Composition of Other Phospholipids. 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 -arachidonoyl-γ-O-hexadecyl, L-α-phosphatidylcholine-β-acetyl-γ-O- (octad-9-cis-enyl), D-α-phosphatidylcholine-β-arachidonoyl-γ-O-palmitoyl, 3 -sn-phosphatidylcholine-2-arachidonoyl-1-stearoyl, L-α-phosphatide lcholine-β-arachidonoyl-γ-stearoyl, L-α-phosphatidylcholinediarachidoyl, L-α-phosphatidylcholindibehenoyl, L-α-phosphatidylcholine-β- (cis-8,11,14-neucosatrienoyl) -γ-O-hexadecyl α-phosphatidylcholine-β-oleoyl-γ-myristoyl, L-α-phosphatidylcholine-β- (pyrene-1-yl) decanoyl-γ-palmitoyl, 3-sn-phosphatidyl-N, N-dimethylethanolamine-1,2-dipalmitoyl , L-α-phosphatidylethanolamine-diheptadecanoyl, 3-sn-phosphatidylethanolamine-1,2-dilauroyl, 3-sn-phosphatidylethanolamine-1,2-dimyristoyl, 3-sn-phosphatidylethanolamine-1,2-dioleyl, 3-sn-phosphatidylethanolamine-1 , 2-dipalmitoyl, L-α-phosphatidylethanolamindipalmitoyl, L-α-f osfatidylethanolamindipalmitoyl-N-dansil, L-α-phosphatidylethanolamindipalmitoyl-N, N-dimethyl, L-α-dimyristoylphosphatidylglycerol (sodium salt) (DMPG), dipalmitoylphosphatidylglycerol (sodium salt) (sodium salt), sodium salt) - (carbonyl-methoxypolyethylene glycol 2000) -1,2, -distearoyl-sn-glycero-3-phosphoethanolamine sodium (MPEG-DSPE), sodium salt of didecanoyl-L-α-phosphatidic acid, sodium salt of diheptadecanoyl-L-α-phosphatidic acid, sodium salt of 1,2-dimyristoyl-3-sn-phosphatidic acid, sodium salt of dioctanoyl-L-α-phosphatidic acid, sol l sodium dioleoyl-L-α-phosphatidic acid, sodium salt of dipalmitoyl-L-α-phosphatidic acid, sodium salt of dimyristoyl-L-α-phosphatidyl-DL-glycerol, sodium salt of dioleyl-L-α-phosphatidyl-DL-glycerol, ammonium salt of dipalmitoyl-L-α-phosphatidyl-DL-glycerol, ammonium salt of distearoyl-L-α-phosphatidyl-DL-glycerol, ammonium salt of L-α-phosphatidyl-DL-glycerol-β-oleoyl-γ-palmitoyl, ammonium salt L-α-phosphatidylinositol, Sodium salt of L-α-phosphatidylinazitol, Sodium salt of L-α-phosphatidyl-L-serinedioleoleil, sodium salt of L-α-phosphatidyl-L-serine and dipalmitoi a. Negatively charged surfactant emulsifier compounds are also suitable as additives, for example sodium cholesteryl sulfate and the like.

A pharmaceutical agent (eg, propofol) can be used alone or dissolved in a water-immiscible solvent. A wide range of water-immiscible solvents such as soybean, safflower, cottonseed, corn, sunflower, peanut, castor or olive oil can be used. Preferred oil is a vegetable oil, of which soybean oil is most preferred. Soybean oil can be used in the composition in the range of 1 to 10 wt.%. Preferably, soybean oil is present in the pharmaceutical composition in an amount of about 3 wt.%

The pharmaceutical composition of the invention can be stabilized with a pharmaceutically acceptable surfactant. As used herein, the term “surfactant” refers to a surfactant group (s) of amphiphilic molecules. Surfactants can be anionic, cationic, nonionic and zwitterionic. Any surfactant may be included in the pharmaceutical composition of the invention. Suitable surfactants include nonionic surfactants such as phosphatides, sorbitan polyoxyethylene esters, and tocopheryl polyethylene glycol succinate. Preferred surfactants are egg lecithin, tween 80, and vitamin E-td-α-tocopheryl polyethylene glycol-1000 succinate (TPGS). For preparations containing soybean oil, egg lecithin is preferred and is not more than 1.2% by weight for a preparation containing 3% soybean oil, preferably at 1.1% by weight of the composition. For non-soybean oil preparations, Tween 80 or Vitamin E-TPGS are preferred surfactants. Typically, 0.1 to 1.5 wt.% Tween 80 or 0.5 to 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: Theory and Practice , Robert E. Krieger Publishing, Malabar, Fla. (1965).

There is a wide variety of suitable formulations of the pharmaceutical composition of the invention (see, for example, US Pat. No. 5,916,596). The following preparations and methods are illustrative only and in no way limiting. Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound, dissolved in solvents, such as water, saline or orange juice, (b) capsules, sachets or tablets, each of which contains a preliminary a certain amount of the active ingredient as solid particles or granules, (c) suspensions in suitable liquids, and (d) suitable emulsions. Tablet forms may include one or more fillers of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid and other fillers, dyes, solvents buffering agents, moisturizing agents, preservatives, flavoring agents and pharmacologically compatible excipients. Forms of medicinal candies may contain the active ingredient in the flavoring, usually sucrose and acacia or tragacanth, as well as lozenges containing the active ingredient in an inert basis, such as gelatin and glycerin or sucrose and acacia, emulsions, gels and the like, containing in addition to the active component such fillers that are known in this field.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions that contain antioxidants, buffers, antimicrobial additives and solutions that render the preparations isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. The preparations can be presented in unit dose or multiple doses, sealed in containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid excipient, for example, water for injection immediately before use. Solutions and suspensions for injection prepared for immediate administration can be prepared from sterile powders, granules and tablets of the previously described type. Injectable drugs are preferred.

Formulations suitable for aerosol administration contain the pharmaceutical composition of the invention, including aqueous and non-aqueous isotonic sterile solutions, which may contain antioxidants, buffers, antimicrobial additives and solutions, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents agents, solubilizers, thickeners, stabilizers and preservatives alone or in combination with other suitable components that can be prepared in aerosol formulations which s to be administered via inhalation. Aerosol formulations may be placed in sealed acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. They can also be formulated as pharmaceuticals for non-pressure preparations such as a nebulizer or atomizer.

Other suitable preparations are possible, for example, suppositories can be prepared using a variety of bases, such as emulsifying bases or water soluble bases. Preparations suitable for vaginal administration can be provided as pessaries, tampons, creams, gels, pastes, foams or aerosol formulations containing, in addition to the active component, carriers which are known in the art to be suitable.

In a preferred embodiment of the invention, the pharmaceutical composition is prepared so that it has a pH in the range from 4.5 to 9.0, and more preferably a pH from 5.0 to 8.0. The pharmaceutical composition can also be prepared so that it is isotonic with blood, adding a suitable tonicity modifier such as glycerin. In addition, the pharmaceutically acceptable carrier preferably also contains pyrogen-free water or water for injection according to USP requirements. Preferably, the pharmaceutical composition of the invention is prepared as a sterile aqueous preparation, a nanoparticle, an oil-in-water emulsion or a water-in-oil emulsion.

For a pharmaceutical composition containing propofol, according to the invention, an oil-in-water emulsion is prepared by dissolving propofol in a water-immiscible solvent alone and preparing an aqueous phase containing albumin, deferoxamine, a surfactant and other water soluble components, and mixing the oil and water phase. The primary emulsion is homogenized at high pressure at a pressure of 10,000 to 25,000 psi and recycled for 5 to 20 cycles to form the perfect emulsion. The preferred pressure is from 15,000 to 20,000 psi, and more preferably 10,000 psi. The primary emulsion can be recycled for 7 to 15 cycles, and preferably it is recycled in 15 cycles. Alternatively, separate passages through the homogenizer can be used.

Preferably, the pharmaceutical composition of the invention may comprise particles or droplets of a size of less than about 200 nanometers (nm). For example, in the case of paclitaxel, docetaxel, rapamycin, cyclosporin, propofol and others, the average size of these dispersions is less than 200 nm.

The invention further relates to a method for reducing one or more side effects associated with the administration of a pharmaceutical composition to a human. The method comprises administering to a human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin and deferoxamine. The descriptions of the pharmaceutical composition, pharmaceutical agent and pharmaceutically acceptable carrier and their components set forth above in connection with the pharmaceutical composition of the invention are also applicable to the same aspects of the method of the invention.

The dose of a pharmaceutical composition of the invention, administered to a human, in the context of the present invention will vary for certain pharmaceutical compositions, route of administration and specific treatment site. The dose should be sufficient to elicit the necessary response, such as a therapeutic or prophylactic response against a particular disease, or when the pharmaceutical agent is an anesthetic, such as propofol, an anesthetic response at the desired time interval.

Although any suitable means of administering the pharmaceutical composition to humans can be used in the context of the invention, preferably, the pharmaceutical composition is administered to humans via 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 composition of the invention may be administered by inhalation to treat pathological conditions of the respiratory tract. There are minimal side effects associated with the inhalation of the pharmaceutical composition according to the invention, since albumin is a natural component of the lining and secretions of the respiratory tract. The composition of the invention can be used to treat respiratory conditions such as pulmonary fibrosis, bronchiolitis obliterans, lung cancer, bronchoalveolar carcinoma, and the like.

The method of the invention results in a reduction of one or more side effects associated with the administration of the pharmaceutical composition to a human. Such side effects include, for example, myelosuppression, neurotoxicity, hypersensitivity, inflammation, vein irritation, phlebitis, soreness, skin irritation, and combinations thereof. These side effects, however, are illustrative only, and other side effects or a combination of side effects associated with various pharmaceutical agents can be reduced or avoided using the new compositions and methods of the present invention.

