CA2303147A1 - Small particle liposome aerosols for delivery of anti-cancer drugs - Google Patents
Small particle liposome aerosols for delivery of anti-cancer drugs Download PDFInfo
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- CA2303147A1 CA2303147A1 CA002303147A CA2303147A CA2303147A1 CA 2303147 A1 CA2303147 A1 CA 2303147A1 CA 002303147 A CA002303147 A CA 002303147A CA 2303147 A CA2303147 A CA 2303147A CA 2303147 A1 CA2303147 A1 CA 2303147A1
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- camptothecin
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
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- 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
- A61K31/02—Halogenated hydrocarbons
- A61K31/025—Halogenated hydrocarbons carbocyclic
- A61K31/03—Halogenated hydrocarbons carbocyclic aromatic
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- 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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
- A61K31/175—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine having the group, >N—C(O)—N=N— or, e.g. carbonohydrazides, carbazones, semicarbazides, semicarbazones; Thioanalogues thereof
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The small particle liposome aerosol compounds and methods of treatment of the present invention involve lipid- or water soluble anti-cancer drugs incorporated into liposomes. The liposomes are administered in aqueous dispersions from a jet nebulizer to the respiratory tract of an individual.
Various anti-cancer drugs may be used, including 20-S-Camptothecin, 9-Nitro-camptothecin, 9-Amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol or its derivatives. Administration of these drugs by inhalation provides faster and more efficient absorption of the anti-cancer drug than does intramuscular administration or oral administration.
Various anti-cancer drugs may be used, including 20-S-Camptothecin, 9-Nitro-camptothecin, 9-Amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol or its derivatives. Administration of these drugs by inhalation provides faster and more efficient absorption of the anti-cancer drug than does intramuscular administration or oral administration.
Description
SMALL PARTICLE LIPOSOME AEROSOLS FOR DELIVERY
OF ANTI-CANCER DRUGS
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates generally to the field of pharmacology and cancer treatment. Specifically, the present invention provides formulations and methods for small particle aerosol delivery by inhalation of aqueous dispersions of liposomes carrying anti-cancer drugs to the respiratory tract.
Description of the Related Art Small particle liposome aerosol treatment consists of lipid-soluble or water-soluble anti-cancer drugs incorporated into liposomes, which are administered from aqueous dispersions in a jet nebulizer (see U.S. Patent No: 5,049,388). Aerosols of 1- 3 ~.m mass median aerodynamic diameter, generated upon nebulization, enable targeted delivery onto surfaces of the respiratory tract. The deposited liposomes subsequently release drug locally within the lung or into the blood circulation with delivery to extra-pulmonary tissue. If the drug is lipid soluble, it will associate with the lipid molecules in a manner specific to the lipid employed, the anti-cancer drug employed and possibly it may be modified further by various soluble constituents which may be included in the suspending aqueous medium. Such soluble constituents may include buffering salts and possibly inositol to enhance the synthesis and secretion of surfactant phospholipid in lung tissue and to minimize respiratory distress already present or that which might result from the aerosol treatment (Hallman, M., et al. Inositol Supplementation in Premature Infants with Respiratory Distress Syndrome, N. Eng. J.
Med. 1992 326:1233-1239).
If the drug is water soluble, it may be incorporated b y appropriate procedures in aqueous vesicles that exist in concentric spaces between lipid bilayers (lamellae) of the multilamellar liposome. Unilamellar liposomes may b a prepared; however, their capacity to entrap either lipid-soluble or water-soluble drugs is diminished since entrapment is restricted to one central vesicle.
Aerosol water droplets may contain one or more drug-liposomes. Nebulization shears liposomes to sizes readily discharged from the nozzle of the nebulizer. Liposomes up to several microns in diameter are typically sheared to diameters of less than 500 nm, and may be considerably smaller than that depending on the operating characteristics of the nebulizer a n d other variables. Shearing of water-soluble drugs contained in Iiposomes will release appreciable amounts of the water soluble compound, perhaps 50 percent. This is not a contraindication to their use, but it means that two forms of the drug preparation is administered, and the effect includes the therapeutic effect that would be produced by both forms if either form had been given alone. Many other details of liposome aerosol treatment are described in U.S. Patent No. 5,049,388. Moreover, it is also possible to incorporate more than one drug in a aerosol liposome treatment, either by mixing different drug-containing liposomes, or by using liposomes wherein the drugs have b a a n combined and incorporated together into liposomes.
The prior art is deficient in formulations and methods for small particle aerosol delivery of aqueous dispersions of liposomes containing anti-cancer drugs. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The small particle aerosol compounds a liposome n d methods of treatment of the presentinvention involve lipid-soluble or water-soluble anti-cancerdrugs incorporated into liposomes. The liposomes then are administered in aqueous dispersions from a jet nebulizer. The present invention demonstrates that speedier and more efficient systemic absorption o f drug is actualized pulmonary administration after by aerosol than is actualized by intramuscular or oral administration.
One abject of the present invention is to provide a method for treating cancer, comprising the step of delivering, via small particle aerosol, aqueous dispersions of anti-cancer drugs to the respiratory tract of an individual in need of such treatment.
Examples of anticancer drugs available for use in this embodiment of the invention include, but are not limited to, 20-S-camptothecin, 9-vitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane, methotrexate, mercaptopurine, lomustine, interferon, 5 -fluorouracil and etopiside. In a more preferred embodiment of this object, the anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-vitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol.
Additionally, in a preferred embodiment of the present objective, the delivery of the anticancer drug is performed by a j et nebulizer.
In another object of the present invention, there is provided a liposome for delivery of anticancer drugs via small particle aerosols comprising an anticancer drug and a lipid, wherein the anticancer drug is at a concentration not exceeding about 10°Io of the total volume of the preparation and a ratio of th a anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the preparation. One specific embodiment of this object includes 9-vitro-camptothecin and dilauroylphosphatidylcholine in a ratio of about 1:10 to 1:50 wt:wt; with a particularly preferred embodiment having a 9-nitro-camptothecin and dilauroylphosphatidylcholine of about 1:50 wt:wt. In another embodiment, there is provided , a liposome for delivery of anticancer drugs via small particle aerosols comprising Taxol a n d dilauroylphosphatidylcholine in a ratio of about 1:30 wt:wt.
In yet another embodiment of the present invention, there is provided a liposome produced by the following steps:
dissolving a lipid-soluble anticancer drug in a solvent suitable for dissolving the anticancer drug to produce dissolved anticancer drug; adding the dissolved anticancer drug to a dissolved lipid suitable for formulation and delivery of drugs by aerosol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of the solution and a ratio of the anticancer drug to the lipid is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the solution; and freezing and lyophilizing the solution. At this point, the solution may be stored frozen for later use or dissolved in sterile water for use, producing a suspension, wherein the concentration of the anticancer drug in the sterile water in the suspension is no more than about 5.0 mg/ml.
A preferred embodiment of the above object provides liposomal preparations of 20-S-camptothecin (CPT), 9 -nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives, produced by the following steps: preparing concentrated stock solutions of said 20-S-camptothecin (CPT), 9 -nitrocamptothecin (9-NC) or other-lipid soluble camptothecin derivatives and lipids in compatible solvents; adding appropriate volumes of the 20-S-camptothecin (CPT), 9-nitrocamptothecin ( 9 -NC) or other-lipid soluble camptothecin derivative and lipid concentrated stock solutions to _ a volume of t-butanol to form a second solution, wherein a concentration of said 20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives does not exceed 10% of said second solution and wherein a ratio of drug to lipid is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 t o about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) in said second solution; freezing said second solution;
and lyophilizing said second solution to produce a p o w de r preparation. At this point, the powder preparation may be stored frozen for later use or dissolved in sterile water producing a suspension, wherein a concentration of said anticancer drug in said suspension is no more than about 5 mg/ml.
A more particular embodiment provides liposomes produced by the following steps: preparing a concentrated stock solutions of anticancer drug, for example 100 mg CPT in 1 ml t -butanol or 100 mg 9-NC in DMSO, preparing a stock solution of 2 0 lipid, for example, 100 mg DLPC in 1 ml butanol; a d d i n g appropriate volumes of said concentrated stock solutions to a volume of t-butanol to form a second solution wherein a final volume is about 10 ml, a volume of DMSO, if any, does not exceed 10% (vol:vol) of said final volume, a concentration of anticancer drug does not exceed 10% (wt:wt) of the total volume, and wherein a ratio of drug to lipid is in a range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, a n d most preferably in a range of about 1:10 to about 1:50 (wt:wt);
freezing said second solution; and lyophilizing said frozen solution to produce a powder preparation. Tthe powder preparation m a y then be stored frozen for later use or dissolved in sterile water producing a suspension. Generally, the concentration of the anticancer drug in the suspension is no more than about 5 mg/ml.
S Another preferred embodiment of the object above provides a liposome produced by the following steps: mixing taxol with synthetic alpha lecithin: dilauroylphosphatidylcholine;
dissolving the taxol-DLPC in t-butanol to produce a preparation;
and freezing and lyophilizing the preparation. Liposomes are produced by adding sterile, pure water at a temperature above 25 °C, wherein the final concentration of taxol t o dilauroylphosphatidylcholine is about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and m o s t preferably in a range of about 1:25 to about 1:40 (wt:wt). I n addition to alpha lecithin, other natural or synthetic lecithins m a y be used, including but not limited to egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dimyristophosphatidylcholine, diolyeolyl dipalmitoyleolylphospha-tidylcholine and dipalmitoyl phosphatidylcholine.
The efficiency of incorporation of 9-NC and other camptothecin derivatives and anticancer drugs into liposomes can be tested by layering an aqueous dispersion of lyophilized drug-liposome preparation over a PercollT"" gradient and centrifuging.
