WO1994026251A1 - Procede d'apport sous-cutane de liposomes - Google Patents

Procede d'apport sous-cutane de liposomes Download PDF

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Publication number
WO1994026251A1
WO1994026251A1 PCT/US1994/005072 US9405072W WO9426251A1 WO 1994026251 A1 WO1994026251 A1 WO 1994026251A1 US 9405072 W US9405072 W US 9405072W WO 9426251 A1 WO9426251 A1 WO 9426251A1
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Prior art keywords
liposomes
compound
liposome
entrapped
administering
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PCT/US1994/005072
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English (en)
Inventor
Theresa M. Allen
Martin C. Woodle
Luke S. S. Guo
Jessica L. Krakow
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Sequus Pharmaceuticals, Inc.
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Priority to AU67850/94A priority Critical patent/AU6785094A/en
Publication of WO1994026251A1 publication Critical patent/WO1994026251A1/fr

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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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers

Definitions

  • the present invention relates to a method for administering therapeutic compounds to a subject.
  • the method includes entrapping a therapeutic compound in a liposome composition and administering the compound subcutaneously.
  • Liposome delivery systems have been proposed for a variety of drugs. These systems provide circulating controlled drug release systems that release drug slowly in the body over an extended period of time (Poznansky) .
  • One limitation of present liposome drug delivery is that liposomes accumulate over time in certain tissues, such as the liver, spleen, and, to a lesser extent, in the lungs and bone marrow (Saba) . Liposome-entrapped drugs concentrated in these tissues may cause cytotoxic effects resulting from drug accumulation.
  • Numerous drugs currently used for treatment of disease are hepatotoxic above a threshold concentration.
  • carmustine is a drug that can be employed in the treatment of Hodgkin's and other lymphomas. In free form this drug shows cytotoxic effects in the liver (Salmon) .
  • Certain antitumor drugs which are nonselective and inhibit growth of highly replicating cells may adversely inhibit the growth of cells in the bone marrow which also contains highly replicating cells.
  • certain drugs may be converted into inactive metabolites by enzymatic processes that occur predominantly in the liver.
  • doxorubicin and daunorubicin frequently used in antitumor therapy, are metabolized to inactive metabolites in the liver (Salmon) .
  • Preventing the accumulation of liposome- entrapped drug in the liver will minimize the conversion of drug to an inactive metabolite and will increase the active drug's lifetime in the bloodstream.
  • the invention provides, in one aspect, a method of delivering a liposome-entrapped compound into the bloodstream, for extended release into the bloodstream over a several-day period.
  • the method includes subcutaneously administering to a subject liposomes with an average diameter of less than about 120 nm, typically between 50 and 120 nn, having a surface coating of polyethylene glycol, at a surface concentration thereof, sufficient to extend the blood circulation time of the liposomes severalfold over that of liposomes in the absence of such coating, and containing a liposome- entrapped compound.
  • Subcutaneous administration of the liposomes results in (i) a total amount of liposomes released in the bloodstream which is a substantial fraction of the total liposome amount subcutaneously administered, and is typically about 50% of the total liposomes administered subcutaneously, and (ii) a peak liposome level in the bloodstream between about 6 to 24 hours after liposome administration which is up to 30% of the total amount of liposome-entrapped compound.
  • the level of the liposomes in the bloodstream is at least about 1% of the total amount of administered liposome-entrapped compound, over a period of at least about 96 hours.
  • the method is used for administering a compound to treat a disease associated with the lymphatic system.
  • the subcutaneously administered liposome ⁇ are prepared to contain a targeting molecule on its outer surface to localize the liposomes at specific sites in the lymphatic system, such as lymph nodes.
  • the present invention in another aspect, provides a method of administering first and second compounds to a subject.
  • the method includes administering subcutaneously to a subject, a composition containing (i) the first compound in a non-entrapped form, and (ii) the second compound entrapped in liposomes having sizes predominantly less than about 120 nm, and a surface coating of polyethylene glycol.
  • Administration of the composition results in bloodstream levels of the first and second compounds which are maximal in the first six hours, and between six and twenty-four hours after said administering, respectively.
  • the method is of use in anti- coagulant or anti-thrombolytic therapy, wherein the first compound is a polysulfonated anti-coagulant compound, such as heparin, and the second compound is a polyamine compound, such as protamine sulfate, effective to neutralize the activity of the anti-coagulant compound.
  • the method is of use in imaging a target site in a subject, wherein the first compound is an imaging agent, such as a' chelated form technetium-99, and the second compound is one effective to reduce the concentration of the imaging agent in the bloodstream.
  • the second compound in this embodiment, is effective to form a multivalent complex with the first compound, to accelerate clearance of the first compound from the bloodstream.
