Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/282—Platinum compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Anticancer agents may be classified according to several criteria, such as class of compound and disease state treated. Certain agents have been developed to take advantage of the rapid division of cancer cells and target specific phases in the cell cycle, providing another method of classification. Agents can also be grouped according to the type and severity of their side effects or method of delivery. However, the most common classification of non-biotherapeutic based anticancer agents is by class of chemical compound, which broadly encompasses the mechanism of action of these compounds.
• Nitrosoureas are often categorized as alkylating agents, and have a similar mechanism of action, but instead of directly alkylating DNA, they inhibit DNA repair enzymes causing replication failure. These compounds have the advantage of being able to cross the blood-brain barrier and therefore can be used to treat brain tumors.
• the antimetabolite class of anticancer agents interfere with the growth of DNA and RNA and are specific to the S-phase of the cell-cycle. They can be broken down further by type of compoimd, which include folic acid analogs, purine analogs, and pyrimidine analogs. They are often employed in the treatment of chronic leukemia, breast, ovary, and gastrointestinal tumors.
• corticosteroid hormones There are two classes of hormones or hormone analogs used as anticancer agents, the corticosteroid hormones and sex hormones. While some corticosteroid hormones can both kill cancer cells and slow the growth of tumors, and are used in the treatment of lymphoma, leukemias, etc., sex hormones function primarily to slow the growth of breast, prostate and endometrial cancers. There are numerous subclasses of hormones and hormone analogs, including, androgens, antiadrenals, antiandrogens, antiestrogens, aromatase inhibitors, estrogens, leutenizing hormone releasing hormone (LHRH) analogs and progestins.
• LHRH leutenizing hormone releasing hormone
• An additional smaller class of anticancer agents is classified as immunotherapy. These are agents that are intended to stimulate the immune system to more effectively attack the neoplastic (cancerous) cells. This therapy is often used in combination with other therapies.
• Combinations of anticancer agents are also utilized in the treatment of a number of cancers.
• Sanofi Syntholabo markets ELOXATINTM (oxaliplatin for injection) for the treatment of colorectal cancer for use in combination with 5-fluorouracil and leuvocorin.
• ELOXATINTM oxaliplatin for injection
• Oxaliplatin is an alkylating agent that is believed to act by inhibiting both DNA replication and transcription. Unlike other platinum agents, oxaliplatin has demonstrated a decreased likelihood of resistance development. Oxaliplatin is further described in U.S. Pat. Nos.
• liposome compositions include U.S. Pat. No. 4,983,397; 6,476,068; 5,834,012; 5,756,069; 6,387,397; 5,534,241 ; 4,789,633; 4,925,661; 6,153,596; 6,057,299; 5,648,478; 6,723,338; 6,627218; U.S. Pat. App.
• liposomes that aimed to specifically target particular cell types by incorporating targeting factors (also referred to as targeting ligands) for particular cell types.
• targeting factors also referred to as targeting ligands
• targeting factors/ligands include asialoglycoprotein, folate, transferrin, antibodies, etc.
• one or more of the constituent lipids could be modified by the attachment of a targeting factor.
• R 1 , R 2 , R 3 and R 4 are each, independently, oleoyl, stearoyl, palmitoyl or myristoyl.
• R 1 and are R are the same, and R 3 and R are the same, hi particular embodiments, R 1 , R 2 , R 3 and R 4 are the same.
• R 1 , R 2 , R 3 and R 4 are oleoyl or stearoyl.
• m and n are 3, where the one or more phospholipids is DMPC or DSPC and the at least one additional lipid is present and is cholesterol.
• m and n are each independently, an integer from 2 to 4. In certain embodiments, m and n are equal and are an integer from 2 to 4. In particular embodiments, m and n are equal are 3.
• the at least one additional lipid is present.
• the at least one additional lipid is cholesterol or a cholesterol derivative.
• DMPC and cholesterol, DSPC and cholesterol, POPC and cholesterol, or DPPC and cholesterol are incorporated.
• DMPC and cholesterol are incorporated.
• the mean diameter of the liposome is from about 50 run to about 250 nm. In others, the mean diameter of the liposome is from about 90 nm to about 200 nm.
• the drug is an anticancer agent.
• the drug is a cytotoxic drug.
• the drug is a topoisomerase I inhibitor.
• the topoisomerase I inhibitor is topotecan or irinotecan.
• the drag is a vinca alkaloid.
• the vinca alkaloid is vincristine, vinblastine, vinleurosine, vinrodisine, vinorelbine or vindesine.
• the drug is a nucleic acid.
• the nucleic acid is an antisense oligonucleotide or a ribozyme.
• the concentration of targeting ligand incorporated in the liposome is from about 1.0 mg/ml to about 3.0 mg/ml. In others, the concentration of targeting ligand incorporated in the liposome is from about 1.0 mg/ml to about 2.5 mg/ml.
• the targeting ligand is transferrin. In particular embodiments, the transferrin is in a holo-form. In some embodiments, feme iron is in a concentration of from about 0.4 to about 3.0 ⁇ g/ml. In other embodiments, ferric iron is in a concentration of from about 0.4 to about 1.5 ⁇ g/ml.
• the formulations further include sucrose.
• compositions described herein are provided pharmaceutical formulations of the lipid-containing compositions described herein.
• Particular embodiments of the liposome-containing compositions, targeted liposomes and blank liposomes include the liposome-containing compositions, targeted liposomes or blank liposomes as described herein and one or more pharmaceutically acceptable carriers, excipients, diluents, stabilizers, or preservatives.
• the liposome-containing compositions, targeted liposomes or blank liposomes as described herein is contained in a first container and one or more pharmaceutically acceptable carriers, excipients, diluents, stabilizers, or preservatives are contained in a second container.
• step (a) adding a drug or labeled compound to the lipid mixture formed in step (a);
• step (d) purifying the liposome of step (c).
• the drug in step (b) is in aqueous solution prior to mixing.
• step (c) comprises sonication or stirring.
• step (c) comprises extrusion.
• succinimidyl ester of the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is represented by Formula 2,
• R 3 and R 4 are each, independently, an acyl group, n is, independently, an integer from 1 to 10;
• step (a) adding drug or labeled compound to the lipid mixture formed in step (a);
• the method also includes a step(e), purifying the liposome of step (d).
• step (b) the drag in step (b) is in aqueous solution prior to mixing.
• step (c) comprises sonication or stirring.
• step (c) comprises extrusion.
• step (c) comprises stirring.
• the method further comprises a step (c), purifying the liposome of step (b).
• step (b) comprises sonication or stirring. In some embodiments, step (b) comprises extrusion.
• succinimidyl ester of the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is represented by Formula 2,
• the methods further include a step (d), purifying the liposome of step (c).
• methods of making the lipid- containing compositions where the at least one additional lipid is present comprising the step of mixing the one or more phospholipids, the at least one additional lipid, the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine.
• step (a) adding a labeled compound to the lipid mixture formed in step (a).
• step (b) comprises sonication or stirring. In some embodiments, step (b) comprises extrusion.
• step (a) in step (a) the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is present.
• the targeting factor-modified N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine is present.
• methods for treating cancer comprising, a) administering a targeted liposome as described herein to an individual in need thereof in an amount effective to treat cancer, wherein the targeted liposome comprises a drug, and the drug is an anticancer agent.
• step (a) is performed prior to, concurrently with or after combined modality cancer therapy.
• the combined modality cancer therapy comprises chemotherapy, radiation therapy, or surgery.
• the one or more additional anticancer agents comprise 5-fluorouracil, leucovorin, capecitabine, UFT/LV (tegafur-uracil and leucovorin), irinotecan, an-anti EGFR antibody, an anti-VEGF antibody, a tyrosine kinase inhibitor, or combinations thereof.
• step (b) comprises detection via a gamma counter.
• R 5 and R 6 are each, independently, an acyl group and p is an integer from 1 to 10, and transferrin is liked linked to the N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine.
• R 5 and R 6 are each, independently, oleoyl, stearoyl, palmitoyl or myristoyl. In particular embodiments, R 5 and R 6 are the same. In some embodiments, R 5 and R 6 are oleoyl or stearoyl. In certain embodiments, R 5 and R 6 are oleoyl and p is 3.
• lipid mixtures comprising a mixture of at least two different neutral lipids, an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and a succinimidyl ester of an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine, wherein the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is represented by Formula 1,
• succinimidyl ester of the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is represented by Formula 2,
• R 1 , R 2 , R 3 and R 4 are each, independently, an acyl group, m and n are, independently, an integer from 1 to 10; and, wherein the mixture does not comprise a non- derivatized phosphatidyl ethanolamine or polyethylene glycol.
• R 1 , R 2 , R 3 and R 4 are each, independently, an acyl group; m and n are, independently, an integer from 1 to 10; and, wherein the mixture does not comprise a non- derivatized phosphatidyl ethanolamine or polyethylene glycol.
• m and n are each independently, an integer from 2 to 4. In certain embodiments, m and n are equal and are an integer from 2 to 4. In other embodiments, m and n are equal and are 3.
• R 1 , R 2 , R 3 and R 4 are each, independently, oleoyl, stearoyl, palmitoyl or myristoyl.
• R 1 and are R 2 are the same, and R 3 and R 4 are the same.
• R 1 , R 2 , R 3 and R 4 are the same, hi some embodiments, R 1 , R 2 , R 3 and R 4 are oleoyl
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is NG-DOPE and the succinimidyl ester of an N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine is NHS-NG-DOPE.
• the increased specificity of the labeled compound formulations with the concomitant reduction in adverse effects should increase the number of individuals who can be successfully diagnosed, for example, able to tolerate the diagnosis formulation, and also increase the accuracy (e.g., sensitivity, etc.) of diagnosis, including allowing for earlier diagnosis of conditions and more effective monitoring of the severity of the disease (e.g., progression or regression with or without therapy).
• the invention also provides transferrin-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines, which can be used in the lipid-containing compositions and formulations thereof described herein.
• N-(co)-dicarboxylic acid-derivatized phosphatidyl ethanolamine refers to the N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamines encompassed by Formula 1 as provided herein.
• N ⁇ PE is used to refer to the N ⁇ (co)-dicarboxylic acid-derivatized phosphatidyl ethanolamines encompassed by Formula 1 (e.g., N ⁇ -DOPE, N ⁇ -DSPE, NG- DOPE, etc.), and, for example NG-PE refers to N-glutaryl phosphatidyl ethanolamine(s) of Formula 1, unless otherwise noted.
• non-derivatized phosphatidyl ethanolamine refers to phosphatidyl ethanolamine, semi-synthetic phosphatidyl ethanolamine(s), synthetic phosphatidyl ethanolamine(s) and/or derivatives thereof that are not encompassed by Formula 1, Formula 2 or Formula 3.