Unincorporated drug collects at the bottom of the tube, but drug incorporated into liposomes collects at the interface between the Percoll gradient and the water phase. One qualitative test of incorporation efficiency is the observation of drug crystals w h a n *rB
the dispersion of drug-liposomes are examined by microscopy under polarized light. Other methods are also available, for example, analytical HPLC methods can be used to quantitatively assess non-encapsulated, crystalized drug.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail.
These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.
Figure 1: shows the effect of treatment with 9-NC
DLPC liposome aerosol on xenografted human breast cancer i n nude mice.
Figure 2: shows the effect of further treatment with 9-NC-DLPC liposome aerosol in mice selected from Figure 1.
Figure 3: shows the effect of treatment with 9-NC
DLl'C liposome aerosol on xenografted human colon cancer (Squires) in nude mice.
Figure 4: shows the effect of treatment with 9-NC-DLPC by liposome aerosol or by oral administration on the growth of human lung cancer xenografts (Spark) in nude mice a s measured by tumor volume.
Figure 5: shows the output of Taxol-DLPC liposomes ( 1:30; 1 mg/ml) with the Aerotech II nebulizer.
Figure 6 : shows the recovery of taxol from t h a preparation described in Figure 5; taxol-DLPC dosage at 15 bpm, 500 ml TV.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are provided. Terms n o t specifically defined are meant to interpreted as is customary i n the art. As used herein, the term "aerosols" refers to dispersions in air of solid or liquid particles, of fine enough particle size a n d consequent low settling velocities to have relative airborne stability (See Knight, V., Viral and Mycoplasmal Infections of the Respiratory Tract. 1973, Lea and Febiger, Phila. Pa., pp. 2).
"Liposome aerosols" consist of aqueous droplets within which are dispersed one or more particles of liposomes or liposomes containing one or more medications intended for delivery to the respiratory tract of man or animals (Knight, V. and Waldrep, J. C
Liposome Aerosol for Delivery of Asthma Medications; see also I n Kay, B., Allergy and Allergic Diseases, 1997, Blackwell Publications, Oxford, England, Vol. I pp. 730-741). The size of the aerosol droplets defined for this application are those described in U.S.
patent 5,049,338, namely mass median aerodynamic diameter (MMAD) of 1-3 ~,m with a geometric standard deviation of about 1.8-2.2. However, with low concentrations of 9-NC and possibly other camptothecin derivatives, the MMAD may be less than 1 ~.m, such as 0.8 p,m. Based on the studies disclosed by the present invention, the liposomes may constitute substantially all of the volume of the droplet when it has equilibrated to ambient relative humidity.
As used herein, the "Weibel Lung Model" refers to a classification of the structure of the human lungs that recognizes 23 consecutive branchings of the airways of humans. The trachea is labeled 0, bronchi and bronchioles extend through branches 16.
These portions of the airways contain ciliated epithelium a n d mucus glands. Together they constitute the mucociliary blanket.
Branchings 17-23 compose the alveolar portion of the lung and do not have a mucociliary blanket. Thus, particles deposited here are not carried up the airway to be swallowed.
As used herein, the terms "20-S-camptothecin" o r "CPT" refers to a plant alkaloid with anti-cancer properties.
As used herein, the terms "9-nitro-camptothecin" o r "9-NC", "9-amino-camptothecin" or "9-AC," and "10, I 1-methylenedioxy- camptothecin" or "MDC" refer to active anti-cancer drugs derived from 20-S-camptothecin that are insoluble in water but are soluble in certain lipid solvents.
As used herein, the terms "dilauroylphosphatidylcholine" or "DLPC" is a lipid used to formulate liposomes.
The present invention is directed to a method for treating cancer, comprising the step of delivering, via small particle aerosol, aqueous dispersions of anti-cancer drugs to the respiratory tract of an individual in need of such treatment. In a preferred embodiment of this object, the anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-nitro camptothecin, 9-amino-camptothecin and 10, 11-methylenedioxy camptothecin. Add-itionally, in a preferred embodiment of the present objective, the delivery of the anticancer drug is performed by a jet nebulizer.
Additionally, there is a liposome for delivery of anticancer drugs via small particle aerosols comprising a n anticancer drug and a lipid, wherein the anticancer drug is at a concentration not exceeding about 10% of the total volume of the preparation and a ratio of the anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100; and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the preparation. One specific embodiment of this object includes 9-nitro-camptothecin and dilauroylphosphatidylcholine in a ratio of about 1:10 to 1:50 wt:wt; with a particularly preferred embodiment having a 9-nitro-camptothecin and dilauroyl-phosphatidylcholine of about 1:50 wt:wt.
Taxol is another anti-cancer drug that is lipid soluble and is incorporated easily into a liposome formulation. The optimal ratio for taxol and dilauroylphosphatidylcholine is a ratio of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and more preferably in a range of about 1:25 to about 1:40 (wt:wt). A preferred form provides a taxol to DLPC
ratio of about 1:30. Taxol is dissolved in t-butanol without use of DMSO as is used for some camptothecins. The Taxol liposomal preparation is otherwise similar to that of the camptothecins.
Further, the present invention is directed to liposomes for delivery of anticancer drugs via small particle aerosols produced by the following steps: dissolving a lipid-soluble anticancer drug in a solvent suitable for dissolving the anticancer drug to produce dissolved anticancer drug; adding the dissolved anticancer drug to a dissolved lipid suitable for formulation a n d delivery of drugs by aerosol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of the solution and a ratio of the anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200 of the solution; and freezing and lyophilizing th a solution. At this point, the solution may be stored frozen for later use or dissolved in sterile water to produce a suspension, wherein the concentration of the anticancer drug in the sterile water in t h a suspension is no more than about S.0 mg/ml. A particular embodiment of the present invention provides a Iiposome produced by the following steps: dissolving a lipid-soluble anticancer drug selected from the group of 20-S-camptothecin, 9 -nitro-camptothecin, 9-amino-camptothecin and 10, 1 1-methylenedioxy-camptothecin in 100% DMSO to produce dissolved anticancer drug; and adding said dissolved anticancer drug to dilauroylphosphatidylcholine dissolved in t-butanol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 5% of the total volume of the solution and the ratio of anticancer drug to dilauroylphosphatidylcholine is about I:50 in the solution. The solution is frozen and lyophilized overnight. For use, the lyophilized solution is suspended in appropriate volumes of sterile, distilled water. In addition, other methods of liposome preparation known in the art may be utilized, for example, rotary evaporation can be used instead of lyophilization.
9-NC-DLPC aerosol is prepared by first dissolving the drug in DMSO; to do so, heating to 50-60°C may be required. This solution is added to a larger volume of t-butanol, such that the DMSO solution does not exceed 5-10% of the total t-butanol a n d DMSO volume combined. The organic solvents DMSO and t-butanol are evaporated from the solution on liquid nitrogen resulting in a slightly yellow powder. For use, distilled sterile water is added to the vials containing the drug at the appropriate concentration a n d added to the reservoir of the nebulizer. The Aerotech IIT""
nebulizer CIS-USA, Inc., Bedford Ma. is employed, but nebulizers with similar aerosol-generating properties may be used.
A particular embodiment of the present invention is directed to a liposome produced by the following steps: mixing taxol with synthetic alpha lecithin: dilauroylphosphatidylcholine;
dissolving the taxol-DLPC in t-butanol to produce a solution; a n d freezing and lyophilizing the solution. Liposomes are produced b y adding sterile, pure water at a temperature above 25°C, wherein the final concentration of taxol to dilauroylphosphatidylcholine is about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and more preferably in a range of about 1:25 to about 1:40 (wt:wt).
It is contemplated specifically that the pharmaceutical compositions of the present invention be used for aerosol delivery of aqueous dispersions of liposomes carrying anti-cancer drugs to the respiratory tract. A person having ordinary skill in this art would readily be able to determine, without a n d a a experimentation, the appropriate dosages of these aerosol formulations. When used in vivo for therapy, the aerosol formulations of the present invention are administered to the patient in therapeutically effective amounts; i.e., amounts that eliminate or reduce the tumor burden. As with all pharmaceuticals, the dose and dosage regimen will depend upon the nature of the cancer (primary or metastatic), the characteristics of the particular drug (e.g., its therapeutic index}, the patient, th a patient's history and other factors. The amount of aerosol formulation administered will typically be in the range of about 8 ~,g/kg of patient weightfday to about 100 ~.g/kg of patient weight/day for 9-NC. Again, dose and dosage regimen will vary depending on factors known to those skilled in the art. See Remington's Pharmaceutical Sci., 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press.
The small particle liposome aerosol compounds a n d methods of treatment of the present invention involve lipid- o r water-soluble anti-cancer drugs incorporated into liposomes. The liposomes are administered in aqueous dispersions from a jet nebulizer. Various anti-cancer drugs may be used, including 2 0 -S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:
Anti-cancer drugs CPT and its 9-NC derivative Representative of the anti-cancer drugs of the present invention are 20-S-camptothecin and its 9-NC derivative. Other camptothecins such as 9-AC and 10, 11-MDC, as well as other anticancer formulations known in the art, also possess properties suitable for use. All these anti-cancer drugs rnay be used i n liposome formulations.
Table 1 shows a comparison of blood or plasma concentrations of 9-NC and the time of peak concentrations following oral administration to humans, dogs and mice. (Hinz, H.
R., et. al. Pharmacokinetics of the in vivo and in vitro conversion of 9-Nitro 20-(S)-camptothecin to 9-Amino-20-(S)-camptothecin in Humans, Dogs and Mice. Cancer Research 1994 54:3096-3100).