  • the method is of use in targeting an agent to a selected site in the subject, wherein the first compound is a targeting agent effective to localize at a site, and said second compound is one effective to reduce the concentration of the targeting agent in the bloodstream.
  • the method is of use in the administration of an anesthetic, wherein the first compound is an anesthetic, such as an opioid, and the second compound, an opioid antagonist, is effective to block the action of the anesthetic in the subject.
  • an anesthetic such as an opioid
  • the second compound an opioid antagonist
  • the method is of use in the administration of a therapeutically active compound, wherein the first compound is an inactive compound which is converted, by interaction with the second compound, to a therapeutically active compound.
  • the subcutaneously administered liposomes with diameters greater than about 120 nm, typically between 120 and 300 nm serve as a depot for the release of a therapeutic compound effective to treat a disease.
  • heat or ultrasound is administered in a pulsatile manner close to the site of subcutaneous administration to cause pulsatile release of a therapeutic compound from liposomes into lymphatic system and bloodstream.
  • Fig. 1 shows liposome peak blood levels as a function of average liposome diameter
  • Fig. 2 shows liposome peak blood levels achieved after subcutaneous administration of different liposome compositions: A-V described below;
  • Fig. 3 shows the percent of injected cpm remaining in vivo at 16 days post injection for different liposome compositions: A-V described below;
  • Figs. 4A-4B shows blood levels for different small sized liposome compositions as a function of time post- injection: HSPC:CH:PEG-DSPE (solid inverted triangles); PC40:CH:PEG-DSPE (solid circle), HSPC:CH:PG (solid squares), and PC40:CH:PG (solid triangles) at a dose of 10 micromoles/mouse (Fig.
  • HSPC:CH:PEG-DSPE solid circles
  • HSPC:PEG-DSPE solid triangles
  • Fig. 4B shows blood levels for different liposome compositions composed of PC40:CH:PEG-DSPE with an average diameter of 79 nm at 10 micromoles/mouse (solid circles) , with an average diameter of 82 nm at 0.5 micromoles/mouse (solid inverted triangles) , with an average diameter of 460 nm at 10 micromoles/mouse (solid squares) , and with an average diameter of 656 nm at 0.5 micromoles/mouse (solid triangles) ;
  • Figs. 5A and 5B show liposome biodistribution at 48 hours post-injection of PC40:CH:PEG-DSPE liposomes by a subcutaneous route (Fig. 5A) or an intravenous route (Fig. 5B) ;
  • Fig. 6 shows PC40:CH:PEG-DSPE liposome (average diameter 76 nm) lymph node levels as a function of time post-injection: open bars, 24 hours post-injection; shaded bars, 48 hours post-injection; and solid bars, 72 hours post-injection;
  • Figs. 7A-7D shows distribution of liposomes as a function of time post injection for intravenous (solid circle) , intraperitoneal (solid triangle) , and subcutaneous (solid square) routes of injection in the blood (Fig. 7A) ; in the liver (Fig. 7B) ; in the spleen (Fig. 7C) ; and in the carcass (Fig. 7D) ;
  • Fig. 8 illustrates inhibition of diuresis in Brattleboro rats after vasopressin administration by the method of the present invention, as a percentage of the predosage diuresis rate, at 0 micrograms (open circles) , 25 micrograms (closed triangles) , 100 micrograms (closed circles) and 400 micrograms (closed squares) ;
  • Fig. 9 illustrates inhibition of diuresis in Brattleboro rats after subcutaneous administration of vasopressin at 0.18 mg/kg, as a percentage of the predosage rate, in an aqueous solution (open circles) , in a liposome composition composed of PS:HEPC:CH (open triangles) , and in a liposome composition composed of PEG:HEPC:CH (closed squares) ; Fig.
  • FIG. 10 shows plasma concentration of vasopressin determined by radioimmunoassay in Brattleboro rats after subcutaneous administration of vasopressin at 0.18 mg/kg in an aqueous solution (open triangles) , in a liposome composition composed of PS:HEPC:CH (open circles), and in a liposome composition composed of PEG-DSPE:HEPC:CH (open squares) ; and
  • Fig. 11 shows plasma pharmacokinetics of vasopressin administered at 0.18 mg/kg in an aqueous solution (open triangles) , in a liposome composition composed of P S :HEP C : C H (open circles) , and in a liposome composition composed of PEG:HEPC:CH (open squares).
  • the present invention provides a method of delivering a liposome-entrapped compound to the bloodstream in a subject over a several-day period.
  • the method includes the subcutaneous administration of the compound in liposome-entrapped form.
  • the method achieves peak liposome blood levels of up to 30% of the a d ministered dose between 6 and 24 hours post-injection.
  • the total amount of liposomes released in the bloodstream is a substantial fraction of the total liposome amount administered subcutaneously, and is typically about 50% of the total liposome amount administered subcutaneously.