The invention further relates to a method for inhibiting the growth of microorganisms in a pharmaceutical composition. “Inhibition of the growth of microorganisms” means either the complete removal of microorganisms from the pharmaceutical composition, or a decrease in the number or rate of growth of microorganisms in the pharmaceutical composition. The method includes preparing a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier in which the pharmaceutically acceptable carrier contains deferoxamine, its salts, its analogues and combinations thereof in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition. Additionally, the invention relates to a method for inhibiting oxidation in a pharmaceutical composition. This method encompasses the preparation of a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier in which the pharmaceutically acceptable carrier contains deferoxamine, its salts, its analogues and combinations thereof in an amount effective to inhibit oxidation in the pharmaceutical composition. The descriptions of the pharmaceutical composition, pharmaceutical agent and pharmaceutically acceptable carrier and their components set forth above in connection with the pharmaceutical composition of the invention are also applicable to the same aspects of the method of the invention.

The amount of deferoxamine or its preferred salt, the deferoxamine mesylate salt included in the composition will depend on the active pharmaceutical agent and other excipients. Preferably, the amount of deferoxamine, its salts and their analogues in the composition is an amount effective to inhibit the growth of microorganisms and / or inhibit oxidation. As described above, typically, the pharmaceutical composition is prepared in liquid form, and deferoxamine, its salts and their analogs are then added to the solution.

Preferably, the pharmaceutical composition in liquid form contains from about 0.0001 to about 0.5 wt.% (For example, about 0.005 wt.%, About 0.1 or about 0.25 wt.%) Of deferoxamine, its salts or their analogues . More preferably, the composition in liquid form contains a similar amount of a preferred salt of deferoxamine, deferoxamine mesylate. Most preferably, the pharmaceutical composition in liquid form contains from about 0.5 wt.% Deferoxamine mesylate. When the composition is prepared in solid form, as described above, such as by wet granulation, fluidized bed drying and other methods known to those skilled in the art, the deferoxamine mesylate is preferably used with the active pharmaceutical agent and other excipients, if present, as a solution . The deferoxamine mesylate solution preferably contains from about 0.0001 to about 0.5 wt.% (For example, about 0.005 wt.%, About 0.1 or about 0.25 wt.%) Deferoxamine.

The invention also relates to a method for increasing the transport of a pharmaceutical agent to a disease focus, where the method comprises administering to a human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, in which the pharmaceutically acceptable carrier contains albumin and in which the ratio of albumin to pharmaceutical agent in the pharmaceutical composition is approximately 18 : 1 or less. The invention further relates to a method for increasing the binding of a pharmaceutical agent to in vitro or in vivo cells, the method comprising administering to said cells an in vitro or in vivo pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier contains albumin and in which the ratio of albumin to pharmaceutical agent in the pharmaceutical composition is approximately 18: 1 or less. The descriptions of the pharmaceutical composition, pharmaceutical agent, pharmaceutically acceptable carrier, methods of administration and their components set forth above in connection with the pharmaceutical composition of the invention are also applicable to the same aspects of the transport and binding methods.

In a method of increasing the transport of a pharmaceutical agent to a disease site or increasing the binding of a pharmaceutical agent to a cell, a pharmaceutically acceptable carrier comprises albumin, most preferably human serum albumin. Without adhering to any particular theory, it is believed that the ratio of a protein, for example human serum albumin, to a pharmaceutical agent in a pharmaceutical composition affects the ability of the pharmaceutical agent to bind and transfer the pharmaceutical agent into the cell. In this regard, a higher ratio of protein to pharmaceutical agent is mainly due to poor cellular binding and transfer of the pharmaceutical agent, which is possibly the result of competition for receptors on the cell surface. The ratio of the protein, for example, albumin and the active pharmaceutical agent, must be such that a significant amount of the pharmaceutical agent binds to it or is transferred into the cell. Illustrative ranges for drug and protein preparations are protein to drug (w / w) ratios of 0.01: 1 to about 100: 1. More preferably, the ratios range from 0.02: 1 to about 40: 1. Although the ratio of protein to pharmaceutical agent should be optimized for various combinations of protein and pharmaceutical agent, in general, the ratio of protein, for example, albumin and pharmaceutical agent is approximately 18: 1 or less (e.g., approximately 15: 1, approximately 10 : 1, approximately 5: 1 or approximately 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 preparation mainly does not contain cremophor and, most preferably, does not contain cremophor EL ^® (BASF). Cremophor EL ^® is a non-ionic emulsifying agent that is a polyester of castor oil and ethylene oxide. As described above, cremophor is typically used as a solvent for paclitaxel, and it is associated with side effects that can be dangerous (see, for example, Gelderblom et al., Above ).

The pharmaceutical agent may be any suitable pharmaceutical agent described herein (eg, propofol, paclitaxel or docetaxel). Additionally, the pharmaceutical agent may be a nucleic acid sequence, most preferably a DNA sequence. In this regard, the pharmaceutical composition of the invention can be used to transport genes into a cell by receptor-mediated caveolar / vesicular transport. In order to transfer DNA sequences such as genes and other genetic material, including but not limited to plasmids or cDNA, to a cell (e.g., an endothelial cell or tumor cell), pharmaceutical compositions containing albumin in combination with genetic material can be prepared. Since tumor cells and other cells in the foci of the disease have the property of increased uptake of proteins, the genetic material is preferably taken by these types of cells and can be introduced into the genetic material of the cell for a successful therapeutic effect. The use of proteins, such as human serum albumin, serves as a non-virus vector for delivering genetic material without the risk of virus-related diseases or side effects. For example, a pharmaceutical composition containing a nucleic acid sequence encoding a β-galactosidase or green fluorescent protein (GFP) and albumin can be prepared and contacted with endothelial cells derived from a human umbilical vein or human lung microvasculature in order to facilitate the introduction of a sequence nucleic acid into endothelial cells. The occurrence of a nucleic acid sequence can be determined by methods known in the art, such as, for example, fluorescence or staining.

In the method of the invention, for increasing the transport of a pharmaceutical agent to a focal point of the disease, the disease can be any suitable disease or condition. Preferably, the disease is cancer, cardiovascular disease or arthritis.

In the method of the invention, in order to increase the binding of a pharmaceutical agent to an in vitro or in vivo cell, the pharmaceutical composition is introduced into the cell in vitro or in vivo . Preferably, the cell is an animal cell. More preferably, the cell is a mammalian cell and, most preferably, the cell is a human cell. The pharmaceutical composition is preferably administered to the cell in vivo . A cell may be any suitable cell that is a necessary target for administration of a pharmaceutical composition. For example, a cell may reside in tissues or originate from tissues of the digestive system, including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. A cell can also be in tissues or come from tissues of the respiratory system, including, for example, the larynx, lungs and bronchi. A cell can reside in tissues or come from tissues, such as the cervix, uterus, ovary, vagina, prostate, testicles and penis, which make up the male and female reproductive systems, and the bladder, kidneys, renal pelvis, and ureter that make up the urine the system. A cell may reside in tissues or originate from tissues of the cardiovascular system, including, for example, endothelial cells and cardiac muscle cells. A cell can also be in tissues or come from tissues of the lymphatic system (e.g., lymph cells), the nervous system (e.g., neurons or glial cells) and the endocrine system (e.g., thyroid cells). Preferably, the cell resides in tissues or originates from tissues of the cardiovascular system. Most preferably, the cell is an endothelial cell. In the context of the method of the invention, in order to increase transport and binding of the pharmaceutical agent to the cell, the pharmaceutical composition desirably contacts more than one cell.

In another aspect of the invention, the method of the invention for increasing transport and increasing the binding of a pharmaceutical agent to a cell can be used to treat tumor cells. Tumor cells exhibit increased uptake of proteins, such as albumin and transferrin, compared to normal cells. Since tumor cells divide at high speed, they require additional sources of nutrients compared to normal cells. Tumor studies with the pharmaceutical composition of the invention containing paclitaxel and human serum albumin showed increased uptake of albumin with paclitaxel into the tumor. It was found that this capture is the result of a previously undetermined phenomenon of albumin transport with the drug via glycoprotein 60 receptors (“gp60”), which are specific for albumin.

Thus, according to another aspect of the present invention, the albumin-specific gp60 receptor and other protein transport receptors that are present on tumor cells can be used as targets for suppressing tumor growth. By blocking the gp60 receptor using antibodies against the gp60 receptor or compounds of large or small molecules that bind, block or inactivate gp60 and other protein transport receptors on tumor cells or tumor endothelial cells, it is possible to block the transport of proteins to these cells and thereby reduce their growth rate and cause cell death. Blocking this mechanism, therefore, leads to the treatment of an individual (eg, a person) with cancer or another disease. The determination of a protein-blocking / binding receptor is carried out by screening any number of compounds for isolated gp60 or other receptors, such as gp16 or gp30, or using whole cell preparations. Additionally, suitable animal models can also be used for this purpose, such as, for example, mice containing knock-out mutations of genes encoding gp60 or caveolin-1 or other proteins that are specific for transport. Thus, a method for determining compounds that block or bind gp60, gp16, gp30 or other protein receptors are within the scope of this invention.

Additionally, compounds that block or bind the gp60 receptor or other receptors can be used to treat certain diseases, including cancer. In relation to the treatment of cancer, a blocking or binding compound can be used as a single agent or in combination with other standard chemotherapy or chemotherapy agents. For example, it is useful to treat cancer with conventional chemotherapy or pharmaceutical albumin compositions with the drug of the invention (which show a high degree of accumulation in tumors), accompanied by the use of compounds that block the transport of proteins to the tumor cell. Blocking compounds may be administered prior to or in conjunction with other chemotherapeutic or anti-cancer agents. Thus, any compounds that block or bind the gp60 receptor or other receptors are within the scope of the present invention.