Single oral doses ranged from 0.1 to 1.0 mg/kg for humans a n d dogs and were 4.1 mg/kg for mice. There may be some differences in pharmacokinetics between CPT and 9-NC, but it is a reasonable possibility that the foregoing differences are predictive of the properties of both agents.
Table 1. Comparison of 9-NC concentrations in blood or plasma following single oral, intranasal or aerosol dosage *Intranasal and aerosol results from Baylor Aerosol Laboraton~. the others from Hinz, H.R. (see teat for reference) Table 2 describes the method of calculating aerosol dosage in the mouse, and this is the basis for determining dosages cited in Table 1. Much of the inhaled drug is deposited in the nasopharynx of the mouse because of the complex nose structure of rodents. Similarly, nasal instillation leads to high nasopharyngeal deposition. Material deposited here is promptly transported to the esophageal orifice and swallowed. About 10 -15 percent of the inhaled aerosol dose, however, will penetrate to the peripheral lungs.
In humans with mouth breathing, only small amounts of the aerosol particles will deposit in the mouth and virtually none will deposit in the nasopharynx. Material deposited in the central airways is returned to the pharynx by muco-ciliary action where it is swallowed. In the case of primary lung tumors which often occur at bronchial bifurcations, drug will deposit on tumor surfaces and be adsorbed directly into the tumor mass. Material deposited beyond the 16th Weibel generation, which is b a y o n d the ciliated epithelium, will not be moved upward; thus, in tumors within the peripheral lung parenchyma, the drug will deposit in adjacent areas and be absorbed directly into the tumor mass. I n addition, a major advantage of the present invention is that drug deposited beyond the ciliated epitheiium is picked up b y capillaries in the interstitial space and the lymphatics of the lungs and will enter the circulation.
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Table 3 shows the tissue distribution of CPT following 15 minutes of nebulization in DLPC liposome aerosol. The deposited dose was calculated to be 486 ng per mouse. The mean concentrations in lungs and liver were similar with smaller concentrations in the other sites examined. Table 4 shows tissue distributions over a period of one hour following intranasal instillation of 7 ~g per mouse (233 ~g/kg). Drug was cleared promptly from the lungs so that by 15 minutes after stopping nebulization only negligible amounts of drug were present in t h a Iungs. Liver, kidney and spleen had substantial amounts of drug initially which gradually diminished through the one hour of study. Interestingly, blood concentrations were the least throughout the study. These studies indicate substantial immediate deposition of drug in the lungs with rapid clearance to the viscera. The amount of drug contributed by absorption from swallowed drug is uncertain.
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Table 3. Tissue distribution of CPT following 15 minutes inhalation of CPT
liposome aerosol The CPT concentration in the liposomal preparation in the nebulizer was 0.2 mg/ml aerosol was generated with an Aerotech II nebulizer operating at a flow rate of 10 L/min.
Table 4. Time dependent organ distribution of CPT after intranasal administration Lung ~. 1287 t 657 19 f 3 36 ~ 23 7 t 3 Liver 651 t 418 255 f 101 66 t I7 34 f 7 Kidney 542 ~ 174 190 f 57 49 ~ 13 24 ~ 21 Spleen 35I t 137 84 t 32 21 t 8 7 ~ 2 Blood 213 ~ 19 53 t 20 8 ~ 3 4 t 2 Remarks:
CPT was administered in liposomal formulation prepared with DLPC with initial drug concentration 0.2 mg/ml.
35 1tL of suspension was installed to each animal (group of 3 animals was treated for each time point).
WO 99/15153 PCT/US98/19851 _ Table 5 shows the distribution of drug in blood and viscera following intramuscular injection of CPT. Drug disappeared very slowly from the site of intramuscular injection in the first I2 hours, with only very small concentrations detected in the liver and virtually no drug present at other sites.
Concentrations in the blood were negligible throughout the study.
The dose administered was the same as that given by intranasal instillation. These findings indicate a speedier and more efficient systemic absorption of drug after pulmonary administration of dru= than by the intramuscular route. It is likely that deposition in organs and vascular spaces will increase the opportunity for exposure to albumin molecules and degradation to the carboxy form of the drug.
Table 5. Time dependent organ distribution of CPT after intramuscufar administration Leg.. 2~.i..... 4~2 3t3 4-~3 . ~~. -.
Liver 3 f 1 87 ~ 74 136 t 107 126 .~
Spleen 2 ~ I 18 t 9 I I t 5 5 '-' 1 ~~ey 2 t 0 40 t 14 26 7 15 ~ 5 Blood 2 t 1 12 ~ 5 8 ~ 1 ~ = 1 Site of 6918 t 265 43091 1548 4609 -~ 1412 154- 1751 ini. -Remarks:
CPT initial stock 5 mg/ml in DMSO was suspended in saline (1.4 ~L stock + 48.6 ~L saline] and total 50 ~tL~of suspension was injected i.m. in each mice.
Group of 3 animals was treated for each time point.
Stability of Linosomes Consisting of DLPC and 9-AC
Table 6 shows the stability of liposomes with fixed weight ratio of 9-NC and DLPC of 1:50 (w/w) but with increasing concentrations of constituents from 0.1 mg/ml to 1.0 mg/ml of drug. The samples were tested under various conditions after vortexing, but before start of nebulization, after nebulization for 1.5 to 2 minutes (sample taken from the fluid in the reservoir of the nebulizer) and from the aerosol that was collected in an all-glass impinger (All-Glass Impinger, Ace Glass Co., Vineland N. J.).
Table 6. Liposome particle size and drug crystal formation in preparations of 9-NC DLPC liposome formulations 2 =~
The most stable preparation was the one with lowest concentration of constituents. A few crystals appeared in the reservoir following nebulization. Nebulization caused a ten-fold reduction in the diameter of the liposome particles, due to the shear forces associated with nebulization. There was further reduction in the diameter of liposome particles recovered from the aerosol. . This finding is consistent with selection of smaller particles for discharge in aerosol. The lack of crystals suggests that crystals may not nebulize as readily as liposomes. With larger dosages of Iiposomes, size reduction following nebulization occurred, but particles recovered from aerosol were not reduced in size compared to particles that had been cycled in the reservoir of the nebulizer.
Kinetics of Lactone Ring Opening The anti-tumor activity of several of the camptothecins are diminished following dissolution in aqueous media. This is due to a hydrolyzable alpha-hydroxy Iactone ring (ring E). The change results from acyl cleavage yielding the biologically inactive carboxylate form of the molecule. The lactone ring form of the drug is sheltered in Iiposomes (Burke, T. G., Biochemistry 19 9 3 32:5352-5364), but the carboxyl form of 'the drug has high affinity for human serum albumin. This leads to rapid conversion of lactone to carboxylate in the presence of human serum albumin, and thus to loss of anti-cancer activity. Deposition within the lungs on alveolar surfaces where there is little albumin and/or interaction with constituents of the liposomes is clearly a factor in preserving the anti-cancer effect of 9-NC.
Effect of 9-NC on Growth of Human Breast Cancer Ex~lants in Mice Figure 1 shows growth in the area of subcutaneous breast cancer xenografts during treatment with 9-NC-DLPC liposome aerosol. There were six 9-NC treated and 5 control mice.
Treatments were given 15 minutes daily, five days per week.
The dose was 8.1 wg/kg per day. The deposited dose in the respiratory tract of each mouse was estimated to be 234 ng p a r day. The data on tumor size was normalized and the divergence of tumor size (% initial tumor growth) in the two groups w a s highly significant by day 17 of treatment (P<0.011). After this time, control mice were sacrificed because of the presence of large necrotic tumor masses. Figure 2 shows the course of events with two treated mice which were subsequently followed with higher doses of drug, following a period of 16 days without treatment. A few days following restart of treatment with a five-fold increase in the dose of 9-NC liposome aerosol, the size of tumors in the treated animals diminished rapidly, and were no longer visible by the 85th day after start of treatment.
Effect of 9 NC on Human ColoRectal Cancer Xenografts in Mice A similar study was performed in nude mice with human colon carcinoma xenografts and is shown in Figure 3.
There were 15 treated and 20 control mice. Ten controls received empty DLPC liposomes and 10 received no treatment. Control animals who received no treatment or DLPC only showed a consistent and rapid increase in tumor size until they were sacrificed on day 36. The overall rate of tumor growth was 7 to 11 times greater in control than in 9-NC-treated mice. The treated animals were divided into two groups of 10 each. One group received 77 wg/kg/daily, five days per week throughout the entire experiment. The other received 77 ~.g/kg per day five days p a r week until day 35 when the dose was increased to 153 ~,g/kg p a r day five days per week until day 46 when it was increased to 3 0 7 ~,g/kg on the same schedule until day 61. There was slightly less increase in tumor size in the group receiving the higher dose, b a t the differences were not statistically significant, and the data are combined in the figure. Four mice in the DLPC treatment group were sacrificed because of large tumors or tumor necrosis before day 61, and six mice in the no treatment group were sacrificed for the same reasons before day 61. In the treatment group five mice were sacrificed because of tumor necrosis or emaciation before day 61. The emaciated mice were in the high dose group, suggesting drug toxicity. One additional treated mouse w a s sacrificed because of rectal prolapse. Based on these findings of reduced rate of tumor growth, day 28, (P<0.007, Student t test, 2 tailed) and reduced mortality there is an unequivocal therapeutic effect of 9-NC treatment (P<0.002~chi square test).
Effect of 9 NC on Human Lung Carcinoma Xenog~afts in Nude Mice Additionally, studies were performed on the effect of treatment with 9-NC-DLPC via liposome aerosol or via oral administration on the growth of human lung cancer xenografts (Spark) in nude mice as measured by tumor volume. Treatment was initiated about two weeks after tumor implantation. Control animals showed a rapid increase in volume of tumors. Animals who received oral dosage with the liposome drug aqueous suspension in doses of 100 ~.g/kg/day--more than twice the aerosol dosage--did not respond to treatment. See Figure 4.