• the acyl groups are derived from oleic acid (oleoyl), stearic acid (stearoyl), palmitic acid (palmitoyl), linoleic acid (linoleoyl, 18 carbons), or myristic acid (myristoyl).
• the acyl groups are derived from oleic acid (oleoyl).
• the acyl groups are derived from stearic acid (stearoyl).
• the acyl groups are derived from palmitic acid (palmitoyl).
• the acyl groups are derived from myristic acid (myristoyl).
• the acyl group is derived from a saturated aliphatic carboxylic acid, such as, but not limited to palmitic acid (16 carbons), stearic acid (18 carbons), or myristic acid (14 carbons).
• R 1 and R 2 are the same acyl group. In other embodiments, R 1 and R 2 are different acyl groups. In certain embodiments, R 1 and R 2 are oleoyl, stearoyl, palmitoyl or myristoyl. In some embodiments, R 1 and R 2 are oleoyl. In other embodiments, R 1 and R 2 are stearoyl. In particular embodiments, R 1 and R 2 are palmitoyl. In other embodiments, R 1 and R 2 are myristoyl.
• N-glutaryl-dimyristoyl phosphatidyl ethanolamine NG-DMPE (i.e., where R 1 and R 2 are myristoyl and m is 3)
• NG-DPPE N-glutaryl-dipalmitoyl phosphatidyl ethanolamine
• NS-DSPE N-succinyl- distearoyl phosphatidyl ethanolamine
• N-adipinyl-distearoyl phosphatidyl ethanolamine (i.e., where R 1 and R 2 are stearoyl and m is 4)).
• N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine of Formula 1 is NG-DOPE or NG-DSPE.
• Phospholipids including phosphatidyl ethanolamines and their derivatives utilized for the purposes described herein, must be of high purity and should ideally be homogeneous.
• Known methods for the preparation of high purity phospholipids include extraction of the lipid from a buffer solution and purification using column chromatography. For example, production methods for N-succinyl dipalmitoylphosphatidylethanolamine are described in International Patent Application Publication WO93/01828 (JPAH7-501316) and U.S. Pat. Nos. 5,804,552 and 5,554,728, the contents of which are hereby incorporated in their entirety.
• DPPE dipalmitoyl phosphatidyl ethanolamine
• N-( ⁇ -carboxy) acylamido-phosphatidyl ethanolamine phospholipid derivatives are described in Japanese published patent application JPA2001-261688, which includes purification by separating a liquid layer after addition of pH3.5 - 7.5 buffer solution to the reaction mixture, and is herein incorporated by reference in its entirety. Li this case, the PE was reacted with dicarboxylic acid anhydride with triethylamine alkali catalyst at 4°C for lhr. This method may not work well for all phosphatidyl ethanolamine derivatives.
• DOPE dioleoyl-phosphatidyl ethanolamine
• lecithin API Starting Material
• DOPC dioleoyl-phosphatidyl choline
• DOPE can be prepared from DOPC by reacting with ethanolamine using phospholipase D.
• SuccN ⁇ PE can be used to refer to the succinimidyl esters of N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines encompassed by Formula 2 ⁇ e.g., SuccN ⁇ -DOPE, SuccN ⁇ -DSPE, SuccNG- DOPE, etc.), and, for example NHS-NG-PE refers to a the succinimidyl ester of N-glutaryl phosphatidyl ethanolamine(s) of Formula 2 formed by reaction with NHS, unless otherwise noted.
• R 3 and R 4 are the same acyl group. In other embodiments, R 3 and R 4 are different acyl groups. In certain embodiments, R 3 and R 4 are oleoyl, stearoyl, palmitoyl or myristoyl. In some embodiments, R 3 and R are oleoyl. In other embodiments, R 3 and R 4 are stearoyl. In certain embodiments, R 3 and R 4 are palmitoyl. In other embodiments, R 3 and R 4 are myristoyl. [0214] In certain embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• n is an integer from 1-8, 1-6, 1-5, 1-7, 1-3, 1-2, 2-8, 2-6, 2-5, 2-4, 2-3, 3-4, 3-5, or 3-6. In some embodiments, n is an integer from 2-4. In other embodiments, n is 1 , 2 or 3.
• n is an integer from 1 to 4.
• n is 3 (glutaric acid).
• the succinimidyl ester of the N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine of Formula 2 is a succinimidyl ester of N-glutaryl-dioleoyl phosphatidyl ethanolamine (NG-DOPE). In other embodiments, it is a succinimidyl ester of N-glutaryl-distearoyl phosphatidyl ethanolamine (NG-DSPE).
• the succinimidyl ester of the N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine of Formula 2 is the succinimidyl ester of the N-glutaryl-dioleoyl phosphatidyl ethanolamine (SuccNG-DOPE (i.e., where R 3 and R 4 are oleoyl and n is 3)).
• it is the succinimidyl ester of N- glutaryl-distearoyl phosphatidyl ethanolamine (SuccNG-DSPE (i.e., where R 3 and R 4 are stearoyl and n is 3).
• succinimidyl ester of N-glutaryl- dimyristoyl phosphatidyl ethanolamine SuccNG-DMPE (i.e., where R 3 and R 4 are myristoyl and n is 3)
• succinimidyl ester of N-glutaryl- dipalmitoyl phosphatidyl ethanolamine SuccNG-DPPE (i.e., where R 3 and R 4 are palmitoyl and n is 3)
• succinimidyl ester of the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine of Formula 2 is SuccNG-DOPE or SuccNG-DSPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is N ⁇ -DOPE and the succinimidyl ester of the N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine is SuccN ⁇ -DOPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is NG-DOPE and the succinimidyl ester of the N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine is SuccNG-DOPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is NG- DSPE and the succinimidyl ester of the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is SuccNG-DSPE.
• succinimidyl esters of N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines described herein can be obtained by derivatization of the N- ( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines described herein, prepared as known in the art and described herein. Preparation of the succinimidyl esters of N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamines is also described in greater detail below, including in the Examples. In view of the teaching provided in the present specification, the skilled artisan will also be able to modify the methods described herein.
• a method for the production of the succinimidyl esters of N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamines of the present inventions is as follows:
• carbodiimide compounds that made be used include, but are not limited to, carbodiimide groups such as N, N'-dicyclohexyl-carbodiimide(DCC), N,N'-diisopropyl-carbodiimide, N-ethyl-N'-(3- dimethylaminopropyl)-carbodiimide hydrochloride(EDC), etc.
• DCC used, hi others EDC is used.
• the above-described reaction may also be performed under conditions that minimize or eliminate production of by-products.
• Unwanted by-products include urea compounds (e.g., N, N'-dicyclohexylurea, N-ethyl-N'-(3- dimethylaminopropyl)urea, etc.), N-acylated urea compounds, carboxyanhydride compounds and 5-oxazolone compounds.
• Conditions and materials which disfavor or minimize the formation of by-products include, 1) slowly dissolving the carbodiimide compounds in organic solvent, 2) performing the reaction below 0 C° to prevent heat evolution by the reaction, etc.
• Other means for optimizing the reaction and minimizing the production of by-products will be understood by those of ordinary skill in the field, particularly in view of the teaching provided herein.
• R 5 and R are, independently, an acyl group, and p is and integer from
• targeting factor-modified N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine refers to the N- ( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines encompassed by Formula 3 and modified with a targeting factor as provided herein.
• p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other embodiments, p is an integer from 1-8, 1-6, 1-5, 1-7, 1-3, 1-2, 2-8, 2-6, 2-5, 2-4, 2-3, 3-4, 3-5, or 3-6. In some embodiments, p is an integer from 2-4. In other embodiments, p is 1,
• TF-NG-DSPE N-glutaryl-distearoyl phosphatidyl ethanolamine
• N-adipinyl-distearoyl phosphatidyl ethanolamine (NA- DSPE (i.e., where R 5 and R 6 are stearoyl and p is 4)).
• N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine of Formula 3 is NG-DOPE or NG-DSPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is N ⁇ -DOPE and the targeting ligand is transferrin (Tf), and the targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is Tf-N ⁇ -DOPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is N ⁇ -DSPE and the targeting ligand is transferrin (Tf) 5 and the targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is Tf-N ⁇ -DSPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is N ⁇ -DOPE and the targeting ligand is transferrin (Tf), and the targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is Tf-N ⁇ -DSPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is N ⁇ -DSPE and the targeting ligand is transferrin (Tf), and the targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is Tf-N ⁇ -DOPE.
• m may be 3 and the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is a NG-PE according to Formula 1.
• the N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine is NG-DOPE and the targeting factor-modified N- ( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is TF-NG-DSPE.
• the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is NG-DSPE and the targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine is TF-NG-DOPE.
• the targeting-factor modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines may be prepared from the SuccN ⁇ PE by reaction with the targeting ligand and other reactants as described herein in greater detail.
• the TF- N ⁇ PE may either be prepared prior to mixture with other lipid components of the lipid-containing compositions described herein (and optionally purified) or may be prepared in situ from the reaction of the pre-prepared SuccN ⁇ PE that has been incorporated in a lipid-containing composition.
• the phospholipid may be a phosphatidylcholine, including naturally occurring, semi-synthetic or synthetic phosphatidylcholines (e.g., DSPC, DMPC, etc.).
• the phosphatidylcholine is a non-naturally occurring phosphatidylcholine (e.g., not egg phosphatidylcholine).
• the phosphatidylcholine is an acyl phosphatidylcholine (e.g., DMPC, DPPC, POPC, DSPC, etc.).
• the phospholipid is cationic.
• the phospholipid is anionic. In still other embodiments, the phospholipid is neutral. In particular embodiments, the one or more phospholipid(s) is not anionic, hi other embodiments, the one or more phospholipid(s) is not cationic. In certain embodiments where more than one phospholipid is present, an anionic and neutral lipid may be included.
• Exemplary phospholipids include, but are not limited to, phosphatidylcholines (PCs), phospliatidic acid, pliosphatidylserine, phosphatidylglycerol, etc. In some embodiments, the lipid-containing compositions do not include phosphatidylserine or phosphatidylglycerol.
• the compositions may additionally comprise an additional neutral, non-phospholipid as the additional lipid.
• additional lipid for example, cholesterol or a cholesterol derivative as described above.
• Phospholipids (non-PEs) for use in the lipid-containing compositions described herein include synthetic, semi-synthetic and naturally occurring phospholipids.
• Exemplary phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid, phosphatidylserine, phosphatidylglycerol, etc.
• the one or more phospholipids include phosphatidylcholine (PC) or phosphatidic acid and do not include phosphatidylserine or phosphatidylglycerol.
• the phospholipid is a phosphatidylcholine.