Albumin-drug compositions of the invention, such as, for example, albumin with paclitaxel, albumin with docetaxel, albumin with epothilone, albumin with camptothecin or albumin with rapamycin and others, are used in the treatment of diseases. It is believed that such drug compositions are effective due to increased receptor-mediated transport of the protein-drug composition to the desired site, for example, a tumor. Not wanting to be attached to any particular theory, transport of a protein composition with a drug using receptor-mediated transport leading to a therapeutic effect is believed to be a transport mechanism, for example, albumin compositions with paclitaxel in the tumor, as well as albumin transport with paclitaxel and albumin with rapamycin through the lungs. Transport is affected by the presence of gp60, gp16, or gp30 in such tissues. Accordingly, drugs and protein compositions with drugs whose transport to the focal point of the disease, for example, inflammation (e.g. arthritis) or tumors are associated with gp60, gp16 or gp30 receptors and which lead to a therapeutic effect, are considered as compositions of the present invention.

In accordance with another aspect of the present invention, endothelial cells can be co-cultured with cells having a specific function. Incubation of endothelial cells with other types of cells, such as insulocytes, hepatocytes, neuroendocrine cells and others, takes into account the necessary transport of components, such as proteins and other beneficial components, into such cells. Endothelial cells provide transport of these components to cultured cell types in order to simulate in vivo conditions , that is, in cases where these cell types would normally be in close proximity to endothelial cells and would depend on endothelial cells for nutrient transport, growth factors, hormonal signals, etc., which are necessary for their proper functioning. Previously, it was impossible to sufficiently cultivate such various types of cells and to obtain their physiological functioning in cases where endothelial cells were absent. The presence of endothelial cells in a culture with the necessary cell type allows differentiation and proper functioning of insulocytes, hepatocytes or neuroendocrine tissues in vitro or ex vivo . Thus, it was found that co-cultures of endothelial cells with insulocytes produce insulocytes with improved physiological properties, for example, the ability to secrete insulin compared to insulocytes cultured in the absence of endothelial cells. Such tissues can then be used ex vivo or transplanted in vivo to treat diseases caused by loss of the corresponding cellular function (e.g. diabetes in the case of insulocytes, impaired liver function in the case of hepatocytes and neuroendocrine disorders or analgesia in the case of neuroendocrine cells). Cells originating from other tissues and organs (as described above) can also be cultured together with endothelial cells to provide the same effect. In addition, co-cultures can be used to introduce genetic material into predetermined cell types. The presence of albumin in such cultures has been found to be very beneficial.

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

EXAMPLE 1

This example demonstrates the preparation of a pharmaceutical composition comprising albumin and paclitaxel. The preparation of compositions of paclitaxel with albumin is described in US patent No. 5439686 and 5916596, which are fully incorporated here by reference. In particular, 30 mg of paclitaxel was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human serum albumin solution (2% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and methylene chloride was quickly removed at 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained paclitaxel particles was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot could easily be restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization.

It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting. When compared with the toxicity of paclitaxel dissolved in cremophor preparations, the pharmaceutical composition of the invention containing albumin showed significantly lower toxicity.

EXAMPLE 2

This example demonstrates the preparation of a pharmaceutical composition comprising amiodarone and albumin. 30 mg of amiodarone was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human serum albumin solution (1% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and methylene chloride was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained amiodarone particles was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization.

It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting. When compared with the toxicity of amiodarone dissolved in tween formulations, the pharmaceutical composition of the invention with albumin showed significantly lower toxicity.

EXAMPLE 3

This example demonstrates the preparation of a pharmaceutical composition comprising lyiotironin and albumin compositions. Lyothyronine (or a suitable salt) was dissolved in a water-alcohol solution or an alkaline solution at a concentration of 0.5-50 mg / ml. An alcohol (or alkaline) solution was added to the albumin solution (0.1-25%) and mixed. Mixing was carried out on a mixer with low shear forces or on an ultrasonic unit or homogenizer with high shear forces. At a low concentration of lyothyronine (5-1000 μg / ml), clear solutions were obtained. As the concentration was increased, a stable milky white suspension was obtained. These solutions or suspensions were filtered through a sterilizing filter. Organic solvents were removed by evaporation or other suitable method.

EXAMPLE 4

This example demonstrates the preparation of a pharmaceutical composition comprising rapamycin and albumin. 30 mg of rapamycin was dissolved in 2 ml of the chloroform / ethanol system. The solution was added to 27.0 ml of human serum albumin solution (3% w / v). The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting.

EXAMPLE 5

This example demonstrates the preparation of a pharmaceutical composition comprising epothilone B and albumin. 30 mg of epothilone B was dissolved in 2 ml of the chloroform / ethanol system. The solution was added to 27.0 ml of human serum albumin solution (3% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 min at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are not limiting. When compared with the toxicity of epothilone B dissolved in cremophor preparations, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 6

This example demonstrates the preparation of a pharmaceutical composition comprising a colchicine dimer and albumin. 30 mg of colchicine dimer was dissolved in 2 ml of the chloroform / ethanol system. The solution was then added to 27.0 ml of human serum albumin solution (3% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are not limiting. When compared with the toxicity of a colchicine dimer dissolved in tween, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 7

This example demonstrates the preparation of a pharmaceutical composition comprising docetaxel and albumin. 30 mg of docetaxel was dissolved in 2 ml of the chloroform / ethanol system. The solution was then added to 27.0 ml of human serum albumin solution (3% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 min at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are not limiting. When compared with the toxicity of docetaxel dissolved in the tween / ethanol system, which is the standard solvent for this drug, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 8

This example demonstrates the preparation of a pharmaceutical composition comprising docetaxel and albumin. 150 mg of docetaxel was dissolved in 1 ml of the ethyl acetate / butyl acetate system and 0.5 ml of an oil, for example soybean oil or vitamin E oil. Other ratios of solvents and oils were used, and these compositions are also considered as part of this invention. A small amount of a negatively charged component, for example benzoic acid (0.001% -0.5%), was also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 min at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are not limiting. When compared with the toxicity of docetaxel dissolved in the tween / ethanol system, which is the standard solvent for this drug, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 9

This example demonstrates the preparation of a pharmaceutical composition comprising taxan IDN5390 and albumin. 150 mg of IDN5390 was dissolved in 1 ml of an ethyl acetate / butyl acetate system and 0.5 ml of an oil, such as soybean oil or vitamin E oil. Other solvent to oil ratios were used and these compositions are also considered as part of this invention. A small amount of a negatively charged component, for example benzoic acid (0.001% -0.5%), was also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w / v). If necessary, deferoxamine was added. The mixture was homogenized for 5 min at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are not limiting. When compared with the toxicity of IDN5390 dissolved in tween, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 10

This example demonstrates the preparation of a pharmaceutical composition comprising taxan IDN5109 and albumin. 150 mg of IDN5109 was dissolved in 2 ml of the chloroform / ethanol system. Other ratios of solvents and oils were used, and these compositions are also considered as part of this invention. A small amount of a negatively charged component, for example benzoic acid (0.001% -0.5%), was also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w / v). If necessary, deferoxamine was added. In order to obtain the primary emulsion, the mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.), and then it was transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents and proteins used in this example are not limiting. When compared with the toxicity of IDN5109 dissolved in tween, the pharmaceutical composition containing albumin showed significantly lower toxicity.

EXAMPLE 11

This example demonstrates the preparation of a pharmaceutical composition comprising 10-hydroxycamptothecin (10HC) and albumin. 30 mg of 10-HC was dissolved in 2 ml of the DMF / methylene chloride / soybean oil system. The solution was then added to 27.0 ml of human serum albumin solution (3% w / v). In order to obtain a primary emulsion, the mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting.

EXAMPLE 12

This example demonstrates the preparation of a pharmaceutical composition comprising cyclosporin and albumin. 30 mg of cyclosporine was dissolved in 3.0 ml of methylene chloride. The solution was then added to 27.0 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 min at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to Rotavap and methylene chloride was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average particle diameter was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization.

EXAMPLE 13

This example demonstrates the preparation of a pharmaceutical composition comprising oil and comprising cyclosporin and albumin. 30 mg of cyclosporine was dissolved in 3.0 ml of a suitable oil (sesame oil containing 10% orange oil). The solution was then added to 27.0 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting dispersion contained a typical average particle diameter in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was directly used or lyophilized for 48 hours, optionally adding a suitable cryoprotectant. The resulting clot was easily restored to the original dispersion by adding sterile water or saline. It should be understood that the amounts, types and quantitative ratios of the drug, solvents and proteins used in this example are in no way limiting.

EXAMPLE 14

This example demonstrates the preparation of a pharmaceutical composition comprising amphotericin and albumin. 30 mg amphotericin was dissolved in 3.0 ml of the methylpyrrolidinone / methylene chloride system. The solution was added to 27.0 ml of human serum albumin solution (1% w / v). The mixture was homogenized for 5 minutes at low rpm (Vitris homogenizer, model Tempest I.Q.) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the solvent was quickly removed at 40 ° C under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained amphotericin particles was in the range between 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot could be easily restored to the original dispersion by adding sterile water or saline. The particle size after recovery remained the same as before lyophilization. It should be understood that the amounts, types and quantitative ratios of the drug, solvents and proteins used in this example are in no way limiting. The addition of other components, such as lipids, bile salts, etc., also led to the preparation of suitable preparations.