Both aerosol and oral doses were doubled on day 13.
The increased dosage was followed by decrease in the size of tumors treated with aerosol, but there was no decrease in size of tumors in mice given oral treatment. Thus, despite the fact that half or more of aerosol dosage administered to mice is deposited in the nose, head, trachea and upper bronchi and is promptly carried by the mucociliary system to the esophagus where it is swallowed, the fraction of inhaled drug that is deposited in the lung is principally responsible for the effect on tumor growth.
The most likely explanation of the clear efficiency of aerosol delivery is the rapid entry of the drug to the circulation where it is returned to the left heart, and then to the aorta a n d peripheral circulation. Thus, the drug would reach the tumors o n "first pass" without having passed through the liver, which would remove large amounts of drug from blood.
Animal Models Nude Mice: Swiss immunodeficient nude mice of the NIH-1 high fertility strain, bred and housed at the Stehlin Institute were used for these experiments (Giovella, B.C., et al., Complete Growth Inhibition of Human Cancer Xenografts in Nude Mice by Treatment with 20-(S)-Camptothecin, Cancer Research 1991 51:3052-3055).
Human Cancer Xenografts: Human heterotransplants were established in nude mice. For an implant, approximately 50 m g of wet weight of finely minced tumor in 0.5 ml of Eagles minimum . essential medium was injected under the skin over the right dorsal chest region. The animals were started on treatment with the experimental drug about 10 days after implantation of tumors. Tumors of breast cancers were measured in tw o dimensions (i.e. area) with calipers, while colon cancers were measured in three dimensions (i.e. volume) with calipers.
Cam~tothecin Liposome Aerosol Formulation and Administration CPT and 9-nitrocamptothecin were provided by Dr.
Beppino Giovanella of the Stehlin Institute, Houston, Texas. DLPC
was obtained from Avanti Polar Lipids, Pelham, AL. Aerotech II
nebulizers were obtained from Cis-USA, Inc., Bedford, MA.
For formulation of liposomes, 9-NC ( I00 mg/ml) or CPT
(10 mg/ml) was dissolved in 100% DMSO, and added to DLPC
dissolved in tertiary butanol (40°C) so the final DMSO
concentration did not exceed 5 percent of the total volume and th a ratio of drug to lipid was 1:50 (w/w). The final suspension was clear. If precipitation occurred, it was reheated to 50-60°C. The preparation was frozen in liquid nitrogen and lyophilized overnight. For use the material was dissolved in sterile water to the appropriate drug concentration, not exceeding I.0 mg/ml for either drug. The efficiency of incorporation of drug in the liposomes was examined qualitatively by microscopic examination under polarized light. Unincorporated drug was seen as b i -refringent crystals. The efficiency of incorporation was examined by centrifugation of aqueous suspensions of liposomes on PercollT«
gradients. One-tenth ml of suspension was layered over 2 ml of gradient and centrifuged at 2000 rpm for 25-30 minutes.
Liposomes layer at the water-Percoll interface, while unincorporated drug was deposited at the bottom of the tube.
Many other lipids may be substituted for DLPC in the formulation and use of liposomes for delivery of drugs by aerosol (Sugarman, S. M., et. al. Lipid-complexed campothecin: formulation and initial biodistribution and anti-tumor activity studies. Cancer Chemothera~v Pharmacol. 1996 37:531-538).
HPLC Analysis The Waters (Milford, MA) 710B Wisp automatic injector and Waters Nova-Pak C18 column at room temperature was used to quantitate CPT and 9-NC. The mobile phase was 30%
acetonitrile and 70% of 0.1% glacial acetic acid. CPT was detected using the Waters 470 scanning fluorescence detector set to an excitation wavelength of 370 nm and an emission wavelength of 440 nm. 9-NC was detected using the Waters 440 absorbence detector and monitoring at 254 nm. The data were analyzed w i th the Waters Millenium software.
Aerosol Droplet Measurement The size of aerosol droplets was measured with t h a Andersen ACFM non-viable ambient particle sizing sampler pCT/US98/19851 (Andersen Instruments, Inc., Atlanta, Ga.) by methods previously described (Waldrep, J. C. et. al., J Aerosol Med. 1994 7:135-145).
Mass median aerodynamic diameters and geometric standard deviations were determined using KaleidaGraph 2.0 (Synergy Software, Reading Pa.). The aerosol droplets consisted of a n aqueous suspension of liposomes. Liposome diameters were measured in aqueous suspension with the Model 3300 NICOMP
Laser Particle Sizer.
Preparation and Efficien~cv of Taxol-DLPC Linosomes Formulated by Aerosol Delivery Taxol is another anti-cancer drug that is lipid soluble and is incorporated easily into liposomal formulation. Taxol i s dissolved directly in t-butanol without use of DMSO similar to the camptothecins. The taxol liposomal preparation is otherwise like that of the camptothecins.
The optimal taxol to DLPC ratio was found to be ab o a t 1:30 wt:wt. Formulations compatible with nebulization and aerosol delivery formulations were produced at 1 mg Taxol and with 30 mg DLPC per ml. For optimized Taxol-DLPC liposomes, 5 mg of Taxol was mixed with 150 mg of synthetic alpha-lecithin:
dilauroylphosphatidylcholine (DLPC). Working at 37°C, the drug/DLPC was mixed in 20 mls of tertiary butanol with stirring.
After mixing, the drug/lipid preparation was pipetted into glass vials, frozen rapidly, and lyophilized overnight to remove the t -butanol leaving a powder. Mufti-lamellar liposomes w a r a produced by adding 5 mls of ultra pure water above the DLPC
phase transition temperature (Tc) at 25°C to deliver a final standard drug concentration of 1 mg Taxol: 30 mg DLPC per m1.
The mixture was incubated for 30 minutes at room temperature with intermittent mixing to produce multilamellar vesicular (MLV) liposomes. Aliquots were removed for determination of drug concentration by HPLC.
Figure 5 shows the particle size distribution of taxol-DLPC liposome aerosol with a MMAD of 1.4 wm and a geometric standard deviation of 2.0 Figure 6 shows the recovery of taxol from the preparation described in Figure 5. In the lung model (Harvard Respirator) used to measure the output of taxol from the nebulizer (Aerotech II), 87.75 liters of aerosol were sampled yielding 3000 ~.g of taxol. There was thus an aerosol concentration of 34.2 ~,g/l. From this information; the dose of aerosol deposited in the respiratory tract following inhalation can be calculated.
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the a r t to which the invention pertains. Further, these patents a n d publications are incorporated by reference herein to the same extent as if each individual publication was specifically a n d individually indicated to be incorporated by reference.
One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and a r a not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined b y the scope of the claims.
OF ANTI-CANCER DRUGS
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates generally to the field of pharmacology and cancer treatment. Specifically, the present invention provides formulations and methods for small particle aerosol delivery by inhalation of aqueous dispersions of liposomes carrying anti-cancer drugs to the respiratory tract.
Description of the Related Art Small particle liposome aerosol treatment consists of lipid-soluble or water-soluble anti-cancer drugs incorporated into liposomes, which are administered from aqueous dispersions in a jet nebulizer (see U.S. Patent No: 5,049,388). Aerosols of 1- 3 ~.m mass median aerodynamic diameter, generated upon nebulization, enable targeted delivery onto surfaces of the respiratory tract. The deposited liposomes subsequently release drug locally within the lung or into the blood circulation with delivery to extra-pulmonary tissue. If the drug is lipid soluble, it will associate with the lipid molecules in a manner specific to the lipid employed, the anti-cancer drug employed and possibly it may be modified further by various soluble constituents which may be included in the suspending aqueous medium. Such soluble constituents may include buffering salts and possibly inositol to enhance the synthesis and secretion of surfactant phospholipid in lung tissue and to minimize respiratory distress already present or that which might result from the aerosol treatment (Hallman, M., et al. Inositol Supplementation in Premature Infants with Respiratory Distress Syndrome, N. Eng. J.
Med. 1992 326:1233-1239).
If the drug is water soluble, it may be incorporated b y appropriate procedures in aqueous vesicles that exist in concentric spaces between lipid bilayers (lamellae) of the multilamellar liposome. Unilamellar liposomes may b a prepared; however, their capacity to entrap either lipid-soluble or water-soluble drugs is diminished since entrapment is restricted to one central vesicle.
Aerosol water droplets may contain one or more drug-liposomes. Nebulization shears liposomes to sizes readily discharged from the nozzle of the nebulizer. Liposomes up to several microns in diameter are typically sheared to diameters of less than 500 nm, and may be considerably smaller than that depending on the operating characteristics of the nebulizer a n d other variables. Shearing of water-soluble drugs contained in Iiposomes will release appreciable amounts of the water soluble compound, perhaps 50 percent. This is not a contraindication to their use, but it means that two forms of the drug preparation is administered, and the effect includes the therapeutic effect that would be produced by both forms if either form had been given alone. Many other details of liposome aerosol treatment are described in U.S. Patent No. 5,049,388. Moreover, it is also possible to incorporate more than one drug in a aerosol liposome treatment, either by mixing different drug-containing liposomes, or by using liposomes wherein the drugs have b a a n combined and incorporated together into liposomes.