• the phosphatidylcholine may be, e.g., distearoyl phosphatidyl choline (DSPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), egg phosphatidylcholine (EPC), hydrogenated soya phosphatidylcholine (HSPC), etc.
• at least one phospholipid is a phosphatidylcholine.
• the phosphatidylcholine is DMPC.
• the phospholipid- containing composition includes a single phospholipid (non-PE phospholipid). In other embodiments where the lipid-containing composition includes a single phospholipid (non-PE phospholipid), the phospholipid is DPPC. In other embodiments where the lipid-containing composition includes a single phospholipid (non- PE phospholipid), the phospholipid is POPC. In other embodiments where the lipid- containing composition includes a single phospholipid (non-PE phospholipid), the phospholipid is EPC. In other embodiments where the lipid-containing composition includes a single phospholipid (non-PE phospholipid), the phospholipid is HSPC.
• the additional lipid component(s) include cholesterol and DSPC. In certain embodiments, the additional lipid component(s) are cholesterol and one of DMPC or DSPC. In certain embodiments, the additional lipid component(s) are cholesterol and DMPC. In other embodiments, the additional lipid component(s) are cholesterol and DSPC. In other embodiments, the additional lipid component(s) include cholesterol and DPPC. In other embodiments, the additional lipid component(s) include cholesterol and POPC. In other embodiments, the additional lipid component(s) include cholesterol and EPC. In other embodiments, the additional lipid component(s) include cholesterol and HSPC.
• the additional lipid component(s) as described herein, and those known to the skilled artisan, are commercially available from a number of suppliers, including, for example Avanti Polar Lipids, Inc. (Alabaster, AK), Northern Lipid Inc. (Canada), Lipoid GmbH (Germany), NOF Corporation (Japan), Nippon Fine Chemical Co., Ltd (Japan).
• the drug may be a nucleic acid, for example, nucleic acid encoding for sequences with anticancer properties.
• nucleic acid for example, nucleic acid encoding for sequences with anticancer properties.
• antisense oligonucleotides for example, but not limited to, antisense oligonucleotides, ribozymes, etc.
• the anticancer agent can be a cytotoxic drug, including those known by skill in the art and medical practitioners.
• exemplary anticancer agents include topoisomerase I inhibitors, vinca alkaloids, alkylating agents (including platinum compounds), taxanes and others known to those of skill in the art.
• the anticancer drug may be a topoisomerase I inhibitor, for example, but not limited to, topotecan, irinotecan, etc.
• Oxaliplatin cis-oxalato complex of trans- 1-1,2- diaminocyclohexane
• platinum is a platinum, more specifically, an oragnoplatinum, complex having a structure represented by the following formula shown below.
• Oxaliplatin is also known as the following: diaminocyclohexane platinum, DACH-platinum, and cis-[(lR, 2R)-1, 2- cyclohexanediamine-N,N'] [oxalato(2)-0,0 J ] platinum (C 8 Hi 4 N 2 O 4 Pt; MW 397.4 g/mol).
• oxaliplatin is the active pharmaceutical ingredient in EloxatinTM.
• the oxaliplatin concentration encapsulated within the liposome is about 1 mg/ml, for example about 0.8 mg/ml.
• the compositions contain from about 1 to about 45 ⁇ g oxaliplatin/mg lipid, from about 1 to about 40 ⁇ g oxaliplatin/mg lipid, from about 1 to about 35 ⁇ g oxaliplatin/mg lipid, from about 1 to about 30 ⁇ g oxaliplatin/mg lipid, from about 1 to about 25 ⁇ g oxaliplatin/mg lipid, from about 1 to about 20 ⁇ g oxaliplatin/mg lipid, from about 1 to about 15 ⁇ g oxaliplatin/mg lipid, from about 1 to about 10 ⁇ g oxaliplatin/mg lipid, from about 1 to about 5 ⁇ g oxaliplatin/mg lipid, from about 5 to about 50 ⁇ g oxaliplatin/mg lipid, from about 5 to about 45 ⁇ g oxaliplatin/mg lipid, from about 5 to about 35 ⁇ g o
• the compositions contain from about 1 to about 145 ⁇ g TF/mg lipid, from about 1 to about 120 ⁇ g TF/mg lipid, from about 1 to about 115 ⁇ g TF/mg lipid, from about 1 to about 100 ⁇ g TF/mg lipid, from about 1 to about 90 ⁇ g TF/mg lipid, from about 1 to about 70 ⁇ g TF/mg lipid, from about 1 to about 60 ⁇ g TF/mg lipid, from about 1 to about 50 ⁇ g TF/mg lipid, from about 1 to about 25 ⁇ g TF/mg lipid, from about 10 to about 150 ⁇ g TF/mg lipid, from about 10 to about 140 ⁇ g TF/mg lipid, from about 10 to about 125 ⁇ g TF/mg lipid, from about 10 to about 100 ⁇ g TF/mg lipid, from about 10 to about 80 ⁇ g TF/mg lipid, from
• the oxaliplatin may be dissolved in a solution ⁇ e.g., an aqueous solution).
• the solution includes a sugar (e.g., trehalose, maltose, sucrose, lactose, mannose, mannitol, glycerol, dextrose, fructose, etc.)
• the concentration of the sugar maybe of several percent.
• sugar concentrations of about 0.1 - 12%; 0.5-12%, 1%-12%, 2%-8%, 2%-6%, 2%-5%, 2%-4%, 2%-5%, 2%-6%, 2%-8%, 2%-9%, 2%-10%, 4%-10%, 4%-9%, 4%-8%, 4%-6%, 3%-4%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10%.
• the solution includes a sugar and is aqueous. It is intended that the solution in which oxaliplatin is dissolved can also contain additional components, including those known to the skilled artisan.
• the concentration of sugar in solution may be, for example about 50 mg/ml to about 150 mg/ml, about 50 mg/ml to about 130 mg/ml, about 50 mg/ml to about 120 mg/ml, about 50 mg/ml to about 100 mg/ml, about 80 mg/ml to about 100 mg/ml, about 90 mg/ml to about 150 mg/ml, about 90 mg/ml to about 130 mg/ml, about 60 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 105 mg/ml, about 120 mg/ml, or about 140 mg/ml.
• the solution may also contain other ingredients known to those of skill in the art, such as, but not limited to, salts, buffers, sugar alcohol, etc.
• the solution in which oxaliplatin is dissolved contains sodium phosphate ⁇ e.g., monobasic and/or dibasic sodium phosphate).
• the concentration of sodium phosphate may be about 5 to about 15 mM.
• the concentration of sodium phosphate may be about 5 to about 15 mM.
• the sugar solution may additionally include about 1.0 to about 1.5 mg/ml sodium phosphate.
• the drug is oxaliplatin, and is contained in a solution of about 9% sucrose.
• the drag is oxaliplatin, and is contained in a solution of about 9% sucrose in an oxaliplatin concentration of about 1 mg/ml.
• the drug is oxaliplatin, and is contained in a solution of about 105 mg/ml sucrose.
• the drug is oxaliplatin, and is contained in a solution of about 105 mg/ml sucrose in an oxaliplatin concentration in liposome solution of about 1 mg/ml.
• the solution further contains sodium phosphate.
• oxaliplatin is in a concentration of about 0.8 +/- 10% mg/ml of liposome solution.
• labeled compounds may also be included in the lipid- containing compositions of the present invention.
• the labeled compound may be an agent useful in carrying out in vivo diagnostic procedures.
• the amount of labeled compound to be included in the lipid-containing compositions, and formulations thereof, as described herein can be readily determined by the skilled artisan in view of the teaching herein provided and depending on the labeled compound selected and the use intended for the composition or formulation, taking into account factors specific to both the labeled compound and the individual to be diagnosed, as described further herein.
• Exemplary labeled compounds include, for example, materials comprising radioisotopes (e.g., 3 H, 14 C 5 67 Ga, 111 In, 125 1, 131 1, 133 Xe, etc.), material comprising fluorescent moieties (e.g., fluorescein, fluorescein isothiocyanate, etc.), material comprising enzyme (e.g., peroxidase, alkaline phosohatase, etc.), as well as additional labeled compounds known to those of skill in the art.
• radioisotopes e.g., 3 H, 14 C 5 67 Ga, 111 In, 125 1, 131 1, 133 Xe, etc.
• fluorescent moieties e.g., fluorescein, fluorescein isothiocyanate, etc.
• material comprising enzyme e.g., peroxidase, alkaline phosohatase, etc.
• lipid-containing compositions e.g., targeted liposomes, liposome-containing compositions, etc.
• lipid-containing compositions e.g., targeted liposomes, liposome-containing compositions, etc.
• 125 I are particularly useful for identifying the presence and determining the severity (e.g., initially, during a course of treatment, after treatment) of various cancers (e.g., breast cancer, gastric cancer, colorectal cancer, colon cancer, etc.) by gamma-counter.
• the lipid-containing compositions described herein are characterized by incorporating an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine modified with a targeting factor (i.e., TF-N ⁇ PE) directed to a particular target cell.
• a targeting factor i.e., TF-N ⁇ PE
• the term "targeting factor” refers to a moiety that can bind to a receptor or a surface antigen present on the surface of a target cell.
• the targeting factors are directed to cell surface receptors on a particular target cell.
• the targeting factor is often a protein or a peptide that can be attached to a lipid component of the lipid-containing composition.
• targeting factors need to selectively deliver the liposomes as described herein (including encapsulated drug) to the tai-geted cells (e.g., pathogenic, unhealthy, etc.).
• Selective delivery of the encapsulated drug to the targeted cells thus reduces the occurrence of adverse effects due to the effect of encapsulated drug or labeled compound on non- targeted (e.g., healthy) cells, thereby also reducing the adverse effects experienced by the individual to whom the composition, or formulation thereof, is administered.
• Exemplary targeting factors include, but are not limited to, transferrin, folic acid, folate, hyaluronic acid, sugar chains (e.g., galactose, mannose, etc.), fragments of monoclonal antibodies, asialoglycoprotein, etc., as well as other targeting factors known to the skilled artisan.
• the targeting factor is directed to an antigen (e.g., fragments of monoclonal antibodies (e.g., Fab, Fab', F(ab') 2 , Fc, etc.)). It is not intended that targeting factors include intact or whole monoclonal antibodies.
• the term "whole antibody” or "intact antibody,” and cognates thereof, as used herein generally refer to antibody IgG of immune globulin.
• a fragment of a monoclonal antibody generally refers to a decomposition product of the monoclonal antibody, for example, a fragment obtained by using protease digestion, such as pepsin, etc.
• the targeting factor is not directed to an antigen (e.g., is not a fragment of a monoclonal antibody, e.g., Fab, Fab', F(ab') 2 , Fc, etc).
• the targeting factor is transferrin.