EXAMPLE 15

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

Several preclinical pharmacokinetic studies have been performed in mice and rats in order to evaluate the possible benefits of pharmaceutical compositions of albumin with paclitaxel over pharmaceutical compositions of cremophor with paclitaxel (taxol). The study data showed: (1) that the pharmacokinetics of albumin with paclitaxel in rats was linear, while the pharmacokinetics of taxol was non-linear in dose, (2) pharmaceutical compositions containing albumin and paclitaxel showed lower plasma AUC and C _max values, indicating on a faster distribution of albumin and paclitaxel compositions over tissues compared to taxol (similar isolation), (3) pharmaceutical compositions containing albumin and paclitaxel showed a lower C _max value, which possibly explains the reduced toxicity associated with maximum blood levels relative to taxol, (4) the half-life shown by pharmaceutical compositions containing albumin and paclitaxel was approximately 2 times higher in rats and 4 times higher in mice with tumors relative to taxol and (5) paclitaxel metabolism in pharmaceutical compositions containing albumin and paclitaxel was slower than in taxol pharmaceutical compositions. 24 hours after injection in rats, 44% of the total radioactivity was still associated with paclitaxel in the case of pharmaceutical compositions containing albumin and paclitaxel, compared with only 22% in the case of taxol. The main effect of the above pharmacodynamics, that is, increased intracellular uptake, prolonged half-life and slowed metabolism shown in the case of pharmaceutical compositions containing albumin and paclitaxel, leads to a 1.7-fold higher AUC of the tumor, 1.2 times higher the C _max value of the tumor and a 1.7-fold longer half-life in the tumor than in the case of taxol in tumor mice.

EXAMPLE 16

This example demonstrates reduced side effects and reduced toxicity associated with pharmaceutical compositions containing paclitaxel and albumin.

Due to the unique nature of pharmaceutical compositions containing paclitaxel and albumin in the absence of cremophor, the toxicity of pharmaceutical compositions containing paclitaxel and albumin is significantly lower than taxol. In preclinical studies in mice and rats, a single dose acute toxicity study showed that the LD ₅₀ dose was approximately 59 times greater for pharmaceutical compositions containing paclitaxel and albumin than for taxol. In a multiple dose toxicity study in mice, the LD ₅₀ dose was approximately 10 times greater for pharmaceutical compositions containing paclitaxel and albumin than for taxol. Further, the study determined the degree of myelosuppression in rats, which were treated with pharmaceutical compositions containing paclitaxel and albumin, and taxol. The results showed that at equal doses, pharmaceutical compositions containing paclitaxel and albumin lead to much less myelosuppression in rats than taxol. In an acute toxicity study in rats, cerebral cortex necrosis or severe neurotoxicity were observed in animals receiving taxol at a dose of 9 mg / kg, but these effects were not present in animals receiving a pharmaceutical composition containing paclitaxel and albumin at a dose of up to 120 mg / kg. Thus, the presence of albumin in a pharmaceutical composition containing paclitaxel leads to a significant reduction in side effects and toxicity compared to conventional compositions containing paclitaxel.

EXAMPLE 17

This example demonstrates the clinical effects of a pharmaceutical composition containing paclitaxel and albumin in humans.

Clinical studies of over 500 sick people to date provide evidence supporting the fact that the toxicity and side effects of a pharmaceutical composition containing paclitaxel and albumin (“albumin with paclitaxel”) are reduced compared to compositions containing cremophor and paclitaxel (taxol). In a study of 19 patients in stage I, the maximum permissible dose of albumin with paclitaxel, which was given every 3 weeks, was 300 mg / m ² . It is significantly higher than the usual dose of cremophor with paclitaxel, which is 175 mg / m ² , which is given once every 3 weeks. Hematological toxicity in these patients was moderate without allergies, with mild neuropathies and without side effects associated with the administration of drugs, such as venous irritation, etc.

In another study of 27 patients in stage I, the maximum allowable dose of albumin with paclitaxel, which was given on a weekly schedule, was 125-150 mg / m ² . It is significantly higher than the usual dose of cremophor with paclitaxel, which is 80 mg / m ² if given on a weekly schedule. Hematological toxicity in these patients was weak without allergies, with mild neuropathies, and without side effects associated with the administration of drugs, such as venous irritation, etc.

In two studies at the stage II albumin with paclitaxel, which were given either 175 or 300 mg / m ² every 3 weeks to 43 and 63 patients, respectively, hematological toxicity was low, only in 7% and 24% of patients ANC <500 / mm ³ at dosages of 175 mg / m ² and 300 mg / m ² , respectively. Severe neuropathies occurred in 0% and 14% of patients at dosages of 175 mg / m ² and 300 mg / m ² , respectively. There were no cases of severe allergies and there were no cases of side effects associated with the administration of the drug, such as venous irritation, pain during injection, etc. These side effects were significantly lower than in the studied patients with taxol.

In stage III trials comparing the compositions of albumin with paclitaxel ABI-007 and taxol (which contains cremophor with paclitaxel), the dosage of ABI-007 was significantly higher (260 mg / m ² versus 175 mg / m ² taxol), indicating that he is better tolerated. Albumin / paclitaxel compositions also show significantly reduced neutropenia compared with cremophor paclitaxel.

EXAMPLE 18

This example demonstrates increased preclinical efficacy using a pharmaceutical composition containing paclitaxel and albumin.

An in vitro cytotoxicity study comparing the effects of albumin with paclitaxel and taxol on cervical squamous cell carcinoma A431 showed an approximately 4-fold increase in cytotoxic activity for albumin with paclitaxel with IC ₅₀ values of 0.0038 and 0.012 μg / ml for albumin with paclitaxel and taxol, respectively .

In five different models of human xenograft tumors in athymic mice (mammary gland MX-1, lung NCI-H522, ovaries SK-OV-3, prostate PC-3 and colon HT-29) MTD or equally toxic dose of ABI-007 was 1.5-3.4 times higher than for taxol and led to a statistically significant improvement in slowing tumor growth (p <0.05) in all tumors except for a lung tumor (p = 0.15).

In the MX 1 breast model, one hundred percent (100%) of the animals treated with paclitaxel albumin survived for 103 days compared to 20-40% of the survivors in the groups treated with the same doses of taxol.

EXAMPLE 19

This example demonstrates enhanced clinical efficacy using a pharmaceutical composition containing intra-arterial albumin and paclitaxel.

In studies at the stage I / II of intraarterial administration of a pharmaceutical composition containing albumin and paclitaxel, as described herein, head and neck cancer (N = 31) and anal canal cancer (N = 12) were recorded in patients. A increasing dose of 120-300 mg / m ² was administered for more than 30 minutes by percutaneous intra-arterial infusion for 3-4 weeks. Patients with head and neck cancer showed a response rate of 76% (N = 29), while patients with anal cancer showed a response rate of 64% (N = 11).

EXAMPLE 20

This example demonstrates 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 glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). The oil phase was prepared by dissolving egg lecithin (0.4 wt.%) And propofol (1 wt.%) In soybean oil (3 wt.%) At about 50 ° C-60 ° C, and shaken until dissolved. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen. The resulting pharmaceutical composition contained the following main ranges of components (wt.%): Propofol 0.5-5%; human serum albumin 0.5-3%; soybean oil 0.5-3.0%; egg lecithin 0.12-1.2%; glycerol 2.25%; water for injection q.s. up to 100; pH 5-8. Optionally suitable chelators are added, for example, deferoxamine (0.001-0.1%).

EXAMPLE 21

This example demonstrates 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 glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). The oil phase was prepared by dissolving egg lecithin (0.8 wt.%) And propofol (1 wt.%) In soybean oil (5 wt.%) At about 50 ° C-60 ° C, and shaken until dissolved. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen. The resulting pharmaceutical composition contained the following main ranges of components (wt.%): Propofol 0.5-5%; human serum albumin 0.5-3%; soybean oil 0.5-10.0%; egg lecithin 0.12-1.2%; glycerol 2.25%; water for injection q.s. up to 100; pH 5-8. Optionally suitable chelators are added, for example, deferoxamine (0.001-0.1%).

EXAMPLE 22

This example demonstrates the preparation of a pharmaceutical composition comprising propofol and albumin and not containing oil.

Using a similar procedure to that described in Example 18, propofol compositions containing albumin and tween 80 were prepared. The aqueous phase was prepared by adding glycerin (2.25 wt.%), Human serum albumin (0.5 wt.%), Tween 80 (1.5 wt.%) And deferoxamine mesylate (0.1 wt.%) In water for injection, and shaken 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 at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen. The resulting pharmaceutical composition contained the following main ranges of components (wt.%): Propofol 0.5-5; human serum albumin 0.5-3%; tween 80 0.1-1.5%; deferoxamine mesylate 0.0001-0.1%; glycerol 2.25%; water for injection q.s. up to 100; pH 5-8.

EXAMPLE 23

This example demonstrates the preparation of a pharmaceutical composition comprising propofol, albumin and vitamin E-TPGS, which does not contain oil.

Using a procedure similar to that described in Example 19, propofol compositions containing albumin and vitamin E-TPGS were prepared. The aqueous phase was prepared by adding glycerin (2.25 wt.%), Human serum albumin (0.5 wt.%), Vitamin E-TPGS (1 wt.%) And deferoxamine mesylate (0.1 wt.%) In water for injection, and shaken 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 at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen. The resulting pharmaceutical composition contained the following main ranges of components (wt.%): 0.5-5 propofol; 0.5-3% human serum albumin; 0.5-4.0% Vitamin E-TPGS; optionally 0.0001-0.1% deferoxamine mesylate; 2.25% glycerol; water for injection q.s. up to 100; pH 5-8.

EXAMPLE 24

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

An emulsion containing 1% (by weight) of propofol was prepared in the following way. The aqueous phase was prepared by adding glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant, for example vitamin E-TPGS (0.5%), was added to the aqueous phase. The oil phase consisted of propofol (1 wt.%) And 1% soybean oil. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for up to 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen.

The resulting pharmaceutical composition contained the following main ranges of components (wt.%): 0.5-5% propofol; 0.01-3% human serum albumin; 0.1-2% Vitamin E-TPGS; soy or other oil (0.1% -5%); 2.25% glycerol; water for injection q.s. up to 100; pH 5-8. Optionally, deferoxamine (0.001-0.1 wt.%) Was added.