The prior art is deficient in formulations and methods for small particle aerosol delivery of aqueous dispersions of liposomes containing anti-cancer drugs. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The small particle aerosol compounds a liposome n d methods of treatment of the presentinvention involve lipid-soluble or water-soluble anti-cancerdrugs incorporated into liposomes. The liposomes then are administered in aqueous dispersions from a jet nebulizer. The present invention demonstrates that speedier and more efficient systemic absorption o f drug is actualized pulmonary administration after by aerosol than is actualized by intramuscular or oral administration.
One abject of the present invention is to provide a method for treating cancer, comprising the step of delivering, via small particle aerosol, aqueous dispersions of anti-cancer drugs to the respiratory tract of an individual in need of such treatment.
Examples of anticancer drugs available for use in this embodiment of the invention include, but are not limited to, 20-S-camptothecin, 9-vitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane, methotrexate, mercaptopurine, lomustine, interferon, 5 -fluorouracil and etopiside. In a more preferred embodiment of this object, the anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-vitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol.
Additionally, in a preferred embodiment of the present objective, the delivery of the anticancer drug is performed by a j et nebulizer.
In another object of the present invention, there is provided a liposome for delivery of anticancer drugs via small particle aerosols comprising an anticancer drug and a lipid, wherein the anticancer drug is at a concentration not exceeding about 10°Io of the total volume of the preparation and a ratio of th a anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the preparation. One specific embodiment of this object includes 9-vitro-camptothecin and dilauroylphosphatidylcholine in a ratio of about 1:10 to 1:50 wt:wt; with a particularly preferred embodiment having a 9-nitro-camptothecin and dilauroylphosphatidylcholine of about 1:50 wt:wt. In another embodiment, there is provided , a liposome for delivery of anticancer drugs via small particle aerosols comprising Taxol a n d dilauroylphosphatidylcholine in a ratio of about 1:30 wt:wt.
In yet another embodiment of the present invention, there is provided a liposome produced by the following steps:
dissolving a lipid-soluble anticancer drug in a solvent suitable for dissolving the anticancer drug to produce dissolved anticancer drug; adding the dissolved anticancer drug to a dissolved lipid suitable for formulation and delivery of drugs by aerosol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of the solution and a ratio of the anticancer drug to the lipid is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the solution; and freezing and lyophilizing the solution. At this point, the solution may be stored frozen for later use or dissolved in sterile water for use, producing a suspension, wherein the concentration of the anticancer drug in the sterile water in the suspension is no more than about 5.0 mg/ml.
A preferred embodiment of the above object provides liposomal preparations of 20-S-camptothecin (CPT), 9 -nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives, produced by the following steps: preparing concentrated stock solutions of said 20-S-camptothecin (CPT), 9 -nitrocamptothecin (9-NC) or other-lipid soluble camptothecin derivatives and lipids in compatible solvents; adding appropriate volumes of the 20-S-camptothecin (CPT), 9-nitrocamptothecin ( 9 -NC) or other-lipid soluble camptothecin derivative and lipid concentrated stock solutions to _ a volume of t-butanol to form a second solution, wherein a concentration of said 20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives does not exceed 10% of said second solution and wherein a ratio of drug to lipid is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 t o about 1:100, and most preferably in a range of about 1:10 to about 1:50 (wt:wt) in said second solution; freezing said second solution;
and lyophilizing said second solution to produce a p o w de r preparation. At this point, the powder preparation may be stored frozen for later use or dissolved in sterile water producing a suspension, wherein a concentration of said anticancer drug in said suspension is no more than about 5 mg/ml.
A more particular embodiment provides liposomes produced by the following steps: preparing a concentrated stock solutions of anticancer drug, for example 100 mg CPT in 1 ml t -butanol or 100 mg 9-NC in DMSO, preparing a stock solution of 2 0 lipid, for example, 100 mg DLPC in 1 ml butanol; a d d i n g appropriate volumes of said concentrated stock solutions to a volume of t-butanol to form a second solution wherein a final volume is about 10 ml, a volume of DMSO, if any, does not exceed 10% (vol:vol) of said final volume, a concentration of anticancer drug does not exceed 10% (wt:wt) of the total volume, and wherein a ratio of drug to lipid is in a range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, a n d most preferably in a range of about 1:10 to about 1:50 (wt:wt);
freezing said second solution; and lyophilizing said frozen solution to produce a powder preparation. Tthe powder preparation m a y then be stored frozen for later use or dissolved in sterile water producing a suspension. Generally, the concentration of the anticancer drug in the suspension is no more than about 5 mg/ml.
S Another preferred embodiment of the object above provides a liposome produced by the following steps: mixing taxol with synthetic alpha lecithin: dilauroylphosphatidylcholine;
dissolving the taxol-DLPC in t-butanol to produce a preparation;
and freezing and lyophilizing the preparation. Liposomes are produced by adding sterile, pure water at a temperature above 25 °C, wherein the final concentration of taxol t o dilauroylphosphatidylcholine is about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and m o s t preferably in a range of about 1:25 to about 1:40 (wt:wt). I n addition to alpha lecithin, other natural or synthetic lecithins m a y be used, including but not limited to egg yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dimyristophosphatidylcholine, diolyeolyl dipalmitoyleolylphospha-tidylcholine and dipalmitoyl phosphatidylcholine.
The efficiency of incorporation of 9-NC and other camptothecin derivatives and anticancer drugs into liposomes can be tested by layering an aqueous dispersion of lyophilized drug-liposome preparation over a PercollT"" gradient and centrifuging.
Unincorporated drug collects at the bottom of the tube, but drug incorporated into liposomes collects at the interface between the Percoll gradient and the water phase. One qualitative test of incorporation efficiency is the observation of drug crystals w h a n *rB
the dispersion of drug-liposomes are examined by microscopy under polarized light. Other methods are also available, for example, analytical HPLC methods can be used to quantitatively assess non-encapsulated, crystalized drug.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
The appended drawings have been included herein so that the above-recited features, advantages and objects of the invention will become clear and can be understood in detail.
These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and should not be considered to limit the scope of the invention.
Figure 1: shows the effect of treatment with 9-NC
DLPC liposome aerosol on xenografted human breast cancer i n nude mice.
Figure 2: shows the effect of further treatment with 9-NC-DLPC liposome aerosol in mice selected from Figure 1.
Figure 3: shows the effect of treatment with 9-NC
DLl'C liposome aerosol on xenografted human colon cancer (Squires) in nude mice.
Figure 4: shows the effect of treatment with 9-NC-DLPC by liposome aerosol or by oral administration on the growth of human lung cancer xenografts (Spark) in nude mice a s measured by tumor volume.
Figure 5: shows the output of Taxol-DLPC liposomes ( 1:30; 1 mg/ml) with the Aerotech II nebulizer.
Figure 6 : shows the recovery of taxol from t h a preparation described in Figure 5; taxol-DLPC dosage at 15 bpm, 500 ml TV.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are provided. Terms n o t specifically defined are meant to interpreted as is customary i n the art. As used herein, the term "aerosols" refers to dispersions in air of solid or liquid particles, of fine enough particle size a n d consequent low settling velocities to have relative airborne stability (See Knight, V., Viral and Mycoplasmal Infections of the Respiratory Tract. 1973, Lea and Febiger, Phila. Pa., pp. 2).
"Liposome aerosols" consist of aqueous droplets within which are dispersed one or more particles of liposomes or liposomes containing one or more medications intended for delivery to the respiratory tract of man or animals (Knight, V. and Waldrep, J. C
Liposome Aerosol for Delivery of Asthma Medications; see also I n Kay, B., Allergy and Allergic Diseases, 1997, Blackwell Publications, Oxford, England, Vol. I pp. 730-741). The size of the aerosol droplets defined for this application are those described in U.S.
patent 5,049,338, namely mass median aerodynamic diameter (MMAD) of 1-3 ~,m with a geometric standard deviation of about 1.8-2.2. However, with low concentrations of 9-NC and possibly other camptothecin derivatives, the MMAD may be less than 1 ~.m, such as 0.8 p,m. Based on the studies disclosed by the present invention, the liposomes may constitute substantially all of the volume of the droplet when it has equilibrated to ambient relative humidity.
As used herein, the "Weibel Lung Model" refers to a classification of the structure of the human lungs that recognizes 23 consecutive branchings of the airways of humans. The trachea is labeled 0, bronchi and bronchioles extend through branches 16.
These portions of the airways contain ciliated epithelium a n d mucus glands. Together they constitute the mucociliary blanket.
Branchings 17-23 compose the alveolar portion of the lung and do not have a mucociliary blanket. Thus, particles deposited here are not carried up the airway to be swallowed.
As used herein, the terms "20-S-camptothecin" o r "CPT" refers to a plant alkaloid with anti-cancer properties.
As used herein, the terms "9-nitro-camptothecin" o r "9-NC", "9-amino-camptothecin" or "9-AC," and "10, I 1-methylenedioxy- camptothecin" or "MDC" refer to active anti-cancer drugs derived from 20-S-camptothecin that are insoluble in water but are soluble in certain lipid solvents.
As used herein, the terms "dilauroylphosphatidylcholine" or "DLPC" is a lipid used to formulate liposomes.
The present invention is directed to a method for treating cancer, comprising the step of delivering, via small particle aerosol, aqueous dispersions of anti-cancer drugs to the respiratory tract of an individual in need of such treatment. In a preferred embodiment of this object, the anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-nitro camptothecin, 9-amino-camptothecin and 10, 11-methylenedioxy camptothecin. Add-itionally, in a preferred embodiment of the present objective, the delivery of the anticancer drug is performed by a jet nebulizer.