• Transferrin is an iron binding protein with a molecular weight of 80,000, which is synthesized in hepatocytes and found in the blood. Transferrin supplies iron (Fe) to the cells through Tf receptors on the surface of each cell.
• the transferrin receptor is generally expressed in tumor tissues in a larger amount compared with normal tissues regardless of the types of the tumors. Tumor cell membranes are known to over- express transferrin receptors to maintain cell proliferation. See Shindelman JE, Ortmeyer AE, Sussman HH. "Demonstration of the transferrin receptor in human breast cancer tissue. Potential marker for identifying dividing cells. " Int J Cancer.
• Transferrin While not being limited to a mechanism of action, the likely route of uptake of transferrin liposomes as described herein is represented schematically in Figs. 2 and 3. Transferrin is commercially available, or can be produced recombinantly as described in, for example, U.S. Pat. No. 5,026,651, which is hereby incorporated by reference in its entirety.
• transferrin (Tf) transferrin
• the molar ratio of Tf to total lipid present in the targeted liposome product is approximately 0.00014:1 mol/mol (Tf:total lipid) (0.015, wt/wt). In other embodiments, the molar ratio of Tf: total lipid is from about 0.016 to about 0.029:about 126 about 158 mM/mM.
• hydrophilic polymer refers to polymers such as polyethylene glycol (PEG) and other polyethoxylated polymers that are used in the liposome field to shield liposomes in an attempt to enhance the circulatory half-life of the liposome. It is intended that this term encompasses free hydrophilic polymers associated non-covalently with the liposomes as well as hydrophilic polymers that are in some way conjugated or covalently linked to a particular component of the liposome (e.g. PEG-modified lipids, etc.). Such hydrophilic polymers are also alternatively referred to in the field as "water-soluble" polymers. Additional exemplary hydrophilic polymers include, but are not limited to, polyvinyl alcohol, polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polyacrylamide, polyglycerol, polyaxozlines, etc.
• liposome-containing composition refers to mixtures of lipids and, optionally, drug(s) or labeled compound(s), in which aqueous solution (e.g., water, buffer (e.g., acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, etc.) or mixture of water and a water-miscible solvent) has been incorporated by mixing (e.g., one or more of, stirring, shaking, etc.).
• aqueous solution e.g., water, buffer (e.g., acetate buffer, phosphate buffer, citrate buffer, borate buffer, tartrate buffer, etc.) or mixture of water and a water-miscible solvent) has been incorporated by mixing (e.g., one or more of, stirring, shaking, etc.).
• the aqueous solution may also include additional components such as one or more sugars (e.g., trehalose, maltose, sucrose, lactose, mannose, dextrose, fructose, etc.) , sugar alcohol (e.g., sorbitol, maltitol, lactitol, mannitol, glycerol, etc.), alcohol (e.g., ethanol, t-butanol, etc.), etc.
• sugars e.g., trehalose, maltose, sucrose, lactose, mannose, dextrose, fructose, etc.
• sugar alcohol e.g., sorbitol, maltitol, lactitol, mannitol, glycerol, etc.
• alcohol e.g., ethanol, t-butanol, etc.
• the aqueous solution may also include organic solvent ((e.g., esters (e.g., ethyl acetate, butyl acetate, etc.), aliphatic hydrocarbons (e.g., hexane, heptane, etc.), aromatic hydrocarbons (e.g., toluene, xylene, etc.), halogenated hydrocarbons (e.g., chloroform, dichloromethane, dichloroethane, etc.), ethers (e.g., THF, dioxane, diethyl ether, isopropyl ether, etc.), cyclic hydrocarbons (e.g., cyclohexaiie, etc.), DMF, DMSO, etc.) or mixtures thereof).
• organic solvent e.g., esters (e.g., ethyl acetate, butyl acetate, etc.), aliphatic hydrocarbons (e.g
• the liposome-containing composition will generally contain a non-homogenous mixture of lipids, aqueous solution, and liposomes having a broad distribution around 100- 10,000 nm and a mean diameter of 500-2,000 nm. Characterization of exemplary liposome-containing compositions is further provided in the Examples.
• the lipid-containing compositions do not incorporate hydrophilic polymers. In particular embodiments, the lipid-containing compositions do not incorporate PEG.
• the intermediate lipid mixtures include at least two different neutral lipids or one or more phospholipids, and an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine, wherein the lipid components are described in greater detail herein and wherein the mixture is free of non-derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• an aqueous solution as herein described may be mixed with the lipid components to form a liposome-containing composition.
• the lipid mixture does not include a drug or labeled compound.
• the lipid-mixture may be treated to form a liposome-containing composition or a liposome formulation.
• the intermediate lipid mixtures include at least two different neutral lipids or one or more phospholipids, an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine and a drug or labeled compound, wherein the lipid components and drag/labeled compound are described in greater detail herein and wherein the mixture is free of non-derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• an aqueous solution as herein described may be mixed with the lipid components to form a liposome-containing composition, for example, as where the drug or labeled compound is added as an aqueous solution of drag/labeled compound.
• the liposome-containing composition can be treated (e.g., by one or more of extrusion, size exclusion chromatography, etc. or methods known in the art) to form a liposome.
• the lipid mixtures include one or more phospholipids or at least two different neutral lipids, an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine and a succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine, wherein the lipid components are described in greater detail herein and wherein the mixture is free of non-derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• the mixtures may also be substantially free of non-NHS starting material, byproduct and/or decomposition product associated with synthesis of the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine (e.g., carbodiimmides (e.g., DCC, EDC, etc.), acylated urea compounds, etc.).
• an aqueous solution as herein described may be mixed with the lipid components to form a liposome-containing composition.
• the mixtures may also be substantially free of non-NHS starting material, byproduct and/or decomposition product associated with synthesis of the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine (e.g., carbodiimmides (e.g., DCC, EDC, etc.), acylated urea compounds, etc.).
• an aqueous solution as herein described may be mixed with the lipid components to form a liposome-containing composition.
• the lipid mixture does not include a drug or labeled compound.
• the lipid mixture may be treated to form a liposome-containing composition or a liposome formulation.
• the intermediate lipid mixtures include one or more phospholipids or at least two different neutral lipids, an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine and a succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and a drug or labeled compound, wherein the lipid and drug/labeled compound components are described in greater detail herein and wherein the composition is free of non -derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• the liposomes may also be substantially free of non-NHS starting material, byproduct and/or decomposition product associated with synthesis of the succinimidyl ester of an N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine (e.g., carbodiimides (e.g., DCC, EDC, etc.), acylated urea compounds, etc.).
• the lipid mixture does not include a drug or labeled compound.
• the lipid mixture may be treated to form a liposome-containing composition or a liposome formulation.
• the lipid mixtures include one or more phospholipids or at least two different neutral lipids, an N-( ⁇ )-dicarboxylic acid- derivatized phosphatidyl ethanolamine, a targeting-factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and an encapsulated drug or labeled compound, wherein the lipid, targeting factor and drug/labeled compound components are described in greater detail herein and wherein the liposome is free of non-derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine may be present in the initial stages in the preparation of the targeted liposomes (e.g., NHS-NG-PEs (e.g., NHS-NG-DOPE, NHS- NG-DSPE, etc.), for example, prior to hydrolysis of the succinimidyl ester which may yield, for example NHS and NG-DOPE in the final formulation.
• NHS-NG-PEs e.g., NHS-NG-DOPE, NHS- NG-DSPE, etc.
• the liposome or liposome-containing composition can also be free or substantially free of NHS, as in when TF-N ⁇ PE is pre-formed and used as starting material.
• the targeted liposomes are substantially free of DCC and EDC. In certain embodiments, the targeted liposomes are substantially free of DCC.
• the liposomes formed from the liposome-containing compositions herein described may incorporate drug or labeled compound or may be free of drug or labeled compound (e.g., for liposomes also referred to herein also as "blank liposomes").
• the liposome-containing compositions, liposomes (including blank liposomes) and targeted liposomes maybe formulated as pharmaceutical formulations and, additionally, may be used in the methods of treatment or diagnosis and/or kits described herein.
• the lipid-containing compositions may contain less than about 0.1%, less than about 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, or less than about 6% by weight of a particular starting material, byproduct or decomposition product associated with synthesis of the succinimidyl ester of an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine to the total lipid content.
• the targeted liposomes include liposomes containing a phosphatidylcholine (e.g., neutral, anionic or cationic), cholesterol or a cholesterol derivative, an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine, a targeting factor-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and an encapsulated drug or labeled compound, wherein the lipid, targeting factor and drug components are described in greater detail herein and wherein the liposome is free of non- derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• the phosphatidyl choline is a neutral phosphatidyl choline.
• the targeted liposomes include liposomes containing a phosphatidyl choline ⁇ e.g., neutral, anionic or cationic), cholesterol or a cholesterol derivative, an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine, a transferrin-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine and encapsulated oxaliplatin, wherein the lipid components are described in greater detail herein and wherein the liposome is free of non-derivatized phosphatidyl ethanolamine and hydrophilic polymers, such as polyethylene glycol.
• the phosphatidyl choline is a neutral phosphatidyl choline.
• the lipid-containing compositions may further contain lipids obtained by derivatizing phosphatidylglycerol, sphingosine, ceramide, a cholesterol derivative or the like with a dicarboxylic acid.
• dicarboxylic acid- derivatives may be prepared as described herein for the N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines and according to preparation methods known to the skilled artisan.
• the lipid-containing compositions do not include anionic lipids ⁇ e.g., phosphatidylserines, phosphatidylinsitosols, phosphatidylglycerols, etc.) or cationic lipids ⁇ e.g., sphingosine, DOTAP, DOTMA, DC- CHOL, etc.).
• anionic lipids e.g., phosphatidylserines, phosphatidylinsitosols, phosphatidylglycerols, etc.
• cationic lipids e.g., sphingosine, DOTAP, DOTMA, DC- CHOL, etc.
• the lipid-containing compositions comprises a drug. In other embodiments, the lipid-containing compositions comprise a labeled compound.
• the drag is oxaliplatin
• the targeting factor (TF) is transferrin (Tf)
• the lipid components include: DMPC or DSPC, and, cholesterol or a cholesterol derivative, and an N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine, and a transferrin-modified N-( ⁇ )- dicarboxylic acid-derivatized phosphatidyl ethanolamine, wherein the transferrin-modified N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine comprises a transferrin linked to an N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamine by a carboxylic acid amide bond.
• the lipid components of the lipid- containing compositions are DMPC, cholesterol, NG-DOPE and TF-modified NG-DOPE.
• the lipid components are DSPC, cholesterol, NG-DOPE and TF- modified NG-DOPE.
• the lipid components are DMPC, cholesterol, NG-DSPE, and TF-modified NG-DSPE.
• the lipid components are DSPC, cholesterol, NG-DSPE, and TF-modified NG-DSPE.