EXAMPLE 25

This example demonstrates the preparation of a pharmaceutical composition comprising propofol, albumin, vitamin E-TPGS, 1% oil and a negatively charged component.

An emulsion containing 1% (by weight) of propofol was prepared in the following way. The aqueous phase was prepared by adding glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant, for example vitamin E-TPGS (0.5%), was added to the aqueous phase. The oil phase consisted of propofol (1 wt.%) And 1% soybean oil. A small amount of a negatively charged component (0.001% -1%), for example, a phospholipid or a bile salt, was added. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for up to 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen.

The resulting pharmaceutical composition contained the following main ranges of components (wt.%): 0.5-5% propofol; 0.01-3% human serum albumin; 0.1-2% Vitamin E-TPGS; soy or other oil (0.1% -5%); 2.25% glycerol; water for injection q.s. up to 100; pH 5-8. Optionally, deferoxamine (0.001-0.1 wt.%) Was added.

EXAMPLE 26

This example demonstrates 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) of propofol was prepared in the following way. The aqueous phase was prepared by adding glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant, for example vitamin E-TPGS (0.5%), was added to the aqueous phase. The oil phase consisted of propofol (1 wt.%) And 1% soybean oil. A small amount of a negatively charged component (0.001% -1%), for example sodium deoxycholate, was added. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for up to 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen.

The resulting pharmaceutical composition contained the following main ranges of components (wt.%): 0.5-5% propofol; 0.01-3% human serum albumin; 0.1-2% Vitamin E-TPGS; soy or other oil (0.1% -5%); 2.25% glycerol; water for injection q.s. up to 100; pH 5-8. Optionally, deferoxamine (0.001-0.1 wt.%) Was added.

EXAMPLE 27

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

An emulsion containing 1% (by weight) of propofol was prepared as follows. The aqueous phase was prepared by adding glycerin (2.25 wt.%) And human serum albumin (0.5 wt.%) To water for injection, and shaken until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant, for example vitamin E-TPGS (0.5%), was added to the aqueous phase. The oil phase consisted of propofol (1 wt.%) And 1% soybean oil. A small amount of a negatively charged component (0.001% -1%), for example, phosphatidylcholine, was added. The oil phase was added to the aqueous phase and homogenized at 10,000 rpm. within 5 minutes The primary emulsion was homogenized at high pressure at 20,000 psi and recycled for up to 15 cycles at 5 ° C. Alternatively, separate passages through the homogenizer were used. The final emulsion was filtered (0.2 μm filter) and stored in nitrogen.

The resulting pharmaceutical composition contained the following main ranges of components (wt.%): 0.5-5% propofol; 0.01-3% human serum albumin; 0.1-2% Vitamin E-TPGS; soybean or other oil (0.1% -5%); 2.25% glycerol; water for injection q.s. up to 100; pH 5-8. Optionally, deferoxamine (0.001-0.1 wt.%) Was added.

EXAMPLE 28

This example demonstrates the binding of propofol to albumin.

The binding of propofol to albumin was determined as follows. The solubility of propofol was tested in water and in a solution containing albumin. 250 μl of propofol was added to 10 ml of water or an albumin solution and shaken for 2 hours in a scintillation vial. The solution was then transferred to a centrifugation tube with 15 ml of polyethylene and kept at 40 ° C for approximately 16 hours. The amount of propofol was analyzed in water samples and albumin solutions. The solubility of propofol in water was determined to be 0.12 mg / ml. The solubility of propofol in albumin solutions depended on the concentration of albumin and increased to 0.44 mg / ml if the concentration of albumin was 2% (20 mg / ml). The solutions were ultrafiltered by means of a filter with an MWCO 30 kD and the amount of propofol was analyzed in the filtrates. It was found that for a propofol / water solution, 61% of propofol could be recovered in the filtrate, while for a propofol / albumin solution, only 14% was recovered in the filtrate, indicating a significant binding of propofol to albumin. Based on these results, the addition of albumin to pharmaceutical compositions containing propofol leads to a decrease in the amount of unbound propofol due to the binding of albumin to propofol.

EXAMPLE 29

This example demonstrates a decrease in unbound propofol in a pharmaceutical composition by filtration / membrane contact.

As observed in the experiments described in Example 28, filtration or ultrafiltration of pharmaceutical compositions containing propofol resulted in a decrease in the amount of unbound propofol. Diprivan and the pharmaceutical composition prepared according to the present invention, containing albumin, each containing 1% propofol (10 mg / ml), were ultrafiltered using a 30 kD membrane. The amount of unbound propofol in the filtrate was measured by HPLC. The concentration of unbound propofol in the filtrate was approximately 17 μg / ml for diprivan, while the concentration of unbound propofol in the filtrate for the pharmaceutical composition of the invention was approximately 7 μg / ml. The results correspond to an effective reduction in unbound propofol, with a coefficient of more than 2 for a pharmaceutical composition containing propofol and albumin.

EXAMPLE 30

This example demonstrates the administration of a pharmaceutical composition containing propofol and albumin to humans.

A random double-blind clinical trial was performed to compare unwanted skin sensations from a pharmaceutical composition containing propofol and albumin with a pharmaceutical composition of a commercially available drug with propofol diprivan. The tests were carried out in accordance with good clinical practice, and consent was obtained from individuals based on complete information. Adult individuals of both sexes were eligible to participate if they had apparently normal skin without damage on the back of their hands.

The preparations initially stored in the refrigerator were brought to room temperature, and 10 μl of the preparations were slowly applied simultaneously to the back of both hands of the individual. The total reaction and sensations on their hands from the preparations were noted. The results of this study are summarized in table 1.

Table 1 Individual Verification Procedure % of individuals with sensations from ABI-propofol % of individuals with diprivan sensation Light warmth or burning or stinging feeling No feeling Light warmth or burning or stinging feeling No feeling 1 degree 0 100.0 75 25

EXAMPLE 31

This example demonstrates the use of deferoxamine as an antioxidant in a pharmaceutical composition containing propofol.

Pharmaceutical compositions comprising propofol and deferoxamine mesylate and containing tween or TPGS were stored at 4 °, 25 ° or 40 ° C in order to examine the effect of deferoxamine mesylate on preventing the oxidation of propofol. For these preparations, the concentration of propofol was measured after a long time to determine the antioxidant activity of deferoxamine. The data are presented below in tables 2 and 3 as% efficiency relative to zero time.

table 2
Albumin / Tween 1 month storage Temperature 4 ° C 25 ° C 40 ° C The control one hundred% 88% 48% 0.01% Def 101% 89% 61% 0.1% Def 103% 89% 64%

Table 3
Albumin / TPGS 1 month storage Temperature 4 ° C 25 ° C 40 ° C The control 99% 73% 42% 0.01% Def 99% 87% 55% 0.1% Def 99% 85% 58%

Under these conditions, deferoxamine was effective in reducing the oxidation state of propofol. The effect was more pronounced at higher temperatures. No significant oxidation occurred at 4 ° C. This study was performed using plugs that were not inert or were coated with Teflon.

EXAMPLE 32

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

The purpose of this study was to determine the period of presence of [ ³ H] ABI-007 in the blood and selected tissues after intratracheal instillation in Sprague Dawley rats.

The target drug volume for the intratracheal dose that was administered to the animals was calculated based on the dose volume of 1.5 ml per kg body weight. The dispenser consisted of a Penn-Century micropulverizer (Model 1A-1B; Penn-Century, Inc., Philadelphia, PA; purchased from DeLong Distributors, Long Branch, NJ) attached to a sealed injector with a 1 ml Luer nozzle. The corresponding volume of the dosed preparation was placed in the dosing device, the filled device was weighed and its mass was recorded. The catheter was inserted into the trachea of the anesthetized animal, the micropulverizing part of the dosing device was placed into the trachea through the catheter and the dose was administered. After the dose was administered, the empty dosing device was re-weighed and the dose administered was calculated as the mass difference of the dosing device before and after the dose. The average dose for all animals was 4.7738 ± 0.0060 (CV 1.5059) mg paclitaxel per kg body weight.

Blood samples in an amount of approximately 250 μl were taken from the jugular vein in the jugular vein of JVC rats at the following predetermined times after administration of the dose: 1, 5, 10, 15, 30 and 45 minutes (min) and 1, 4, 8 and 24 hours (h). Blood samples taken after 24 hours, as well as blood samples taken from euthanized animals after 10 minutes, 45 minutes and 2 hours, were taken by heart puncture anesthetized during sacrifice. All blood samples analyzed for total radioactivity were distributed into pre-weighed sample tubes, and these sample tubes were re-weighed, and the weight of each sample was calculated by subtraction. In blood samples collected from the jugular vein, as well as in blood aliquots of 250 μl collected from animals during killing, the total tritium content was analyzed.

For all rats, the maximum concentration of tritium in the blood was observed 5 min (0.0833 h) after the dose. The half-life of tritium, determined in the time interval from 4 hours to 24 hours, ranged from 19.73 hours to 43.02 hours. It should be noted that this interval includes only three data processing results that can be calculated for the variability of this parameter. The magnitude of the pronounced cleansing of blood from tritium was about 0.04 l / h. The results of these experiments are shown below in table 4.