  • the method of the present invention reduces accumulation of a liposome-entrapped compound in tissues, such as the liver, spleen, lungs and bone marrow, and in the bloodstream, while it increases liposome-entrapped compound accumulation in the lymph nodes.
  • the present invention also provides a method for administering sequentially first and second therapeutic compounds to a subject.
  • the method involves administering subcutaneously to a subject, a composition containing (i) a first compound in free form, and (ii) a second compound entrapped in liposomes, to obtain bloodstream levels of the first and second compounds which are maximal in the first six hours after drug administration for the first compound, and between six and twenty-four hours after drug administration for the second compound.
  • the liposomes used in the present invention are typically prepared to include a majority of saturated or unsaturated phospholipids, a liposome-entrapped compound, and polyethylene glycol
  • PEG-derivatized phospholipids which form a coating of polyethylene glycol on the liposome surface.
  • the inclusion of PEG-derivatized phospholipid in a liposome increases the long term stability of the liposome and extends liposome bloodstream lifetime. Including cholesterol in the liposome further contributes to liposome stabilization. Liposomes are then sized to the desired selected size.
  • the liposome of the present invention is usually composed of at least two different lipid components.
  • the first one which will form the bulk of the liposome vesicle structure includes any amphipathic lipids having hydrophobic and polar head group moieties, and which (a) can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids, or (b) are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the mem ⁇ brane.
  • the vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC) , PE, phosphatidic acid (PA) , phosphatidylglycerol (PG) , phosphatidylinositol (PI) , and sphingomyelin (SM) , where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • PC phosphatidylcholine
  • PE phosphatidic acid
  • PG phosphatidylglycerol
  • PI phosphatidylinositol
  • SM sphingomyelin
  • unsaturated phospholipids such as partially hydrogenated soy phosphatidylcholine, iodine number 40 (PC40)
  • saturated phospholipids such as hydrogenated egg phosphatidylcholine (HEPC) or hydrogenated soy phosphatidylcholine (HSPC)
  • HEPC hydrogenated egg phosphatidylcholine
  • HSPC hydrogenated soy phosphatidylcholine
  • a second lipid component includes a vesicle-forming lipid which is derivatized with a hydrophilic polymer chain.
  • the vesicle-forming lipids which can be used are any of those described above for the first vesicle-forming lipid component.
  • Vesicle-forming lipids with diacyl chains, such as phospholipids, are preferred.
  • One exemplary phospholipid is phosphatidylethanolamine
  • PE with a reactive amino group which is convenient for coupling to the activated polymers.
  • An exemplary PE is distearoyl PE (DSPE) .
  • a preferred hydrophilic polymer in the derivatized lipid is polyethylene glycol (PEG) , preferably as a PEG chain having a molecular weight between 1,000-10,000 daltons, more preferably between 2,000 and 5,000 daltons.
  • PEG polyethylene glycol
  • Incorporation of vesicle-forming lipids derivatized with polyethylene glycol in a liposome results in the polymer forming a hydrophilic coating around the surface of the liposome which prevents the approach of plasma and lymph components that may destabilize the liposome and cause liposome-entrapped compound to be released rapidly.
  • hydrophilic polymers which may be suitable for preparing the derivatized lipid include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.
  • a suitable lipid such as PE
  • block copolymers or random copolymers of these polymers may be suitable.
  • Methods for preparing lipids derivatized with hydrophilic polymers, such as PEG, are well known, e.g., as described in co-owned U.S. Patent No. 5,013,556.
  • the lipids may include a lipid that can stabilize a vesicle or liposome composed predominantly of phospholipids.
  • the most frequently employed lipid is cholesterol at between 25 to 40 mole percent.
  • cholesterol at between 0 to 20 mole percent cholesterol in a bilayer, separate domains exist containing cholesterol and phospholipids and pure phospholipid (Mabrey) . These bilayers show an increased permeability to water (Tsong) . At mole percentages above 50% cholesterol starts destabilizing the bilayer.
  • Liposomes described in the present invention typically include phospholipids, cholesterol and phospholipids derivatized with PEG, such as PEG-DSPE. These liposomes are characterized by drug leakage half- lives which are about 5 fold greater than that for liposomes having the same lipid components but substituting for the derivatized lipid another vesicle- forming lipid containing the same net charge.
  • Liposome compositions are typically composed of lipid components present in a molar ratio of about 40-74 percent vesicle-forming lipids, 25-40 percent cholesterol, and 1-20 percent polymer derivatized lipid.
  • One exemplary liposome formulation includes underivatized hydrogenated soy phosphatidylcholine (HSPC) or hydrogenated egg phosphatidylcholine (HEPC) and cholesterol at 2:1 molar ratio, and 5 mole percent of DSPE-PEG as a ratio of DSPE-PEG to total phospholipid.