• the targeting factor (TF) is transferrin and the drag is oxaliplatin.
• the lipid components are DPPC, cholesterol, NG-DOPE and TF-modified NG-DOPE.
• the lipid components are POPC, cholesterol, NG-DOPE and TF-modified NG-DOPE.
• the lipid components are DPPC, cholesterol, NG-DSPE, and TF-modified NG-DSPE.
• the lipid components are POPC, cholesterol, NG-DSPE, and TF-modified NG-DSPE.
• the targeting factor (TF) is transferrin and the drag is oxaliplatin.
• the molar ratio of (e g., DMPC: Choi :NG-DOPE:NHS-NG-DOPE) may be 43.0:38.5:3.42:1, which can also be expressed as 50:45:4:1 by mol%.
• the relative mol% of additional lipid(s) to N ⁇ PE to SuccN ⁇ PE (e.g., at least two neutral lipids:N ⁇ PE:SuccN ⁇ PE or one or more phospholipids :N ⁇ PE: SuccN ⁇ PE or (one or more phospholipids + neutral lipid(s)):N ⁇ PE: SuccN ⁇ PE) maybe from about 98 mol% to about 87 mol% additional lipids: from about 1 mol% to about 12 mol% N ⁇ PE: about 0.5 mol% to about 1% SuccN ⁇ PE; where the total mol% of all components is 100 mol%.
• additional lipids:N ⁇ PE:SuccN ⁇ PE maybe approximately 95:4:1, 90:9:1, 92:7:1, 93:6:1, etc.
• the additional lipids include a phospholipid and another lipid such as cholesterol, cholesterol derivatives, etc.
• the range of mol% for each lipid component is from about 30 mol% to about 64%, where the total of additional lipids is from about 98 mol% to about 87 mol%.
• the range of mol% for each neutral lipid is from about 30 mol% to about 64%, where the total of neutral lipids is from about 98 mol% to about 87 mol%.
• the mol% of the phosphatidyl choline is from about 30 to about 70 mol%, (e.g., from about 50 to about 64 mol%, from about 40 to about 65 mol%, from about 40 to about 60 mol%, from about 50 to about 62 mol %, from about 55 to about 60 mol%, from about 35 to about 55 mol%, about 30 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%) and the mol% of the cholesterol or cholesterol derivative is from about 30 to about 60 mol% (e.g., from about 32 to about 45 mol%, from about 32 to about 42 mol%, from about 32 to about 40 mol%, from about 40 to about 60 mol%, from about 35 to about
• the phosphatidyl choline is about 50 mol%, about 52 mol%, about 55 mol%, about 58 mol%, about 60 mol%, about 62 mol% and the cholesterol or cholesterol derivatives is from about 30 mol%, about 32 mol%, about 34 mol%, about 35 mol%, about 37 mol%, about 38 mol%, about 40 mol%, about 42 mol%, about 43 mol%, about 45 mol%.
• the mol% of N ⁇ PE is about 1 to about 11 mol%, about 1 to about 10 mol%, about 1 to about 8 mol%, about 1 to about 6 mol%, about 1 to about 5 mol %, about 1 to about 4 mol%, about 1 to about 3 mol%, about 1 to about 2 mol%, about 2 to about 10 mol%, about 2 to about 5 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 7 mol%, about 8 mol%, about 9 mol% about 10 mol%, about 11 mol%, or about 12 mol%.
• the relative mol% of the first additional lipidrsecond additional lipid :N ⁇ PE:SuccN ⁇ PE is, for example 50:45:4:1.
• the first additional lipid is a phosphatidylcholine (e.g., DMPC, DOPC, DPPC, DSPC, etc.) and the second additional lipid is cholesterol.
• the PE of the N ⁇ PE is DOPE or DSPE.
• the N ⁇ PE is NG- DOPE or NG-DSPE.
• the lipids are DMPC:Chol:NG-DOPE:NHS- NG-DOPE and their relative mol% is 50:45:4:1.
• the lipids are DSPC:Chol:NG-DSPE:NHS-NG-DSPE and their relative mol% is 50:45:4:1. In other embodiments, the lipids are DSPC:Chol:NG-DSPE:NHS-NG-DSPE and their relative mol% is 62:33:4:1. [0326] In some embodiments, the total mol% of N ⁇ PE and TF-N ⁇ PE (N ⁇ PE + TF-N ⁇ PE) is about 2 to about 13 mol% of total lipid content.
• the total mol% of TF-N ⁇ PE is about 0.002 to about 0.2 mol% relative to total lipid content.
• the total mol% of TF-N ⁇ PE is about 0.002 to about 0.15 mol%
• total mol% of TF-N ⁇ PE is about 0.002 to about 0.1 mol%, 0.002 to about 0.05 mol%, about 0.01 to about 0.03 mol%, about 0.005 to about 0.2 mol%, about 0.007 to about 0.2 mol%, about 0.007 to about 0.05 mol%, about 0.01 to about 0.025 mol%, about 0.015 to about 0.025 mol%, about 0.01 to about 0.2 mol%, about 0.02 to about 0.2 mol%, about 0.04 to about 0.2 mol%, about 0.06 to about 0.2 mol%, about 0.08 to about 0.2 mol%, about 0.002 mol%, about 0.008 mol%, about 0.01 mol%
• the liposome-containing compositions as described herein may also be characterized ⁇ e.g., physicochemical properties, etc.) using standard analytical methods, as will be appreciated by the skilled artisan.
• Such analytical methods include, but are not limited to and where appropriate, determination of mean diameter, encapsulated volume, net charge (zeta potential), amount of entrapped ⁇ i.e., encapsulated) drug, particle size, stability under various conditions ⁇ e.g., in storage, as prepared for administration, in vitro), osmotic properties, amount of conjugated targeting factor, etc.
• Exemplary analytical methods for such characterization are set forth below, as well as in the Examples, and additional methods known to the skilled artisan may also be used to characterize the compositions.
• the drug content of liposomes can be determined using established methods for HPLC analysis, with appropriate controls, as routinely practiced in the art and, additionally, as described in the Examples. Using appropriate controls, the identity of the encapsulated drug can also be determined by HPLC or, for certain drugs (e.g., platinum containing drugs) by analytical methods such as ICP-MS (Inductively Coupled Plasma-Mass Spectrometry) as is practiced by those of skill in the field.
• ICP-MS Inductively Coupled Plasma-Mass Spectrometry
• the amount of drug (e.g., oxaliplatin, etc.) or labeled compound encapsulated within the liposome or liposome-containing composition may be from about 0.1 mg/ml to about 15 mg/ml within the liposome.
• the drug concentration may be from about 0.5 mg/ml to about 15 mg/ml, about 0.5 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 10 mg/ml, about 0.5 mg/ml to about 5 mg/ml; about 0.5 mg/ml to about 3 mg/ml, about 0.5 mg/ml to about 2 mg/ml, about 0.5 mg/ml to about 1.5 mg/ml, about 0.8 mg/ml to about 3 mg/ml, about 0.8 mg/ml to about 2 mg/ml, about 0.8 mg/ml to about 1.5 mg/ml, about 0.7 mg/ml to about 3 mg/ml, about 0.7 mg/ml to about 2 mg/ml, about 0.7 mg/ml to about 1.7 mg/ml, about 0.7 mg/ml to about 1.5 mg/ml, about 0.7 mg/ml to about 1.4 mg/ml, about 0.7 mg/ml to about 1.3 mg/ml, about 0.5 mg/
• the electric potential at the shear plane is called the zeta potential of a liposome.
• the zeta potential of liposomes can be experimentally determined using appropriate instrumentation, for example as measured by an ELS-6000 (Otsuka Electronics, Japan) with the laser-Doppler microelectrophoresis method or other instrumentation and protocols available to the skilled artisan.
• ELS-6000 Oletsuka Electronics, Japan
• the laser-Doppler microelectrophoresis method or other instrumentation and protocols available to the skilled artisan.
• J. Colloid and Interface ScL 39, 670-675(1972), nition.com/en/products/zeecom_s.htm, etc.
• the liposomes (including the targeted liposomes and the liposomes of the liposome-containing composition) as described herein will exhibit an overall net negative zeta potential.
• the zeta potential is from about -10 mV to about -200 mV.
• Liposome particle size can be obtained from the correlation function by using various algorithms using Photon Correlation Spectroscopy (PCS; Dynamic Light Scattering or Quasi-Elastic Light Scattering (QELS)).
• PCS Photon Correlation Spectroscopy
• QELS Quasi-Elastic Light Scattering
• the particle size obtained by these techniques is comparable to the mean diameter determined by PCS.
• PCS uses standard deviation and ⁇ 2 to describe the size distribution. In PCS systems, ⁇ 2 determines whether the system is unimodal (Gaussian distribution) or multimodal (Nicomp distribution).
• the mean particle size can be determined using intensity weighted measurements and is reported from the Gaussian distribution if ⁇ 2 ⁇ 5. If ⁇ 2 > 5, then the mean of the principal peak in a Nicomp distribution.
• Such analysis will be familiar to the skilled artisan, as will suitable hardware, for example Nicomp QELS Particle Sizer, PSS Model 380ZLS, S/N 0103301; ps
• the liposome mean diameter will be from about 50 to about 275 nm.
• the liposome mean diameter may be from about 50 to about 200 nm, from about 50 to about 265 nm, from about 50 to about 250 nm, from about 50 to about 225 nm, from about 50 to about 175 nm, from about 50 to about 150 nm, from about 50 to about 120 nm, from about 50 to about 100 nm, from about 75 to about 250 nm, from about 75 to about 200 nm, from about 75 to about 175 nm, from about 75 to about 150 nm, from W
• the targeted liposome will be approximately about 15 to about 25 nm greater in diameter than the a liposome formed of the same components but with the targeting factor not incorporated.
• confirmation of transferrin content in liposomes can be evaluated using two assays for the content and/or identity of transferrin conjugated to liposomes.
• the electrophoretic migration of transferrin the liposome as analyzed by SDS-PAGE can be compared to the migration pattern of purified conjugated transferrin, e.g., TF-N ⁇ PE.
• the electrophoretic migration of conjugated transferrin in can also be compared to free transferrin reference standard. Additional support for the identity of transferrin in liposomes can be obtained using ELISA, a research-grade formulation that demonstrates specific binding of anti-transferrin antibody to the targeted liposomes.
• the concentration of transferrin-targeted liposomes can be measured using colorimetric protein quantification assays, such as a BCA, an assay well known to the skilled artisan.
• colorimetric protein quantification assays such as a BCA, an assay well known to the skilled artisan.