Table 4
Non-compartmental analysis of the concentration of tritium in the blood (mg × equiv. / L) compared with the time profiles in rats after intratracheal infusion of [ ³ H] ABI-007 Parameter Mean ± SD C _max (mg × equiv. / L) 1.615 ± 0.279 T _max (h)
t½beta (h) 0.0833 ± 0.0
33.02 ± 1.99

AUClast (mg × equiv. × h / l) 7,051 ± 1,535 Cl / F (l / h) 0.0442 ± 0.0070 Fa (bioavailability) 1.229 ± 0.268

The average blood concentration of radioactivity derived from [ ³ H] ABI-007 after an intravenous dose to rats was analyzed as a function of time in order to evaluate the bioavailability of tritium obtained by intratracheal administration of [ ³ H] ABI-007. This analysis resulted in an AUC value of 24 hours (AUClast) of 6.1354 mg × equiv. × h / l. Based on such data, radioactivity resulting from intratracheal administration of a dose of [ ³ H] ABI-007 is extremely bioavailable. These analyzes are based on total radioactivity.

Tritium originating from [ ³ H] ABI-007 is rapidly absorbed after intratracheal infusion. The mean absorption and half-lives (half-life of k01 and half-life of k10, respectively) of tritium in the blood after intratracheal administration of a dose of [ ³ H] ABI-007 (mean ± SD) were 0.0155 ± 0.0058 h and 4.738 ± 0.366 h, respectively. The average expressed clearance of tritium in the blood was 0.1235 ± 0.0180 l / h (see table 4 above).

Tritium originating from [ ³ H] ABI-007 after intratracheal administration was absorbed and distributed. The dynamics of tritium concentrations in the blood is well described by a two-compartment model with an average absorption and half-life of 0.0155 and 4.738 hours, respectively. Approximately 28% of the administered dose was found in the lungs 10 minutes after intratracheal administration of the dose. A maximum of less than 1% of the dose was found in other tissues, with the exception of the gastrointestinal tract, at all time points studied.

Based on the results of a preliminary study of an intravenous dose of [ ³ H] Capxol ^™ ([ ³ H] Capxol ™]), the bioavailability of tritium derived from an intratracheal dose was 1.229 ± 0.268 (mean ± SD) for three animals in this dose group. It should be noted, however, that this bioavailability assessment is based on total radioactivity. Surprisingly, paclitaxel delivered by the pulmonary route using the albumin compositions of the invention quickly became bioavailable, indicating superior transport through the pulmonary endothelium. No toxicity was observed in animals, which was unexpected since pulmonary delivery of cytotoxic drugs is known to cause pulmonary toxicity.

A fair amount of radioactivity was present in the gastrointestinal tract (including contents) 24 hours after dosing (27% of the intratracheal dose). The presence of tritium in the gastrointestinal tract is possibly due to bile secretion or clearance of tritium from the respiratory tract through mucociliary clearance resulting from swallowing.

EXAMPLE 33

This example demonstrates the study of Aerotech II and Pari nebulizers for pulmonary delivery of pharmaceutical compositions containing paclitaxel and albumin.

The study was carried out using the pharmaceutical composition of paclitaxel with albumin ABI-007 under the following conditions: room temperature (20-23 ° C), relative humidity (48-54%), ambient pressure (629 mm Hg), spray flow (10 l / min for Aerotech II; 7 l / min for Pari), the total feed flow (28.3 l / min), the pressure drop in the nebulizer (23 lb / ⁱⁿ² for Aerotech II; 32 lb / ⁱⁿ² for Pari ), duration of work (from 15 to 60 seconds), sample volume (1.5 ml), paclitaxel ABI-007 concentration (5, 10, 15 and 20 mg / ml).

Both Aerotech II and Pari nebulizers provide acceptable overall efficiencies (30% -60%) when ABI-007 is reduced to a concentration in the range of 5-15 mg / ml. The effectiveness of the Pari atomizer had a higher atomizer efficiency than the Aerotech II atomizer. The performance of the Pari nebulizer decreased to some extent as the concentration of ABI-007 increased. An excellent fraction of fine particles was observed (74% -96%). The Aerotech II atomizer contained a higher order fraction of fine particles than the Pari atomizer. The fraction of fine particles was independent of concentration.

The Pari nebulizer delivered 100 mg of paclitaxel in less than 30 minutes using an ABI-007 solution at a concentration of 15 mg / ml. An Aerotech II nebulizer delivered 100 mg paclitaxel in approximately 65 minutes using an ABI-007 solution at either 10 mg / ml or 15 mg / ml. Performance stability was tested on both Aerotech II and Pari nebulizers. The aerosol concentration and efficacy of both nebulizers were stable until the drug ran out. At a concentration of 15 mg / ml, the Pari nebulizer consumed the drug twice as fast as the Aerotech II nebulizer and produced higher aerosol concentrations than the aerosol aerosol concentration from the Aerotech II nebulizer.

In conclusion, a paclitaxel / albumin nanoparticle preparation (ABI-007) showed excellent bioavailability in rats given by pulmonary administration. There were no pronounced signs of early toxicity from the administered dose. Pulmonary delivery of paclitaxel in nanoparticles (ABI-007) can be accomplished using conventional nebulizers.

EXAMPLE 34

This example describes the intrapulmonary delivery of a pharmaceutical composition comprising albumin and rapamycin. The purpose of this study was to determine the pulmonary absorption of rapamycin after intratracheal infusion to Sprague-Dawley rats compared with intravenous infusion.

The target drug volume for the intratracheal dose that was administered to the animals was calculated based on the dose volume of 1 ml per kg body weight. The intratracheal dosing device consisted of a Penn-Century micropulverizer (Model 1A-1B; Penn-Century, Inc., Philadelphia, PA; purchased from DeLong Distributors, Long Branch, NJ) attached to a sealed injector with a 1 ml Luer nozzle. The corresponding volume of the dosed preparation was placed in the dosing device, the filled device was weighed and its mass was recorded. The catheter was inserted into the trachea of the anesthetized animal, the micropulverizing part of the dosing device was placed into the trachea through the catheter and the dose was administered. After the dose was administered, the empty dosing device was re-weighed and the dose administered was calculated as the mass difference of the dosing device before and after the dose.

Samples with a volume of 250 μl were taken from the cannula located in the jugular vein in rats at the following predefined time points after administration of the dose: 1, 5, 10, 15, 30, and 45 minutes (min) and 1, 4, 8, and 24 hours ( h). All analyzed blood samples were distributed into pre-weighed sample tubes, and these sample tubes were re-weighed, and the weight of each sample was calculated by subtraction. The collected blood samples analyzed the total concentration of rapamycin using LC / MS / MS.

Unexpectedly, the results did not show a significant difference in the concentration in the blood of rapamycin delivered by the pulmonary route compared with the intravenous route. The bioavailability of rapamycin delivered by the pulmonary route using a pharmaceutical composition containing albumin was calculated to be 109%, indicating excellent transport through the pulmonary endothelium.

EXAMPLE 35

This example demonstrates tissue distribution of albumin with rapamycin after intrapulmonary administration of a pharmaceutical composition comprising rapamycin and albumin prepared according to the present invention. The purpose of this study was to determine the pulmonary absorption of rapamycin by tissue after intratracheal infusion to Sprague-Dawley rats compared with intravenous infusion.

The target drug volume for the intratracheal dose that was administered to the animals was calculated based on the dose volume of 1 ml per kg body weight. The dispenser consisted of a Penn-Century micropulverizer (Model 1A-1B; Penn-Century, Inc., Philadelphia, PA; purchased from DeLong Distributors, Long Branch, NJ) attached to a sealed injector with a 1 ml Luer nozzle. The corresponding volume of the dosed preparation was placed in the dosing device, the filled device was weighed and its mass was recorded. The catheter was inserted into the trachea of the anesthetized animal, the micropulverizing part of the dosing device was placed into the trachea through the catheter and the dose was administered. After the dose was administered, the empty dosing device was re-weighed and the dose administered was calculated as the mass difference of the dosing device before and after the dose.

Samples were taken from the brain, lungs and liver from three rats per group at time points of 10 minutes, 45 minutes, 2 hours and 24 hours. Samples were collected and their total concentration of rapamycin was analyzed using LC / MS / MS. The results indicate that the concentration of rapamycin in the lung tissue is greater in cases where pulmonary delivery was compared to intravenous delivery. However, the total concentration of rapamycin in the brain is lower when intratracheal (IT) delivery was performed compared to intravenous (IV) delivery. In the liver, as it turned out, there was no difference in the concentration of rapamycin during IT or IV delivery. Based on these results, pulmonary rapamycin delivery may be suitable for treating conditions (i.e., lung transplantation) when a high local concentration of rapamycin would be beneficial.

EXAMPLE 36

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

Tritium-labeled ABI-007 was used to determine the oral bioavailability of paclitaxel after oral compulsory feeding of rats. After an overnight food restriction, 5 rats were given paclitaxel as part of ABI-007 at a dose of 5.5 mg / kg (Group A), and another 5 rats (Group B) were preliminarily given cyclosporine (5.0 mg / kg), followed by taking paclitaxel as part of ABI-007 at a dose of 5.6 mg / kg. Pharmacokinetic analysis of blood samples obtained after 0.5, 1, 2, 3, 4, 5, 6, 8, 12 and 24 hours was carried out after determining the radioactivity in the blood samples by oxidation. Oral bioavailability was determined by comparison with previously obtained data on intravenous administration. The results are shown below in table 5.

Table 5
The average value of AUC 0-24, C _max , T _max and% absorption derived from ³ H-paclitaxel radioactivity after oral administration Group Treatment Dose / way mg / kg AUC 0-24 (μg × equiv. × h / ml) Absorption (%) C _max (mg / kg) (μg × equiv / ml) T _max (h) BUT ABI-007 in physiological saline 5.5 / PO (P) 2.92 44.3 0.245 one

AT ABI-007 in physiological saline with CsA 5 / PO (C),
5.6 / PO (P) 8.02 121.1 0.565 0.5

An AUC value of 0-24 IV (6.06 μg × h / ml) and a dose of IV (5.1 mg / kg) were used to calculate percent absorption (data based on dose IV ABI-007).