Additionally, there is a liposome for delivery of anticancer drugs via small particle aerosols comprising a n anticancer drug and a lipid, wherein the anticancer drug is at a concentration not exceeding about 10% of the total volume of the preparation and a ratio of the anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100; and most preferably in a range of about 1:10 to about 1:50 (wt:wt) of the preparation. One specific embodiment of this object includes 9-nitro-camptothecin and dilauroylphosphatidylcholine in a ratio of about 1:10 to 1:50 wt:wt; with a particularly preferred embodiment having a 9-nitro-camptothecin and dilauroyl-phosphatidylcholine of about 1:50 wt:wt.
Taxol is another anti-cancer drug that is lipid soluble and is incorporated easily into a liposome formulation. The optimal ratio for taxol and dilauroylphosphatidylcholine is a ratio of about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and more preferably in a range of about 1:25 to about 1:40 (wt:wt). A preferred form provides a taxol to DLPC
ratio of about 1:30. Taxol is dissolved in t-butanol without use of DMSO as is used for some camptothecins. The Taxol liposomal preparation is otherwise similar to that of the camptothecins.
Further, the present invention is directed to liposomes for delivery of anticancer drugs via small particle aerosols produced by the following steps: dissolving a lipid-soluble anticancer drug in a solvent suitable for dissolving the anticancer drug to produce dissolved anticancer drug; adding the dissolved anticancer drug to a dissolved lipid suitable for formulation a n d delivery of drugs by aerosol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of the solution and a ratio of the anticancer drug to the suitable solvent is in the range of about 1:1 to about 1:200 of the solution; and freezing and lyophilizing th a solution. At this point, the solution may be stored frozen for later use or dissolved in sterile water to produce a suspension, wherein the concentration of the anticancer drug in the sterile water in t h a suspension is no more than about S.0 mg/ml. A particular embodiment of the present invention provides a Iiposome produced by the following steps: dissolving a lipid-soluble anticancer drug selected from the group of 20-S-camptothecin, 9 -nitro-camptothecin, 9-amino-camptothecin and 10, 1 1-methylenedioxy-camptothecin in 100% DMSO to produce dissolved anticancer drug; and adding said dissolved anticancer drug to dilauroylphosphatidylcholine dissolved in t-butanol to produce a solution, wherein the dissolved anticancer drug is at a concentration not exceeding about 5% of the total volume of the solution and the ratio of anticancer drug to dilauroylphosphatidylcholine is about I:50 in the solution. The solution is frozen and lyophilized overnight. For use, the lyophilized solution is suspended in appropriate volumes of sterile, distilled water. In addition, other methods of liposome preparation known in the art may be utilized, for example, rotary evaporation can be used instead of lyophilization.
9-NC-DLPC aerosol is prepared by first dissolving the drug in DMSO; to do so, heating to 50-60°C may be required. This solution is added to a larger volume of t-butanol, such that the DMSO solution does not exceed 5-10% of the total t-butanol a n d DMSO volume combined. The organic solvents DMSO and t-butanol are evaporated from the solution on liquid nitrogen resulting in a slightly yellow powder. For use, distilled sterile water is added to the vials containing the drug at the appropriate concentration a n d added to the reservoir of the nebulizer. The Aerotech IIT""
nebulizer CIS-USA, Inc., Bedford Ma. is employed, but nebulizers with similar aerosol-generating properties may be used.
A particular embodiment of the present invention is directed to a liposome produced by the following steps: mixing taxol with synthetic alpha lecithin: dilauroylphosphatidylcholine;
dissolving the taxol-DLPC in t-butanol to produce a solution; a n d freezing and lyophilizing the solution. Liposomes are produced b y adding sterile, pure water at a temperature above 25°C, wherein the final concentration of taxol to dilauroylphosphatidylcholine is about 1:1 to about 1:200, preferably in a range of about 1:10 to about 1:100, and more preferably in a range of about 1:25 to about 1:40 (wt:wt).
It is contemplated specifically that the pharmaceutical compositions of the present invention be used for aerosol delivery of aqueous dispersions of liposomes carrying anti-cancer drugs to the respiratory tract. A person having ordinary skill in this art would readily be able to determine, without a n d a a experimentation, the appropriate dosages of these aerosol formulations. When used in vivo for therapy, the aerosol formulations of the present invention are administered to the patient in therapeutically effective amounts; i.e., amounts that eliminate or reduce the tumor burden. As with all pharmaceuticals, the dose and dosage regimen will depend upon the nature of the cancer (primary or metastatic), the characteristics of the particular drug (e.g., its therapeutic index}, the patient, th a patient's history and other factors. The amount of aerosol formulation administered will typically be in the range of about 8 ~,g/kg of patient weightfday to about 100 ~.g/kg of patient weight/day for 9-NC. Again, dose and dosage regimen will vary depending on factors known to those skilled in the art. See Remington's Pharmaceutical Sci., 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press.
The small particle liposome aerosol compounds a n d methods of treatment of the present invention involve lipid- o r water-soluble anti-cancer drugs incorporated into liposomes. The liposomes are administered in aqueous dispersions from a jet nebulizer. Various anti-cancer drugs may be used, including 2 0 -S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion:
Anti-cancer drugs CPT and its 9-NC derivative Representative of the anti-cancer drugs of the present invention are 20-S-camptothecin and its 9-NC derivative. Other camptothecins such as 9-AC and 10, 11-MDC, as well as other anticancer formulations known in the art, also possess properties suitable for use. All these anti-cancer drugs rnay be used i n liposome formulations.
Table 1 shows a comparison of blood or plasma concentrations of 9-NC and the time of peak concentrations following oral administration to humans, dogs and mice. (Hinz, H.
R., et. al. Pharmacokinetics of the in vivo and in vitro conversion of 9-Nitro 20-(S)-camptothecin to 9-Amino-20-(S)-camptothecin in Humans, Dogs and Mice. Cancer Research 1994 54:3096-3100).
Single oral doses ranged from 0.1 to 1.0 mg/kg for humans a n d dogs and were 4.1 mg/kg for mice. There may be some differences in pharmacokinetics between CPT and 9-NC, but it is a reasonable possibility that the foregoing differences are predictive of the properties of both agents.
Table 1. Comparison of 9-NC concentrations in blood or plasma following single oral, intranasal or aerosol dosage *Intranasal and aerosol results from Baylor Aerosol Laboraton~. the others from Hinz, H.R. (see teat for reference) Table 2 describes the method of calculating aerosol dosage in the mouse, and this is the basis for determining dosages cited in Table 1. Much of the inhaled drug is deposited in the nasopharynx of the mouse because of the complex nose structure of rodents. Similarly, nasal instillation leads to high nasopharyngeal deposition. Material deposited here is promptly transported to the esophageal orifice and swallowed. About 10 -15 percent of the inhaled aerosol dose, however, will penetrate to the peripheral lungs.
In humans with mouth breathing, only small amounts of the aerosol particles will deposit in the mouth and virtually none will deposit in the nasopharynx. Material deposited in the central airways is returned to the pharynx by muco-ciliary action where it is swallowed. In the case of primary lung tumors which often occur at bronchial bifurcations, drug will deposit on tumor surfaces and be adsorbed directly into the tumor mass. Material deposited beyond the 16th Weibel generation, which is b a y o n d the ciliated epithelium, will not be moved upward; thus, in tumors within the peripheral lung parenchyma, the drug will deposit in adjacent areas and be absorbed directly into the tumor mass. I n addition, a major advantage of the present invention is that drug deposited beyond the ciliated epitheiium is picked up b y capillaries in the interstitial space and the lymphatics of the lungs and will enter the circulation.
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Table 3 shows the tissue distribution of CPT following 15 minutes of nebulization in DLPC liposome aerosol. The deposited dose was calculated to be 486 ng per mouse. The mean concentrations in lungs and liver were similar with smaller concentrations in the other sites examined. Table 4 shows tissue distributions over a period of one hour following intranasal instillation of 7 ~g per mouse (233 ~g/kg). Drug was cleared promptly from the lungs so that by 15 minutes after stopping nebulization only negligible amounts of drug were present in t h a Iungs. Liver, kidney and spleen had substantial amounts of drug initially which gradually diminished through the one hour of study. Interestingly, blood concentrations were the least throughout the study. These studies indicate substantial immediate deposition of drug in the lungs with rapid clearance to the viscera. The amount of drug contributed by absorption from swallowed drug is uncertain.
*rB
Table 3. Tissue distribution of CPT following 15 minutes inhalation of CPT
liposome aerosol The CPT concentration in the liposomal preparation in the nebulizer was 0.2 mg/ml aerosol was generated with an Aerotech II nebulizer operating at a flow rate of 10 L/min.
Table 4. Time dependent organ distribution of CPT after intranasal administration Lung ~. 1287 t 657 19 f 3 36 ~ 23 7 t 3 Liver 651 t 418 255 f 101 66 t I7 34 f 7 Kidney 542 ~ 174 190 f 57 49 ~ 13 24 ~ 21 Spleen 35I t 137 84 t 32 21 t 8 7 ~ 2 Blood 213 ~ 19 53 t 20 8 ~ 3 4 t 2 Remarks:
CPT was administered in liposomal formulation prepared with DLPC with initial drug concentration 0.2 mg/ml.
35 1tL of suspension was installed to each animal (group of 3 animals was treated for each time point).
WO 99/15153 PCT/US98/19851 _ Table 5 shows the distribution of drug in blood and viscera following intramuscular injection of CPT. Drug disappeared very slowly from the site of intramuscular injection in the first I2 hours, with only very small concentrations detected in the liver and virtually no drug present at other sites.
Concentrations in the blood were negligible throughout the study.
The dose administered was the same as that given by intranasal instillation. These findings indicate a speedier and more efficient systemic absorption of drug after pulmonary administration of dru= than by the intramuscular route. It is likely that deposition in organs and vascular spaces will increase the opportunity for exposure to albumin molecules and degradation to the carboxy form of the drug.