• BCA colorimetric protein quantification assay
• the concentration of targeting ligand incorporated in the liposome will be from about 0.5 mg/ml to about 5.0 mg/ml, from about 0.5 mg/ml to about 2.0 mg/ml, from about 1.0 mg/ml to about 2.0 mg/ml, from about 1.0 mg/ml to about 3.0 mg/ml, from about 1.0 mg/ml to about 2.5 mg/ml, from about 1.0 mg/ml to about 2.0 mg/ml, or from about 1.3 mg/ml to about 2.5 mg/ml.
• ferric ion The role of ferric ion is very important in binding transferrin to the surface of tumor cells. Therefore, the ferric ion content of targeted liposomes incorporating Tf is another meaningful way of characterizing the liposomes. While a number of methods for determining ferric ion content will be known to those of skill in the art, one method is ICP-MS.
• the liposomes may also be characterized by their osmotic pressure at a given temperature.
• the osmotic pressure at a given temperature depends upon the molar concentration of sugar (sucrose) solution. And it also depends on the total ion density and the size of the molecules within the solution. Normally osmotic pressure can be measured using an instrument known as an osmometer, which measures osmotic pressure in suitable pressure units, as will be appreciated by the skilled artisan.
• the osmotic pressure of the liposomes, particularly the targeted liposomes and blank liposomes, at room temperature will be from about 310 to about 410 m ⁇ sm/kg.
• the osmotic pressure may be from about from about 310 to about 400 mOsm/kg, from about 310 to about 380 mOsm/kg, from about 320 to about 360 mOsm/kg, from about 315 to about 375 mOsm/Kg, from about 320 to about 375 mOsm/Kg, from about 315 to about 370 mOsm/Kg, from about 320 to about 370 mOsm/Kg, about 360 mOsm/Kg, about 350 mOsm/Kg, about 340 mOsm/Kg, about 370 mOsm/Kg or about 380 mOsm/Kg at room temperature.
• the osmotic pressure may vary less than about 25%, less than about 20%, less than about 15% when monitored over a particular time period associated with the various conditions as described herein. For example, 360 +/- 50 m ⁇ sm/kg.
• compositions described herein that can be used to consistently produce high quality (e.g., of high purity, homogeity, etc.) targeted liposomes (and intermediates thereof) and blank liposomes. These methods are also represented schematically in Figs. 4 (production method A) and 5 (production method B).
• Use of the general methods described herein, including in the Examples, to produce the targeted liposomes also encompasses methods for the production of the other lipid-containing compositions (e.g., lipid mixtures, liposome-containing compositions, blank liposomes, and intermediate liposomes), as described herein.
• the other lipid-containing compositions e.g., lipid mixtures, liposome-containing compositions, blank liposomes, and intermediate liposomes
• succinimidyl esters of N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines (SuccN ⁇ PE) and N-( ⁇ )-dicarboxylic acid-derivatized phosphatidyl ethanolamines (N ⁇ PE) as described herein are mixed with additional lipid(s) ((e.g., at least one phospholipid(s) (e.g.
• the SuccN ⁇ PE and N ⁇ PE used as described in the preceding paragraph can be prepared and purified by methods described herein or by methods known to the skilled artisan.
• the SuccN ⁇ PE and N ⁇ PE, as well as other components described herein for the production of the targeted liposomes and intermediates thereof, should be of sufficient purity and homogeneity to ultimately yield targeted liposomes of sufficient purity and homogeneity to fall within regulatory guidelines for the administration of the targeted liposomes to individuals and in accordance with good laboratory practice (GLP) and good manufacturing practice (GMP) guidelines.
• GLP laboratory practice
• GMP good manufacturing practice
• the ratio of phospholipid(s) to other additional lipid(s) is about 2:1.
• a mixing ratio of the SuccNoPE derivatives to the phospholipids is from about 1 to about 12% (1:99 to 12:88), or about 3-6% (3:97 to 6:94) of the total concentration/ratio of (NHS-NGPE + NGPE) to (CHOL +Phospholi ⁇ id).
• exemplary ratio include 50:45:4:1 (e.g., PC:Chol:NG-PE:NHS-NG-PE), where, for example 17.5mg of NHS-NG-DOPE, 63.1mg of NG-DOPE, 312mg of Choi and 607mg of phospholipids in Ig of mixture.
• the N ⁇ PE:SuccN ⁇ PE:additional lipid mixture prepared in step A is then mixed with aqueous solution (e.g., buffer, etc.) containing the drug or labeled compound to be encapsulated (e.g., anticancer agent (e.g., oxaliplatin, topoisomerase I inhibitor, vinca alkaloid, etc.)) to obtain the drug:N ⁇ PE:SuccN ⁇ PE:neutral lipid mixture (Intermediate 2).
• aqueous solution e.g., buffer, etc.
• anticancer agent e.g., oxaliplatin, topoisomerase I inhibitor, vinca alkaloid, etc.
• the concentration of the oxaliplatin solution is about 8 mg/ml in an approximately 9% sucrose solution.
• the concentration of oxaliplatin in the targeted liposome is about 0.8 mg/mL +/- 10%.
• the drug:N ⁇ PE:SuccN ⁇ PE:additional lipid mixture (Intermediate 2) obtained in step B is then sonicated or stirred followed by evaporation of the solvent to form the drug:N ⁇ PE:SuccN ⁇ PE:additional lipid liposome (Intermediate 3).
• Methods and conditions for performing sonication, stirring and evaporation and means for accomplishing these steps are well understood by the skilled artisan and are also further described in the Examples. See for example, methods of production by reverse phase vesicle (REV) methods, U.S. Patent No. 4,235,871 (incorporated by reference in its entirety).
• General liposome production methods such as simple hydration methods and ethanol injection methods, known to the skilled artisan, can be also used.
• the drug :N ⁇ PE:SuccN ⁇ PE: additional lipid liposome formed as described above is then extruded by size and the drug:N ⁇ PE:SuccN ⁇ PE: additional lipid liposome is isolated.
• ultrafiltration can then be used to concentrate the liposome solution.
• the liposome contains /-OHP (drug), DMPC (additional lipid/phospholipid (neutral)), cholesterol (CHOL, additional lipid/neutral lipid), N-glutaryl- DOPE (NG-DOPE) and NHS-NG-DOPE
• a liposome with a mean diameter of about 0.2 micrometer (200 nm) can be isolated.
• sized liposomes can also be obtained for liposomes containing /-OHP (drug), DSPC (additional lipid/phospholipid (neutral), cholesterol, N-glutaryl-DSPE (NG-DSPE) and NHS-NG-DSPE.
• targeted liposomes incorporating apo-transferrin can be treated with ferric compounds, such as ferric citrate, iron (III) chloride, etc., to produce targeted liposomes incorporating holo-transferrin derivatized liposome.
• ferric compounds such as ferric citrate, iron (III) chloride, etc.
• the targeted liposomes as described herein can also be produced by dissolving the additional lipids and N ⁇ PE in a suitable solvent ⁇ e.g., ethanol, t-BuOH, chloroform, isopropylether, etc.), dispersing the resultant solution in an aqueous solution optionally containing a drug or labeled compound, and then performing ultrasonication or reverse phase vesicle of the resultant dispersion to form a liposome (drug:N ⁇ PE:neutral lipids).
• a suitable solvent e.g., ethanol, t-BuOH, chloroform, isopropylether, etc.
• the liposome solution may be concentrated by ultrafiltration.
• this liposome-containing composition can subsequently be treated with NHS and TF to also yield a additional lipid component(s):N ⁇ PE:TF-N ⁇ PE:drug/labeled compound liposome- containing composition.
• C-2 (optionally containing drug or labeled compound) or C2-A, may then be treated to form a liposome (C-3).
• C-3 does not include a drug or labeled compound, the liposome will be an blank liposome, as previously described (e.g., additional lipid component(s):N ⁇ PE:TF-N ⁇ PE liposome).
• C-3 includes a drug or labeled compound, the liposome will be a targeted liposome as described herein.
• lipid mixture D-I e.g., components: additional lipid component(s); N ⁇ PE; or components: additional lipid component(s):N ⁇ PE:drug or labeled compound
• Lipid mixture D-I can then be mixed with aqueous solution to form a liposome-containing composition D-2 (additional lipid component(s):N ⁇ PE: (optionally containing drug or labeled compound).
• Liposome- containing composition D-2 can then be mixed with TF- N ⁇ PE to form a liposome- containing composition D-3 (additional lipid component(s):N ⁇ PE:TF-N ⁇ PE (optionally containing drug or labeled compound)), where the TF-NcoPE is prepared and optionally purified prior to admixture.
• the drug or labeled compound may be added after formation of the liposome-containing composition (D3-A).
• the liposome-containing composition can be formed simultaneously upon the mixing of all the components.
• D-3 (optionally containing drug or labeled compound) or D3-A, may then be treated to form a liposome (D-4).
• D-4 does not include a drug or labeled compound, the liposome will be an blank liposome, as previously described (e.g., additional lipid component(s):N ⁇ PE:TF-N ⁇ PE liposome).
• D-4 includes a drug or labeled compound, the liposome will be a targeted liposome as described herein.
• D- 4 is an blank liposome
• a drug or labeled compound may, as previously described, be added to the blank liposome in a subsequent step to form a targeted liposome, which may be performed immediately after preparation of D-4 or after a delay, which may include storage of the D-4 blank liposome for a period of time.
• the liposome or liposome-containing composition can also be free or substantially free of NHS, as in when TF-N ⁇ PE is preformed and used as starting material.
• the lipid-containing compositions prepared by production method C or method D are substantially free of DCC and EDC.
• the lipid-containing compositions prepared by production method C or method D are substantially free of DCC.
• the additional lipid component(s) includes one or more phospholipid ⁇ e.g., a phosphatidyl choline, etc.) and a cholesterol or cholesterol derivative.
• the phosphatidyl choline is DMPC, POPC, DSPC, etc. as herein described.
• the phosphatidyl choline is DMPC or DSPC.
• the additional lipid(s) are a phospholipid and cholesterol.
• the phospholipid is a neutral phospholipid.
• the N ⁇ PE is an NG-PE.
• the N ⁇ PE is N ⁇ -DOPE or N ⁇ -DSPE.
• the N ⁇ PE is NG-DOPE or NG-DSPE.
• the TF is for example, asialoglycoprotein, folate, transferrin, etc.
• the TF is transferrin (Tf).
• the TF-N ⁇ PE is a Tf-N ⁇ PE (e.g., Tf-NG-DOPE or Tf-NG- DSPE).
• the present invention provides pharmaceutical formulations for treatment or diagnosis of individuals in need thereof, comprising lipid- containing compositions as described herein and one or more pharmaceutically acceptable carriers, excipients, diluents, stabilizers, preservatives, or other inactive ingredients, including combinations of the foregoing, known to skilled artisans and described further herein.