44% of oral bioavailability was shown for ABI-007 alone. This bioavailability is much greater than has been shown for other paclitaxel preparations. Bioavailability increased to 121% if the animals were given cyclosporin (CsA). This is expected since CsA is a known p-glycoprotein pump suppressant that typically prevents the absorption of compounds such as paclitaxel from the LC tract. More than 100% bioavailability can be explained by reabsorption after biliary excretion of paclitaxel into the LC tract. Other known absorption suppressing or enhancing agents can also be used for this purpose.

EXAMPLE 37

This example demonstrates improved penetration of paclitaxel into red blood cells and tumor cells when a pharmaceutical composition comprising paclitaxel and albumin is administered.

MX-1 human breast tumor fragments were implanted subcutaneously in athymic mice. A pharmaceutical composition containing paclitaxel and albumin (“paclitaxel with albumin”) as described previously and taxol were prepared using ³ H-paclitaxel to a specific activity of 25 μCi / mg paclitaxel. 20 mg / kg of isotope-labeled paclitaxel with albumin or taxol was administered intravenously in physiological saline when the tumor volume increased to approximately 500 mm ³ . Plasma, blood, and tumor tissue were collected and radioactivity analyzed in them at 5, 15, and 30 minutes and at 1, 3, 8, and 24 hours after administration. Tumor pharmacokinetics (AUC and absorption constant) were analyzed using WinNonlin, Pharsight, USA.

Paclitaxel with albumin showed a rapid distribution in red blood cells (RBC), as shown by a rapid decrease in the plasma / blood radioactivity ratio to less than unity after intravenous administration of the drug. Complete distribution in RBC occurred 1 h after administration of paclitaxel with albumin. In contrast, the distribution of paclitaxel prepared as taxol in RBC was much slower and incomplete up to more than 8 hours.

Paclitaxel with albumin showed a rapid distribution into the tumor tissue with an absorption constant (K _a ), which was 3.3 times greater than for taxol. K _a were 0.43 h ^-1 and 0.13 h ^-1 for paclitaxel with albumin and taxol, respectively. Rapid uptake of paclitaxel resulted in a 33% higher tumor AUC for paclitaxel with albumin than for taxol. The AUC values were 3632 nCi * h / g and 2739 nCi * h / g for paclitaxel with albumin and taxol, respectively.

EXAMPLE 38

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

Nude mice received increasing doses of paclitaxel with albumin or taxol daily for 5 consecutive days. Survival was plotted against the dose axis to determine the LD ₅₀ . Survival was significantly increased for paclitaxel with albumin compared to taxol (p = 0.017, ANOVA). The LD ₅₀ for paclitaxel with albumin and taxol was calculated to be 47 mg / kg / day and 30 mg / kg / day for the once-daily regimen × 5, respectively. At a dose level of 13.4 mg / kg / day, paclitaxel with albumin and taxol were well tolerated with 1% mortality (1 death from 72 mice) and 4% (2 deaths from 47 mice), respectively. At a dose level of 20 mg / kg / day, 1% mortality was observed for paclitaxel with albumin (1 death from 72 mice) compared with 17% mortality for taxol (8 deaths from 47 mice) (p = 0.0025). At a dose level of 30 mg / kg / day, 4% mortality was observed for paclitaxel with albumin (3 deaths from 72 mice) compared with 49% mortality for taxol (23 deaths from 47 mice) (p <0.0001).

EXAMPLE 39

This example demonstrates a new mechanism for the transport of paclitaxel through endothelial cells (EC) of microvessels for paclitaxel albumin compositions.

Nanoparticles and compositions of albumin with paclitaxel can accumulate in the tumor tissue due to the EPR effect resulting from the "germination" of blood vessels in the tumor. Albumin-specific receptor gp60 (albondin), transporting albumin through the EU by transcytosis of receptors in caveola on the cell surface. This mechanism of transcytosis allows the transport of albumin with paclitaxel into the underlying interstial space. In contrast, cremophor in taxol inhibited the binding of paclitaxel to albumin, significantly reducing the transport of paclitaxel into the tumor. Additionally, the gp16 and gp30 receptors were 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 effects compared to taxol.

EXAMPLE 40

This example demonstrates an increase in endothelial transcytosis of pharmaceutical compositions containing paclitaxel and albumin compared to taxol.

Endothelial cells of human lung microvasculature (HLMVEC) were grown until fixed in a transcytosis plate well. The pharmaceutical composition of the invention containing paclitaxel and albumin or taxol containing fluorescent paclitaxel (flutax) at a concentration of 20 μg / ml was added to the top of the well of the transcytosis plate.

The transport of paclitaxel by transcytosis from the upper compartment to the lower compartment was continuously recorded using a fluorimeter. A control containing only flutax without albumin was also used. The flutax control showed no transport, confirming the integrity of the fixed HLMVEC monolayer. The transport of paclitaxel from the albumin-paclitaxel composition was significantly faster than paclitaxel from taxol in the presence of 5% HAS (physiological concentration). The transport rate constants (Kt) for the albumin composition with paclitaxel and taxol were 1.396 h ^-1 and 0.03 h ^-1 , respectively. The total amount of paclitaxel transported through the monolayer was three times higher for the composition of albumin with paclitaxel than for taxol

EXAMPLE 41

This example demonstrates improved binding of endothelial cells (EC) to pharmaceutical compositions containing paclitaxel and albumin compared to taxol.

Human umbilical vein endothelial cells (HUVEC) were grown on a 96-well microtiter plate. In one experiment, paclitaxel (paclitaxel labeled with flutax-oregon green) was reacted with HUVEC in the presence of increasing concentrations of cremophor EL / EtOH, which is a filler for taxol. In another experiment, a pharmaceutical composition containing albumin and flutax and a Taxol-flutax composition with HUVEC at various final concentrations were reacted. The binding of paclitaxel to cells was inhibited by cremophor. Inhibition was manifested with an IC _{50 of} 0.02% for Cremophor EL / EtOH.

It has been shown that this concentration of cremophor is maintained during taxol chemotherapy for at least 24 hours. Therefore, this corresponds to the in vivo process. At all concentrations analyzed, significant amounts of paclitaxel from the albumin composition with paclitaxel bound to the cells. In comparison, for Taxol, slight binding was observed or no binding was observed.

EXAMPLE 42

This example demonstrates improved albumin binding by pharmaceutical compositions containing paclitaxel and albumin compared to taxol.

Human serum albumin (HSA) was immobilized on a plastic ELISA plate. Paclitaxel (paclitaxel labeled with flutax-oregon green) was reacted with immobilized HSA in the presence of increasing concentrations of Cremophor EL / EtOH. In another experiment, a pharmaceutical composition containing albumin with paclitaxel and flutax and a taxol composition with flutax with immobilized HSA were reacted at a final concentration of 20 mg paclitaxel / ml. The binding of paclitaxel to albumin was inhibited by cremophor. Inhibition was manifested with an IC _{50 of} 0.003% for Cremophor EL / EtOH. This concentration of cremophor has been shown to persist during taxol chemotherapy for at least 24 hours. Therefore, this corresponds to the in vivo process. At an appropriate pharmacological concentration of paclitaxel (20 μg / ml), a significant amount of paclitaxel from the albumin-paclitaxel composition was associated with immobilized HSA. In comparison, no binding for taxol was observed.

EXAMPLE 43

This example demonstrates an increased transfer of paclitaxel with albumin for pharmaceutical compositions containing paclitaxel and albumin compared to taxol.

The compositions of taxol with flutax and albumin with paclitaxel and flutax were mixed either with 5% HSA in Hanks buffer or with serum at a concentration of 20 μg / ml, 40 μg / ml and 80 μg / ml. The mixtures were immediately separated in a native 3-14% polyacrylamide gel and the amount of paclitaxel bound to albumin was determined using a scanning fluorimeter. The transfer of paclitaxel with HSA was faster for the composition of albumin with paclitaxel compared to taxol.

More paclitaxel moved with HAS during electrophoresis, when either serum or 5% HSA was incubated with the albumin composition with paclitaxel and flutax or taxol and flutax composition. Under the influence of 5% HSA, 45%, 60% and 33% more paclitaxel was transferred with HSA for the albumin composition with paclitaxel and flutax than for the composition of taxol with flutax at 20 μg / ml, 40 μg / ml and 80 μg / ml , respectively. Under the action of human serum, 121%, 31% and 83% more paclitaxel was transferred with HSA for the albumin composition with paclitaxel and flutax than for the taxol composition with flutax at 20 μg / ml, 40 μg / ml and 80 μg / ml, respectively . The C _max value for ABI-007 at a concentration of 260 mg / m ^{2 is} approximately 20 μg / ml, therefore, this process is an important in vivo process.

EXAMPLE 44

This example demonstrates that the gp60 glycoprotein receptor is responsible for the binding and transcytosis of albumin to paclitaxel.

Compositions of fluorescently labeled paclitaxel (flutax) with albumin interacted with microvessel endothelial cells in culture. Fluorescence staining was observed under a microscope based on flickering regions, which were postulated to be the gp60 receptor that binds albumin to paclitaxel. These results were confirmed using rhodamine-labeled albumin, which was located in conjunction with the flickering fluorescence of paclitaxel.

EXAMPLE 45

This example demonstrates that increasing amounts of albumin can compete for paclitaxel binding.

Albumin was immobilized on a microtiter plate. Fluorescent paclitaxel was added to the wells and paclitaxel binding was measured using a scanning fluorimeter. Increasing amounts of albumin were added to the wells and the level of inhibition of binding of paclitaxel to immobilized albumin was measured. The data showed that as the amounts of added albumin were increased, a corresponding decrease in binding was observed. A similar effect was observed upon binding to endothelial cells. Such data indicate that a higher concentration of albumin inhibits the binding of paclitaxel. Thus, compositions of the invention containing lower amounts of albumin are preferred.