Table 5. Time dependent organ distribution of CPT after intramuscufar administration Leg.. 2~.i..... 4~2 3t3 4-~3 . ~~. -.
Liver 3 f 1 87 ~ 74 136 t 107 126 .~
Spleen 2 ~ I 18 t 9 I I t 5 5 '-' 1 ~~ey 2 t 0 40 t 14 26 7 15 ~ 5 Blood 2 t 1 12 ~ 5 8 ~ 1 ~ = 1 Site of 6918 t 265 43091 1548 4609 -~ 1412 154- 1751 ini. -Remarks:
CPT initial stock 5 mg/ml in DMSO was suspended in saline (1.4 ~L stock + 48.6 ~L saline] and total 50 ~tL~of suspension was injected i.m. in each mice.
Group of 3 animals was treated for each time point.
Stability of Linosomes Consisting of DLPC and 9-AC
Table 6 shows the stability of liposomes with fixed weight ratio of 9-NC and DLPC of 1:50 (w/w) but with increasing concentrations of constituents from 0.1 mg/ml to 1.0 mg/ml of drug. The samples were tested under various conditions after vortexing, but before start of nebulization, after nebulization for 1.5 to 2 minutes (sample taken from the fluid in the reservoir of the nebulizer) and from the aerosol that was collected in an all-glass impinger (All-Glass Impinger, Ace Glass Co., Vineland N. J.).
Table 6. Liposome particle size and drug crystal formation in preparations of 9-NC DLPC liposome formulations 2 =~
The most stable preparation was the one with lowest concentration of constituents. A few crystals appeared in the reservoir following nebulization. Nebulization caused a ten-fold reduction in the diameter of the liposome particles, due to the shear forces associated with nebulization. There was further reduction in the diameter of liposome particles recovered from the aerosol. . This finding is consistent with selection of smaller particles for discharge in aerosol. The lack of crystals suggests that crystals may not nebulize as readily as liposomes. With larger dosages of Iiposomes, size reduction following nebulization occurred, but particles recovered from aerosol were not reduced in size compared to particles that had been cycled in the reservoir of the nebulizer.
Kinetics of Lactone Ring Opening The anti-tumor activity of several of the camptothecins are diminished following dissolution in aqueous media. This is due to a hydrolyzable alpha-hydroxy Iactone ring (ring E). The change results from acyl cleavage yielding the biologically inactive carboxylate form of the molecule. The lactone ring form of the drug is sheltered in Iiposomes (Burke, T. G., Biochemistry 19 9 3 32:5352-5364), but the carboxyl form of 'the drug has high affinity for human serum albumin. This leads to rapid conversion of lactone to carboxylate in the presence of human serum albumin, and thus to loss of anti-cancer activity. Deposition within the lungs on alveolar surfaces where there is little albumin and/or interaction with constituents of the liposomes is clearly a factor in preserving the anti-cancer effect of 9-NC.
Effect of 9-NC on Growth of Human Breast Cancer Ex~lants in Mice Figure 1 shows growth in the area of subcutaneous breast cancer xenografts during treatment with 9-NC-DLPC liposome aerosol. There were six 9-NC treated and 5 control mice.
Treatments were given 15 minutes daily, five days per week.
The dose was 8.1 wg/kg per day. The deposited dose in the respiratory tract of each mouse was estimated to be 234 ng p a r day. The data on tumor size was normalized and the divergence of tumor size (% initial tumor growth) in the two groups w a s highly significant by day 17 of treatment (P<0.011). After this time, control mice were sacrificed because of the presence of large necrotic tumor masses. Figure 2 shows the course of events with two treated mice which were subsequently followed with higher doses of drug, following a period of 16 days without treatment. A few days following restart of treatment with a five-fold increase in the dose of 9-NC liposome aerosol, the size of tumors in the treated animals diminished rapidly, and were no longer visible by the 85th day after start of treatment.
Effect of 9 NC on Human ColoRectal Cancer Xenografts in Mice A similar study was performed in nude mice with human colon carcinoma xenografts and is shown in Figure 3.
There were 15 treated and 20 control mice. Ten controls received empty DLPC liposomes and 10 received no treatment. Control animals who received no treatment or DLPC only showed a consistent and rapid increase in tumor size until they were sacrificed on day 36. The overall rate of tumor growth was 7 to 11 times greater in control than in 9-NC-treated mice. The treated animals were divided into two groups of 10 each. One group received 77 wg/kg/daily, five days per week throughout the entire experiment. The other received 77 ~.g/kg per day five days p a r week until day 35 when the dose was increased to 153 ~,g/kg p a r day five days per week until day 46 when it was increased to 3 0 7 ~,g/kg on the same schedule until day 61. There was slightly less increase in tumor size in the group receiving the higher dose, b a t the differences were not statistically significant, and the data are combined in the figure. Four mice in the DLPC treatment group were sacrificed because of large tumors or tumor necrosis before day 61, and six mice in the no treatment group were sacrificed for the same reasons before day 61. In the treatment group five mice were sacrificed because of tumor necrosis or emaciation before day 61. The emaciated mice were in the high dose group, suggesting drug toxicity. One additional treated mouse w a s sacrificed because of rectal prolapse. Based on these findings of reduced rate of tumor growth, day 28, (P<0.007, Student t test, 2 tailed) and reduced mortality there is an unequivocal therapeutic effect of 9-NC treatment (P<0.002~chi square test).
Effect of 9 NC on Human Lung Carcinoma Xenog~afts in Nude Mice Additionally, studies were performed on the effect of treatment with 9-NC-DLPC via liposome aerosol or via oral administration on the growth of human lung cancer xenografts (Spark) in nude mice as measured by tumor volume. Treatment was initiated about two weeks after tumor implantation. Control animals showed a rapid increase in volume of tumors. Animals who received oral dosage with the liposome drug aqueous suspension in doses of 100 ~.g/kg/day--more than twice the aerosol dosage--did not respond to treatment. See Figure 4.
Both aerosol and oral doses were doubled on day 13.
The increased dosage was followed by decrease in the size of tumors treated with aerosol, but there was no decrease in size of tumors in mice given oral treatment. Thus, despite the fact that half or more of aerosol dosage administered to mice is deposited in the nose, head, trachea and upper bronchi and is promptly carried by the mucociliary system to the esophagus where it is swallowed, the fraction of inhaled drug that is deposited in the lung is principally responsible for the effect on tumor growth.
The most likely explanation of the clear efficiency of aerosol delivery is the rapid entry of the drug to the circulation where it is returned to the left heart, and then to the aorta a n d peripheral circulation. Thus, the drug would reach the tumors o n "first pass" without having passed through the liver, which would remove large amounts of drug from blood.
Animal Models Nude Mice: Swiss immunodeficient nude mice of the NIH-1 high fertility strain, bred and housed at the Stehlin Institute were used for these experiments (Giovella, B.C., et al., Complete Growth Inhibition of Human Cancer Xenografts in Nude Mice by Treatment with 20-(S)-Camptothecin, Cancer Research 1991 51:3052-3055).
Human Cancer Xenografts: Human heterotransplants were established in nude mice. For an implant, approximately 50 m g of wet weight of finely minced tumor in 0.5 ml of Eagles minimum . essential medium was injected under the skin over the right dorsal chest region. The animals were started on treatment with the experimental drug about 10 days after implantation of tumors. Tumors of breast cancers were measured in tw o dimensions (i.e. area) with calipers, while colon cancers were measured in three dimensions (i.e. volume) with calipers.
Cam~tothecin Liposome Aerosol Formulation and Administration CPT and 9-nitrocamptothecin were provided by Dr.
Beppino Giovanella of the Stehlin Institute, Houston, Texas. DLPC
was obtained from Avanti Polar Lipids, Pelham, AL. Aerotech II
nebulizers were obtained from Cis-USA, Inc., Bedford, MA.
For formulation of liposomes, 9-NC ( I00 mg/ml) or CPT
(10 mg/ml) was dissolved in 100% DMSO, and added to DLPC
dissolved in tertiary butanol (40°C) so the final DMSO
concentration did not exceed 5 percent of the total volume and th a ratio of drug to lipid was 1:50 (w/w). The final suspension was clear. If precipitation occurred, it was reheated to 50-60°C. The preparation was frozen in liquid nitrogen and lyophilized overnight. For use the material was dissolved in sterile water to the appropriate drug concentration, not exceeding I.0 mg/ml for either drug. The efficiency of incorporation of drug in the liposomes was examined qualitatively by microscopic examination under polarized light. Unincorporated drug was seen as b i -refringent crystals. The efficiency of incorporation was examined by centrifugation of aqueous suspensions of liposomes on PercollT«
gradients. One-tenth ml of suspension was layered over 2 ml of gradient and centrifuged at 2000 rpm for 25-30 minutes.
Liposomes layer at the water-Percoll interface, while unincorporated drug was deposited at the bottom of the tube.
Many other lipids may be substituted for DLPC in the formulation and use of liposomes for delivery of drugs by aerosol (Sugarman, S. M., et. al. Lipid-complexed campothecin: formulation and initial biodistribution and anti-tumor activity studies. Cancer Chemothera~v Pharmacol. 1996 37:531-538).
HPLC Analysis The Waters (Milford, MA) 710B Wisp automatic injector and Waters Nova-Pak C18 column at room temperature was used to quantitate CPT and 9-NC. The mobile phase was 30%
acetonitrile and 70% of 0.1% glacial acetic acid. CPT was detected using the Waters 470 scanning fluorescence detector set to an excitation wavelength of 370 nm and an emission wavelength of 440 nm. 9-NC was detected using the Waters 440 absorbence detector and monitoring at 254 nm. The data were analyzed w i th the Waters Millenium software.