• the lipid-containing composition is a targeted liposome as described herein. In other embodiments, the lipid-containing composition is a liposome-containing composition. In some embodiments, the lipid-containing composition is an blank liposome. In certain embodiments, the composition comprises a drug. In other embodiments, the composition comprises a labeled compound.
• the pharmaceutical formulations may further comprise one or more of different salts, sugars, proteins, starch, gelatin, plant oils, polyethylene glycol and the like, including combinations of two or more of the foregoing.
• the methods may practiced as a diagnostic approach towards the diagnosis of the conditions described herein.
• the labeled compound-containing lipid-containing compositions or pharmaceutical formulations may be used to diagnosis the conditions described herein in individuals in need thereof, including humans.
• the methods generally comprise administering to the individual an amount of a composition, or formulation described herein, effective to diagnosis the condition. Such administration is generally undertaken in conjunction with methods to detect the condition.
• a pharmaceutically or therapeutically effective amount refers to an amount of a composition sufficient to treat a specified disorder, condition or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder.
• a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause a tumor to shrink or to decrease the growth rate of the tumor.
• an amount effective to diagnose or “diagnostically effective amount” or “amounts effective for diagnosis” cognates thereof, refer to an amount of a composition sufficient to diagnose a specified disorder, condition or disease, and/or one or more of its manifestations, where diagnosis includes identification of the existence of the disease and/or detection of the extent or severity of the disease.
• a “diagnostically effective amount” comprises an amount sufficient to detect, for example, the presence and/or concentration of one or more of malignant cells, tumor(s) or other manifestation of the cancer.
• diagnosis will be carried out with reference to a baseline or background detection level observed for individuals without the condition. Levels of detection above background or baseline levels (elevated levels of detection) are indicative of the presence and, in some cases, the severity of the condition.
• an individual "in need thereof may be an individual who has been diagnosed with or previously treated for the condition to be treated.
• an individual "in need thereof maybe an individual who is suspected to have a condition, is at risk for a condition (e.g., a family history of the condition, life-style factors indicative of risk for the condition (e.g., smoking as a risk factor for lung cancer, etc.)) or has previously been diagnosed with the condition (e.g., diagnosis can include monitoring of the severity (e.g., progression/regression) of the disease over time and/or in conjunction with therapy).
• a condition e.g., a family history of the condition, life-style factors indicative of risk for the condition (e.g., smoking as a risk factor for lung cancer, etc.)
• diagnosis can include monitoring of the severity (e.g., progression/regression) of the disease over time and/or in conjunction with therapy).
• the condition to be treated or diagnosed is cancer.
• the cancer may be a gastric, colon, colorectal or breast cancer.
• the cancer is a colon cancer.
• the cancer is a breast cancer.
• the cancer is a gastric cancer.
• the cancer is cancer of the pancreas, non small cell lung cancer, small cell lung cancer, brain cancer, liver cancer, renal cancer, prostate cancer, bladder cancer, ovarian cancer, or hematological malignancies (e.g., leukemia, lymphoma, multiple myeloma, etc.).
• compositions including formulations described herein, may be used alone or in conjunction with (e.g., prior to, concurrently with, or after) other modes of treatments (e.g., adjunctive cancer therapy, combined modality treatments).
• other therapeutic agents e.g., cancer chemotherapeutic agents as described herein and known to those of skill in the art (e.g., alkylating agents, taxanes, metabolic antagonist, antitumour antibiotic, plant alkaloids, hormone therapy drug, molecular target drug, etc.)
• surgery and/or radiation therapy.
• the targeted liposomes and formulations thereof described herein may also be administered in conjunction with (e.g., prior to, concurrently with, or after) drugs to alleviate the symptoms associated with the condition or the treatment regimen (e.g., drugs to reduce vomiting, hair loss, immunosuppression, diarrhea, rash, sensory disturbance, anemia, fatigue, stomatitis, hand foot syndrome, etc.).
• drugs to alleviate the symptoms associated with the condition or the treatment regimen e.g., drugs to reduce vomiting, hair loss, immunosuppression, diarrhea, rash, sensory disturbance, anemia, fatigue, stomatitis, hand foot syndrome, etc.
• the targeted liposomes may also be administered at more than one stage of (including throughout) the treatment regimen (e.g., after surgery and concurrently with and after radiation therapy, etc.).
• the compositions are administered prior to or after surgery (e.g., removal of a tumor or lymph nodes, etc.). In other embodiments, the compositions are administered after surgery and prior to, concurrently with or after radiation therapy.
• surgery and/or radiation therapy in conjunction with administration of the compositions described herein, and, optionally, additional one or more chemotherapeutic agents, can be determined by an attending physician based on the individual and taking into consideration the various factors effecting the particular individual, including those described herein.
• Continuous intravenous infusion may be administered over a period of minutes or hours. For example, but not limited to, from about 10 minutes to about 5 hours, from about 15 minutes to about 4 hours; from about 30 minutes to about 4 hours; from about 45 minutes to about4 hours, from about 60 minutes to about 4 hours, from about 45 minutes to about 3 hours, from about 60 minutes to about 2 hours, from about 90 minutes to about 3 hours, from about 90 minutes to about 2 hours, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 50 minutes, about 60 minutes, 80 minutes, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 5 hours, about 12 hours, about 24 hours, about 36 hours, or about 48 hours.
• lipid-containing compositions and pharmaceutical formulations as described herein may be administered to individuals in need thereof for the treatment or diagnosis of conditions as described herein in conjunction with the methods of use described herein.
• an effective amount is an amount sufficient to reduce tumor growth (e.g., as measured by rate of increase of mean tumor volume prior to and/or after treatment). In certain embodiments, an effective amount is an amount sufficient to decrease mean tumor volume (e.g., where mean tumor volume after treatment is reduced compared to mean tumor volume prior to treatment).
• compositions administered in order to administer an effective amount of encapsulated drug will depend upon a variety of factors, including, for example, the particular condition being treated, the mode of administration, the severity of the condition being treated and the age and weight of the patient, the bioavailability of the composition, the adverse effects experienced by the individual being treated, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art in view of the teachings provided herein.
• the dose of encapsulated oxaliplatin administered at a particular time point will be in the range from about 1 to about 400 mg/m 2 /day.
• the dose of encapsulated oxaliplatin administered at a particular time point will be in the range from about 1 to about 400 mg/m 2 /day.
• the range from about 1 to about 350 mg/m 2 /day, 1 to about 300 mg/m 2 /day, 1 to about 250 mg/m 2 /day, 1 to about 200 mg/m 2 /day, 1 to about 150 mg/m 2 /day, 1 to about 100 mg/m 2 /day, from about 5 to about 80 mg/m 2 /day, from about 5 to about 70 mg/m 2 /day, from about 5 to about 60 mg/m 2 /day, from about 5 to about 50 mg/m 2 /day, from about 5 to about 40 mg/m 2 /day, from about 5 to about 20 mg/m 2 /day, from about 10 to about 80 mg/ni 2
• the does admintestered at a particular time point may also be about 130 mg/m 2 /day, about 120 mg/m /day, about 100 mg/m 2 /day, about 90 mg/m 2 /day, about 85 mg/m 2 /day, about 80 mg/m 2 /day, about 70 mg/ni 2 /day, about 60 mg/m 2 /day, about 50 mg/m 2 /day, about 40 mg/m 2 /day, about 30 mg/m 2 /day, about 20 mg/m 2 /day, about 15 mg/m 2 /day, or about 10 mg/m 2 /day.
• Dosages may also be estimated using in vivo animal models, as will be appreciated by those skill in the art.
• compositions as described herein may also be administered to individuals in need thereof of the course of hours, days, weeks, or months. For example, but not limited to, daily, every other day, every 10 days, weekly, monthly, twice weekly, three times a week, twice monthly, three times a month, four times a month, five times a month, every other month, every third month, every fourth month, etc.
• kits for administration of the compositions described herein including pharmaceutical formulations comprising the compositions.
• the kits may include a dosage amount (e.g., as used for therapy or diagnosis) of at least one lipid-containing composition, or pharmaceutical formulation thereof, as disclosed herein.
• Kits may further comprise suitable packaging and/or instructions for use of the composition.
• Kits may also comprise a means for the delivery for the composition, or pharmaceutical formulation thereof, such as a syringe for injection or other device as described herein and known to those of skill in the art.
• kits may include a dosage amount (e.g., as used for therapy or diagnosis) of a blank liposome, or pharmaceutical formulation thereof, as disclosed herein. Kits may further comprise suitable packaging and/or instructions for use of the composition. Kits may also comprise a means for the delivery for the composition, or pharmaceutical formulation thereof, such as a syringe for injection or other device as described herein and known to those of skill in the art. Additionally, in certain embodiments, the kit may contain a separate dosage amount of the drug or labeled compound to be incorporated into the blank liposome.
• the lipid-containing composition, or pharmaceutical formulation thereof may be assembled in the form of kits.
• the kit provides the lipid- containing composition, or pharmaceutical formulation thereof and reagents to prepare a composition for administration.
• the composition may be in a dry or lyophilized form, or in a solution, particularly a sterile solution.
• the reagent may comprise a pharmaceutically acceptable diluent for preparing a liquid formulation.
• diluents include those known to those of skill in the art, for example, sugar solutions, e.g., dextrose, sucrose, etc.
• the lipid-containing composition may be, for example, a targeted liposome, blank liposome, lipid mixture, or liposome-contaming composition (optionally containing drug or labeled compound).
• the kit may also contain a device for administration or for dispensing the compositions, including, but not limited to syringe, pipette, or other device known to those of skill. When in a wet form, the composition may be stored in an ampoule or other sterile sealed container, including those known to persons of skill in the art.
• kits may include other therapeutic compounds for use in conjunction with the compounds described herein.
• the therapeutic agents are other anticancer agents. These agents may be provided in a separate form, or mixed with the compounds of the present invention, provided such mixing does not reduce the effectiveness of either the additional therapeutic agent of the compositions and formulations described herein.
• the kits may include additional agents for adjunctive therapy. For example, agents to reduce the adverse effects of the drug (e.g., anti-nausea agents, anti-alopecia agents, immuno-enhancing agents, etc.).
• kits will include appropriate instructions for preparation and administration of the composition, side effects of the compositions, and any other relevant information.
• the instructions may be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc.
• kits for treating an individual who suffers from or is susceptible to the conditions described herein comprising a first container comprising a dosage amount of a lipid-containing composition or formulations thereof as disclosed herein, and instructions for use.
• the container may be any of those known in the art and appropriate for storage and delivery of intravenous formulations, hi certain embodiments the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the composition to be administered to the individual.
• Kits may also be provided that contain sufficient dosages of the compositions or formulations thereof as disclosed herein to provide effective treatment for an individual for an extended period, such as a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or more.