EXAMPLE 46

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

In order to investigate whether lower amounts of albumin can affect the stability of the pharmaceutical composition of the invention, albumin compositions with paclitaxel with small amounts of albumin were prepared. It was found that these compositions are also stable, as well as compositions with higher amounts of albumin, when tested for several months at various temperatures (2-8 ° C, 25 ° C and 40 ° C), the effectiveness of paclitaxel, pollution, particle size , pH and other typical stability parameters. Thus, compositions with lower amounts of albumin are preferred since they can significantly reduce the cost and also allow increased binding to the cell and transport to the cell.

EXAMPLE 47

In this example, a pharmaceutical composition comprising albumin and paclitaxel with a high albumin to paclitaxel ratio is demonstrated.

30 mg of paclitaxel was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of a solution of human serum albumin (3% w / v) (which corresponds to a ratio of albumin to paclitaxel of 27). If necessary, deferoxamine was added. The mixture was homogenized for 5 minutes at low rpm. (Vitris homogenizer, Tempest I.Q. model) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi, along with the recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and methylene chloride was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained paclitaxel particles was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or physiological saline. The particle size after recovery remained the same as before lyophilization.

It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting. When compared with the toxicity of paclitaxel dissolved in cremophor preparations, the pharmaceutical composition of the invention containing albumin showed a significantly lower toxicity.

EXAMPLE 48

In this example, a pharmaceutical composition comprising albumin and paclitaxel with a low albumin to paclitaxel ratio is demonstrated.

In particular, 300 mg of paclitaxel was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human serum albumin solution (5% w / v%) (corresponding to a ratio of albumin to paclitaxel of 4.5). If necessary, deferoxamine was added. The mixture was homogenized for 5 minutes at low rpm. (Vitris homogenizer, Tempest I.Q. model) in order to obtain a primary emulsion, and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi along with recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and methylene chloride was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained paclitaxel particles was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or physiological saline. The particle size after recovery remained the same as before lyophilization. The final albumin / paclitaxel ratio was achieved by adding the required amount of albumin to obtain the final albumin / paclitaxel ratio of 9: 1. This ratio can be determined by HPLC.

It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting. When compared with the toxicity of paclitaxel dissolved in cremophor preparations, the pharmaceutical composition of the invention containing albumin showed a significantly lower toxicity.

EXAMPLE 4 9

In this example, a pharmaceutical composition comprising albumin and paclitaxel is shown with an intermediate ratio of albumin and paclitaxel.

In particular, 135 mg of paclitaxel was dissolved in 3.0 ml of methylene chloride. The solution was added to 27.0 ml of human serum albumin solution (5% w / v). If necessary, deferoxamine was added. In order to obtain a primary emulsion, the mixture was homogenized for 5 minutes at low rpm. (Vitris homogenizer, Tempest I.Q. model) and then transferred to a high pressure homogenizer (Avestin). Emulsification was carried out at 9000-40000 psi along with recirculation of the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator, and methylene chloride was quickly removed at a temperature of 40 ° C, under reduced pressure (30 mmHg) for 20-30 minutes. The resulting dispersion was transparent, and a typical average diameter of the obtained paclitaxel particles was in the range of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was then lyophilized for 48 hours. The resulting clot was easily restored to the original dispersion by adding sterile water or physiological saline. The particle size after recovery remained the same as before lyophilization. The calculated ratio (w / w) of albumin and paclitaxel in this composition of the invention is approximately 10.

It should be understood that the amounts, types and quantitative ratios of the drug, solvents, proteins used in this example are in no way limiting. When compared with the toxicity of paclitaxel dissolved in cremophor preparations, the pharmaceutical composition of the invention containing albumin showed a significantly lower toxicity.

EXAMPLE 50

This example demonstrates the treatment of rheumatoid arthritis in animal models with a combination of albumin and paclitaxel.

The collagen-induced Louvain rat arthritis model was used to test the therapeutic effect of the albumin-paclitaxel composition on arthritis. In order to assess the severity of arthritis, the paw sizes of the experimental animals were monitored.

After arthritis developed completely (usually 9-10 days after collagen administration), the experimental animals were divided into two groups, which were given intraperitoneally either 1 mg / kg paclitaxel albumin given once daily (qod) or paclitaxel albumin 0.5 mg / kg + prednisone 0.2 mg / kg given once a day (qod) (combination treatment) in 6 doses, then one dose per week for three weeks. Paw sizes were measured at the start of treatment (day 0) and each time a drug was administered. One group received only saline as a control. By the end of the experiment, a paw size reduction of 42% was achieved in the group receiving albumin with paclitaxel, a 33% decrease in paw size was shown in the combination treatment group, while a paw size increase of approximately 20% was observed in the control group compared to meanwhile, when treatment began.

In conclusion, albumin compositions with paclitaxel have shown a therapeutic effect on arthritis. The combination of albumin with paclitaxel is likely to be localized in the focus of arthritic damage due to transport using a receptor-mediated mechanism similar to gp60.

EXAMPLE 51

This example demonstrates the use of paclitaxel albumin compositions for the treatment of cardiovascular restenosis.

Stents eluting paclitaxel cause incomplete healing in animals and, in some cases, loss of stable inhibition of neointimal growth in arteries. This study tested the effectiveness of the new albumin compositions with paclitaxel according to the invention with systemic delivery to reduce restenosis in the stent.

Albumin with paclitaxel, reconstituted with saline, was analyzed in 38 New Zealand white rabbits that had bilateral stents inserted into the iliac arteries. Dosages of albumin with paclitaxel (paclitaxel dose from 1.0 to 5.0 mg / kg) were administered as an intra-arterial infusion over 10 minutes; control animals received vehicle (0.9% saline).

In a chronic experiment followed by observation, 5.0 mg / kg of albumin with paclitaxel was given with stenting with or without an intravenous repeated dose of albumin with paclitaxel 3.5 mg / kg after 28 days; these studies were completed after 3 months. After 28 days, the average non-optimal thickness decreased (p≤0.02) with a dose of paclitaxel with albumin ≥2.5 mg / kg with signs of slow healing. The effectiveness of a single dose of albumin with paclitaxel 5.0 mg / kg, however, was lost in 90 days. In contrast, the second repeated dose of albumin with paclitaxel 3.5 mg / kg, which was given 28 days after stenting, led to a stable decrease in the non-optimal thickness after 90 days (p≤0.009 compared to a single dose of albumin with paclitaxel 5.0 mg / kg and control) with almost complete neointimal healing.

Although systemic administration of albumin with paclitaxel reduces neointimal growth after 28 days, a single repeated dose was necessary for stable neointimal suppression. Thus, the composition of the invention is suitable for treating cardiovascular diseases such as restenosis. A composition of the invention containing a pharmaceutical agent other than paclitaxel, for example rapamycin, other taxanes, epothilones, etc. are all suitable for treating restenosis in blood vessels or transplants of artificial blood vessels, such as vessels used for arterial-venous access patients who need hemodialysis.

Claims (24)

1. A pharmaceutical composition comprising a water-insoluble pharmaceutical agent and a pharmaceutically acceptable carrier in which the pharmaceutical agent is paclitaxel, the pharmaceutically acceptable carrier comprises albumin, wherein the ratio (w / w) of albumin to the water-insoluble pharmaceutical agent is 9 : 1, wherein the pharmaceutical composition comprises nanoparticles comprising a water-insoluble pharmaceutical agent and albumin, where the nanoparticles have a particle size of less than 200 nm. 2. The pharmaceutical composition according to claim 1, which is dehydrated. 3. The pharmaceutical composition according to claim 2, which is lyophilized. 4. The pharmaceutical composition according to claim 1, which is liquid. 5. The pharmaceutical composition according to claim 4, which further comprises physiological saline. 6. The pharmaceutical composition according to claim 1, which is sterile. 7. The pharmaceutical composition according to claim 1, which is in a standard dose. 8. The pharmaceutical composition according to claim 1, which is in several doses. 9. The pharmaceutical composition according to claim 1, which is contained in a sealed container. 10. The pharmaceutical composition according to claim 1, which further comprises deferoxamine. 11. The pharmaceutical composition according to claim 1, wherein the albumin is human serum albumin. 12. The pharmaceutical composition according to claim 1, in which paclitaxel is present in an amount of from 0.1 to 1 wt.%. 13. The pharmaceutical composition according to any one of claims 1 to 12, where the pharmaceutical composition is prepared for use in the treatment of cancer. 14. The pharmaceutical composition of claim 13, wherein the cancer is breast cancer. 15. The pharmaceutical composition according to item 13, where the cancer is lung cancer. 16. The pharmaceutical composition according to any one of claims 1 to 12, where the pharmaceutical composition is intended for parenteral administration. 17. The pharmaceutical composition according to any one of claims 1 to 12, where the pharmaceutical composition is intended for intravenous administration, intraarterial administration, intrapulmonary administration, oral administration, inhalation, intravesical administration, intramuscular administration, intratracheal administration, subcutaneous administration, intraocular administration, intrathecal administration or percutaneous administration. 18. The pharmaceutical composition according to 17, where the pharmaceutical composition is intended for intravenous administration. 19. The pharmaceutical composition according to 17, where the pharmaceutical composition is intended for intravesical administration. 20. The pharmaceutical composition according to 17, where the pharmaceutical composition is intended for intrathecal administration. 21. The pharmaceutical composition according to any one of claims 1 to 12, where the pharmaceutical composition is intended for use in humans. 22. The pharmaceutical composition according to any one of claims 1 to 12, where the pharmaceutical composition is for injection. 23. A method of delivering a water-insoluble pharmaceutical agent, which is paclitaxel, to an individual, comprising administering to the individual a pharmaceutical composition according to any one of claims 1-12. 24. The method according to item 23, where the individual has cancer.