Aerosol Droplet Measurement The size of aerosol droplets was measured with t h a Andersen ACFM non-viable ambient particle sizing sampler pCT/US98/19851 (Andersen Instruments, Inc., Atlanta, Ga.) by methods previously described (Waldrep, J. C. et. al., J Aerosol Med. 1994 7:135-145).
Mass median aerodynamic diameters and geometric standard deviations were determined using KaleidaGraph 2.0 (Synergy Software, Reading Pa.). The aerosol droplets consisted of a n aqueous suspension of liposomes. Liposome diameters were measured in aqueous suspension with the Model 3300 NICOMP
Laser Particle Sizer.
Preparation and Efficien~cv of Taxol-DLPC Linosomes Formulated by Aerosol Delivery Taxol is another anti-cancer drug that is lipid soluble and is incorporated easily into liposomal formulation. Taxol i s dissolved directly in t-butanol without use of DMSO similar to the camptothecins. The taxol liposomal preparation is otherwise like that of the camptothecins.
The optimal taxol to DLPC ratio was found to be ab o a t 1:30 wt:wt. Formulations compatible with nebulization and aerosol delivery formulations were produced at 1 mg Taxol and with 30 mg DLPC per ml. For optimized Taxol-DLPC liposomes, 5 mg of Taxol was mixed with 150 mg of synthetic alpha-lecithin:
dilauroylphosphatidylcholine (DLPC). Working at 37°C, the drug/DLPC was mixed in 20 mls of tertiary butanol with stirring.
After mixing, the drug/lipid preparation was pipetted into glass vials, frozen rapidly, and lyophilized overnight to remove the t -butanol leaving a powder. Mufti-lamellar liposomes w a r a produced by adding 5 mls of ultra pure water above the DLPC
phase transition temperature (Tc) at 25°C to deliver a final standard drug concentration of 1 mg Taxol: 30 mg DLPC per m1.
The mixture was incubated for 30 minutes at room temperature with intermittent mixing to produce multilamellar vesicular (MLV) liposomes. Aliquots were removed for determination of drug concentration by HPLC.
Figure 5 shows the particle size distribution of taxol-DLPC liposome aerosol with a MMAD of 1.4 wm and a geometric standard deviation of 2.0 Figure 6 shows the recovery of taxol from the preparation described in Figure 5. In the lung model (Harvard Respirator) used to measure the output of taxol from the nebulizer (Aerotech II), 87.75 liters of aerosol were sampled yielding 3000 ~.g of taxol. There was thus an aerosol concentration of 34.2 ~,g/l. From this information; the dose of aerosol deposited in the respiratory tract following inhalation can be calculated.
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the a r t to which the invention pertains. Further, these patents a n d publications are incorporated by reference herein to the same extent as if each individual publication was specifically a n d individually indicated to be incorporated by reference.
One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and a r a not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined b y the scope of the claims.
Claims (21)
1. A method for treating cancer, comprising the step of delivering, via small particle aerosol, aqueous dispersions of anti-cancer drugs in liposomes to the respiratory tract of an individual in need of such treatment.
2. The method of claim 1, wherein said anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane, methotrexate, mercaptopurine, lomustine, interferon, 5-fluorouracil and etopiside.
3. The method of claim 2, wherein said anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin and taxol.
4. The method of claim 1, wherein said delivering step is performed by a jet nebulizer.
5. The method of claim 4, wherein said liposomes are sheared to a diameter of less than 500 nm by said jet nebulizer.
6. The method of claim 1, wherein a final concentration of said anticancer drug in said liposomes is no greater than 5.0 mg/ml.
7. The method of claim 6, wherein a final concentration of said anticancer drug in said liposomes is no greater than 1.0 mg/ml.
8. A liposome for delivery of anticancer drugs via small particle aerosol, wherein a ratio of anticancer drug to lipid is about 1:1 to about 1:200 wt:wt.
9. The liposome of claim 8, wherein a ratio of anticancer drug to lipid is about 1:10 to about 1:100 wt:wt.
10. The liposome of claim 9, wherein a ratio of anticancer drug to lipid is about 1:10 to about 1: 50 wt:wt.
11. The liposome of claim 8, wherein said anticancer drug is camptothecin or a derivative of camptothecin, said lipid is dilauroylphosphatidylcholine, and said ratio is about 1:10 to about 1:50.
12. The liposome of claim 8, wherein said anticancer drug is taxol or its derivatives, said lipid is dilauroylphosphatidylcholine and said ratio is about 1:25 to about 1:40.
13. A liposome produced by the following steps:
dissolving a lipid-soluble anticancer drug in a n appropriate solvent to produce dissolved anticancer drug;
dissolving a lipid suitable for the formulation and delivery of anticancer drugs by aerosol in an appropriate solvent to produce dissolved lipid;
combining said dissolved anticancer drug and said dissolved lipid to produce a solution, wherein said dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of said solution and a ratio of said anticancer drug to said suitable lipid solution and a ratio of said anticancer said solution; and evaporating said solvents from said solution to produce a powder.
dissolving a lipid-soluble anticancer drug in a n appropriate solvent to produce dissolved anticancer drug;
dissolving a lipid suitable for the formulation and delivery of anticancer drugs by aerosol in an appropriate solvent to produce dissolved lipid;
combining said dissolved anticancer drug and said dissolved lipid to produce a solution, wherein said dissolved anticancer drug is at a concentration not exceeding about 10% of the total volume of said solution and a ratio of said anticancer drug to said suitable lipid solution and a ratio of said anticancer said solution; and evaporating said solvents from said solution to produce a powder.
14. The liposome of claim 13, further comprising the step of dissolving said powder in sterile water to produce a suspension, wherein a concentration of said anticancer drug i n said sterile water is no more than about 5.0 mg/ml.
15. The liposome of claim 13 wherein said suitable lipid for formulating and delivering drugs by aerosol i s dilauroylphosphatidylcholine.
16. The liposome of claim 13, wherein said anti-cancer drug is selected from the group consisting of 20-S-camptothecin, 9-nitro-camptothecin, 9-amino-camptothecin, 10, 11-methylenedioxy-camptothecin, taxol, taxol-A, mitotane, methotrexate, mercaptopurine, lomustine, interferon, 5-fluorouracil and etopiside.
17. The liposome of claim 13, wherein said anti-cancer drug is selected from the group consisting of 20-S-Camptothecin, 9-Nitro-camptothecin, 9-Amino-camptothecin and 10, 11-methylenedioxy-camptothecin, and taxol.
18. The liposome of claim 13, wherein a ratio of anticancer drug to lipid is about 1:10 to about 1:100 wt:wt.
19. The liposome of claim 18, wherein a ratio of anticancer drug to lipid is about 1:10 to about 1: 50 wt:wt.
20. A liposome produced by the following steps:
dissolving a lipid-soluble anticancer drug selected from the group of 20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives in a volume of DMSO to produce dissolved anticancer drug;
mixing said dissolved anticancer drug with an appropriate solvent;
dissolving a lipid suitable for the formulation and delivery of drugs by aerosol in an appropriate solvent to produce a dissolved lipid;
combining said dissolved anticancer drug and said dissolved lipid to produce a solution, wherein said DMSO is at a concentration not exceeding about 10% of the total volume of said solution and a ratio of said anticancer drug to said suitable lipid is in a range of 1:1 to 1:200 of said solution; and evaporating said solvents from said solution to produce a powder.
dissolving a lipid-soluble anticancer drug selected from the group of 20-S-camptothecin (CPT), 9-nitrocamptothecin (9-NC) and other lipid soluble camptothecin derivatives in a volume of DMSO to produce dissolved anticancer drug;
mixing said dissolved anticancer drug with an appropriate solvent;
dissolving a lipid suitable for the formulation and delivery of drugs by aerosol in an appropriate solvent to produce a dissolved lipid;
combining said dissolved anticancer drug and said dissolved lipid to produce a solution, wherein said DMSO is at a concentration not exceeding about 10% of the total volume of said solution and a ratio of said anticancer drug to said suitable lipid is in a range of 1:1 to 1:200 of said solution; and evaporating said solvents from said solution to produce a powder.
21. The liposome of claim 20, wherein said lipid is dilauroylphosphatidylcholine.
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US08/933,254 US6090407A (en) | 1997-09-23 | 1997-09-23 | Small particle liposome aerosols for delivery of anti-cancer drugs |
PCT/US1998/019851 WO1999015153A1 (en) | 1997-09-23 | 1998-09-23 | Small particle liposome aerosols for delivery of anti-cancer drugs |
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CA2303147A1 true CA2303147A1 (en) | 1999-04-01 |
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CA002303147A Abandoned CA2303147A1 (en) | 1997-09-23 | 1998-09-23 | Small particle liposome aerosols for delivery of anti-cancer drugs |
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TW (1) | TWI224509B (en) |
WO (1) | WO1999015153A1 (en) |
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AU9503198A (en) | 1999-04-12 |
NZ503129A (en) | 2001-08-31 |
TWI224509B (en) | 2004-12-01 |
US6090407A (en) | 2000-07-18 |
WO1999015153A1 (en) | 1999-04-01 |
KR100588248B1 (en) | 2006-06-13 |
KR20010024232A (en) | 2001-03-26 |
EP1011638A1 (en) | 2000-06-28 |
RU2223749C2 (en) | 2004-02-20 |
IL134910A0 (en) | 2001-05-20 |
US7341739B2 (en) | 2008-03-11 |
ZA200105505B (en) | 2003-08-27 |
CN1093399C (en) | 2002-10-30 |
JP2001517614A (en) | 2001-10-09 |
US20040208935A1 (en) | 2004-10-21 |
EP1011638A4 (en) | 2006-06-28 |
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