• Kits may also include multiple doses of the lipid-containing composition or formulations thereof and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
• oxaliplatin (/-OHP) solution was prepared by dissolving oxaliplatin in a 9% sucrose solution (sucrose/distilled water) at a concentration of 8 mg/ml.
• the cell viability was determined using a commercially available cytotoxicity assay kit (WST-I kit, Wako Pure Chemical Industries, Ltd., Japan).
• AsPC-I cells (provided by Dr. Hironobu Yanagie of Research Center for Advanced Science and Technology, the University of Tokyo, Japan) cultured in RPMI 1640 medium supplemented with 10% FCS (fetal calf serum; SIGMA, USA) were treated with concentrations of /-OHP solutions [200 x (l/2) ((M0) nM] at 37°C in 5% CO 2 for 48 hours. Then, the medium was removed and a substrate (WST-I, Cell Counting Kit, Dojindo Laboratories, Japan) was added to the cells which were incubated at 37°C in 5% CO 2 for 2 hours to develop the colored product. The developed color was measured at an absorbance of 450 ran (reference wavelength: 620 nm) on an Immuno Mini NJ-2300 (Cosmo Bio Co., Ltd., Japan).
• Tf (transferrin) receptors The number of Tf (transferrin) receptors on the cell surface of each was determined by Scatchard analysis (Comp. Biochem. Physiol., 116B, 137-160 (1949), using Microsoft Excel). A solution of 125 I-labeled Tf (Na- 125 I (PerkinElmer Japan Co., Ltd., Japan) and h-Tf (T-4132, SIGMA, USA) were combined by iodogen method (Biochem. Biophys. Res. Commun., 122, 319-325 (1984)) was added to each cell culture at different concentrations ranging from [300 x (l/2) (0 ⁇ 9) nM] at 4°C and incubated for 1 hour.
• the concentration of 125 I-labeled Tf was determined by protein quantification assay by the Lowry method (J. Biol. Chem., 193, 265-270 (1951)) and the radioactivity was measured using a gamma counter (Auto Well Gamma System ARC-300, Aloka Co., Ltd., Japan]. Briefly, the solution was centrifuged to precipitate the cells, and the cell fraction was washed with an ice-cooled PBS (180 x g (gravity), for 3 rnin, which was repeated 3 times, followed by the measurement of radioactivity with a gamma counter to determine the concentration of Tf bound to the cell surface. The number of cells was determined by protein quantification assay, using the Lowry method (J. Biol. Chem., 193, 265-270 (1951)).
• the concentration of unbound Tf was determined by subtracting the concentration of bound Tf from the known concentration of Tf added.
• the Scatchard plot was drawn by plotting the concentration of bound Tf on the horizontal axis and the ratio of the concentration of bound Tf to the concentration of unbound Tf on the vertical axis.
• the number of the bound Tf was determined from the x intercept of the graph, as described in Proc. Natl. Acad. ScL USA, 80 2263-2266 (1983); J. Cell Physiol, 132, 492-500 (1987); Proc. Natl. Acad. ScL USA, 92 3318-3322 (1995); J. Pharm.
• a solution of DCC (99%, Aldrich, USA, MW: 206.33g/mol) was prepared by dissolving 70 mg of DCC in 5 mL ethyl acetate. The DCC dissolved quickly in the solvent to yield a clear solution. The DCC solution so prepared (approximately 5mL) was then added dropwise to the lipid/NHS reaction mixture over 10 - 15 minute period. The reaction mixture became more cloudy upon addition of DCC.
• TLC was performed on the control (lipid/NHS) and on an aliquot of lipid/NHS/DCC at time 0 for reference, as follows.
• Sample was spotted 50 ⁇ g (2.5 ⁇ L of 20 mg/mL) on TLC plate (aluminum sheet - silica gel 60F 254 from EM Science (Gibbstown, NJ, USA) Cat No. SP05554M), dried and then placed in the developing chamber where solvent (70% chloroform, 28% methanol, 2% water) was allowed to migrate.
• the solvent front was marked and then the TLC plate was dipped in ammonium molybdate (5% ammonium molybdate in 10% H 2 SO 4 ) and dehydrated with dryer.
• Silica gel for purification of the suspended paste was prepared using silica (400 mesh) 4 g - hydrated in chloroform. The silica gel was packed onto a 1 cm x 28 cm column with stopcock. The approximate size of the bed was 1 cm x 14 cm. The column was equilibrated with chloroform (gravity packed).
• Fractions 6-15 were assayed (5 ⁇ L aliquot) by TLC as described above. [0463] Following TLC of fractions 6-15, fractions 7-11 were pooled and dried to a thin film using rotary evaporation. The final product obtained after evaporation was 130mg (65% yield), as determined by by TLC against the unpurified reaction product and comparison with a standard product (NHS-NG-DOPE) obtained from NOF (Japan).
• NG-DOPE 200 mg
• NHS N-hydroxysulfosuccinimide
• the flask was then placed under vacuum and filled with nitrogen gas flowing gently (repeat at three times). The flask was then kept under nitrogen using a nitrogen balloon.
• reaction mixture continued with stirring under nitrogen flow and the formation of product (Rf 0.3 - 0.4) was monitored over time.
• reaction was allowed to proceed over a time period of 2-3 days at ambient temperature with nitrogen flow and stirring. [0471] The reaction mixture was then filtered through a Bruchner funnel and washed twice with 2 x 5 mL chloroform. The entire solution was collected and dried by rotary-evaporation. A semi-solid paste was obtained.
• Fraction 1 Added 10O mL chloroform/methanol (90/10, vol/vol) to column. Collected 10O mL fractions. (Fraction 2)
• a powder of the lyophilized lipid mixture as obtained above was mixed with 20mg of powdery Tf-NG-DOPE (as prepared in Example 29) and crashed. A homogeneous powder of lipid mixture was thus obtained, with a lipid ratio of 50:45:5 (DMPC:Chol:NG-DOPE+Tf-NG-DOPE).
• Tf-NG-DSPE liposome (( ⁇ ); Example 5), Tf/PEG-NG-DSPE liposome (O ); Example 6) and Tf-PEG-DSPE liposome (( ⁇ ); Example 6) were used.
• Tf-PEG-liposomes prepared in Example 9 Tf-NG-DSPE:NG- DSPE:DSPC:CH liposomes prepared in Example 8, Tf/PEG-NG-DSPE liposomes prepared in Example 9; 9 mice in each group) and for each of the liposome compositions to which transferrin is not bound ((-)TF; 6 mice in each group).
• Tf-NG-DSPE liposome prepared from DSPC (64 parts), CH (32 parts) and NG-DSPE (4 parts), and Tf-NG-DSPE + DSPE liposome was prepared from DSPC (64 parts), CH (32 parts), NG-DSPE (4 parts) and DSPE (10 parts) in the same manner as in Example 8.
• Figs. 17 and 18 show that while the Tf-NG- DOPE:NG-DOPE liposomes show a lower accumulation in the blood (Fig. 17) than the Tf- PEG-DSPE liposomes, they were able to deliver a greater amount of oxaliplatin to the tumor (Fig. 18). Lower accumulation of liposomes in the blood is likely to reduce the adverse systemic effects of the oxaliplatin.
• NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes were administered intravenously (i.v.) every four days for four injections (q4d x 4) as doses of 15 and 10 mg/kg/injection.
• Oxaliplatin was administered on the same schedule at a dose of 15 mg/kg/injection.
• Vehicle about 10.3% sucrose
• blank liposome control groups were injected on the same schedule.
• the mean tumor volume for the HT-29 colon tumor model, following treatment with NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes was 66.3% of the control tumor volume for the 6.7 mg/kg group and 39.5% of the control tumor volume for the 10 mg/kg group (p value ⁇ 0.01).
• mice were administered NG-DOPE:Tf-NG- DOPE:DMPC:CH liposomes at doses of 6.7, 10 or 15 mg/kg, or vehicle control.
• the vehicle and 6.7 and 10 mg/kg treated groups were injected on days 7, 12 and 24 and the 15 mg/kg treated group was injected on days 7 and 24.
• the mean tumor volume for the MKN45 gastric tumor model following treatment with NG-DOPE:Tf-NG- DOPE:DMPC:CH liposomes was 65.4% of the control tumor volume for the 6.7 mg/kg group (p value ⁇ 0.05), 49.6% of the control tumor volume for the 10 mg/kg group (p value ⁇ 0.01), and 48.5% of the control tumor volume for the 15 mg/kg group (p value ⁇ 0.01; delivered every 17 days).
• Example 24 Liposome Antitumor Effects on Xenograft COLO 205 Colon Tumor
• the level of oxaliplatin was determined following membrane filtration using HPLC analysis to quantify the levels of unencapsulated (free) drug.
• the pH of targeted liposomes can be determined by place the liposomes of the invention in distilled water and measuring with a standard pH meter as described below.
• Liposome was dissolved and denatured at 95 °C for 5 min in sample buffer containing 2.5% of SDS and 5% of 2-mercaptoethanol. The samples were then applied to 5-10% gradient polyacrylamide gel and then they were electrophoresed in the presence of SDS. Migrated protein bands were visualized by using a brilliant blue G- colloidal (B2025, SIGMA 5 USA).
• the transferrin in the liposome was detected as transferrin conjugated to NG-DOPE, which showed a higher molecular weight than that of intact transferrin (see Fig. 24). A minor band with a lower molecular weight was detected as free transferrin.
• the osmotic pressure at a given temperature depends on sucrose and salts such as sodium chloride and phosphate buffer. It does not depend on the solute, but on the total ion density and the size of the molecules within the solution. Normally osmotic pressure can be measured using an instrument known as an osmometer, which measures osmotic pressure in suitable pressure units.
• osmotic pressure NG-DOPE:Tf-NG-DOPE:DMPC:CH liposomes prepared as in Example 7 at room temperature was measured using an osmometer (Vapro Vapor Pressure Osmometer Model5520,Wescor, Inc., USA).
• the osmolarity values for 3 preparations of liposomes ranged from 360 -370 m ⁇ sm/kg, as reported in Table 4.
• the filtrate was poured into a vial and frozen for about 8 hours on a shelf at -40 0 C.
• the sample was depressurized to about O.lmmHg and kept under reduced pressure for 2 days with rising temperature from -40 0 C to 25 0 C stepwise. Approximately 444mg of Tf-NG-DOPE (about 45% of transferrin content of the blank liposome was thus obtained.
• reaction product was then fractioned to about 1.7 mL/tube by TOYOPEARL HW-55S column (1.5 cm x 45 cm, 0.9% NaCl, Tosoh Bioscience LLC, US A).
• Tf-NG-DSPE was estimated by mass spectrometry (MALDI-TOF/MS) and SDS- PAGE with CBB (Coomasie Brilliant Blue, Wako Pure Chemical Industries, Ltd., Japan) staining.

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