  • HSPC does not contain any unsaturated bonds in its acyl chains and its incorporation in bilayers results in a relatively rigid bilayer structure.
  • Another exemplary liposome formulation includes partially hydrogenated soy phosphatidylcholine, iodine number 40 (PC40) instead of HSPC at a similar molar ratio.
  • PC40 contains some degree of acyl chain unsaturation and its incorporation in bilayers results in greater membrane fluidity.
  • the use of unsaturated phospholipids, such as PC40, instead of saturated phospholipids, such as HEPC or hydrogenated soy phosphatidylcholine (HSPC) , in a liposome composition permits higher compound leakage rates, and increases the therapeutic effectiveness of the liposome-entrapped compound.
  • saturated phospholipids such as HEPC or hydrogenated soy phosphatidylcholine (HSPC)
  • HSPC hydrogenated soy phosphatidylcholine
  • a variety of therapeutically active compounds are suitable for subcutaneous delivery in liposome-entrapped form.
  • the compound may be a compound with a suitable therapeutic effect in the bloodstream.
  • the compound may also be one suitable for treatment of a disease associated with the lymph.
  • O ne general class of compounds include water- soluble, liposome-permeable compounds which are characterized by a tendency to partition into the aqueous compartment of the liposome composition, and to equilibrate over time into the outer bulk phase of the liposome composition.
  • Preferred compounds are those that have a low liposome permeability.
  • Representative drugs of this kind include cytarabine, cyclophosphamide, methotrexate, gentamicin, and bleomycins, among others.
  • a second general class of compounds are those which are water-soluble, but liposome-impermeable. These compounds include peptide or protein molecules, such as peptide hormones, enzymes, enzyme inhibitors, and higher molecular weight carbohydrates. Representative compounds include peptide hormones, such as vasopressin (VP) , cytokines, such as interferons (alpha, beta, gamma) , interleukins, and colony stimulating factors (macrophage, granulocyte, granulocyte and macrophage) , viral or bacterial vaccines, melanoma vaccine, str ⁇ ptokinase, or other antithrombolytic peptides, protamine sulfate, superoxide dismutase, and asparaginase.
  • VP vasopressin
  • cytokines such as interferons (alpha, beta, gamma) , interleukins, and colony stimulating factors (macrophage, granulocyte, granulocyte
  • a third class of compounds are lipophilic molecules that tend to partition into the lipid bilayer phase of the liposomes, and which are therefore associated with the liposomes predominantly in a membrane-entrapped form.
  • the compounds in this class include adrenocorticosteroids, prostaglandins, amphotericin B, nitroglycerin, polymyxins, vincristine, carmustine, d acarbazine, dexamethasone, daunomycin and doxorubicin.
  • the liposomes may be prepared by a variety of techniques, such as those detailed in Szoka et al , 1980.
  • Multilamellar vesicles can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium. The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
  • liposomes are prepared by vortexing dried lipid films in a buffered aqueous solution (Olson) .
  • a therapeutic drug is incorporated into liposomes by adding the drug to the vesicle-forming lipids during or prior to liposome formation, as described below, to entrap the drug in the formed liposome. If the drug is hydrophobic the drug is added directly to the hydrophobic mixture. If the drug is hydrophilic the drug can be added to the .aqueous medium which covers the thin film of evaporated lipids.
  • One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 30 to 200 nm, typically 50, 80, 100, or 200 nm.
  • the pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down-sizing liposomes to sizes of lOOnm or less (Martin) .
  • Liposomes with an average diameter greater than about 120 nm administered subcutaneously are not released in the bloodstream. If liposomes have an average diameter of less than about 120 nm the liposomes are released slowly into the bloodstream with a half-life of about 9 hours after subcutaneous administration.
  • the liposomes are therefore prepared to have substantially homogeneous sizes in a selected size range, typically above 120 nanometers if the liposomes are to be localized outside of the bloodstream and sizes between about 50 to 120 nanometers, if the liposomes are to be released in the bloodstream.
  • Subcutaneous administration of a liposome-entrapped compound has been demonstrated to be a viable method for delivering a compound to the bloodstream or the lymph.
  • the biodistribution of liposomes following subcutaneous administration is discussed in Part C.
  • Fig. 1 illustrates peak blood levels of liposomes as a function of average diameter after subcutaneous administration. Blood levels of liposomes at different times post-injection were measured following subcutaneous administration of liposomes to mice. Liposome blood levels were usually followed by tracking radiolabelled tyraminylinulin entrapped within a liposome composition. Tyraminylinulin is metabolically inert, and is rapidly excreted from the body, even following sc administration, once the tyraminylinulin is no longer entrapped in liposomes. The marker, therefore provides a measure of intact liposomes in blood (Kim) .
  • liposomes with sizes less than about 120 nanometers are released to the bloodstream over time. Once the liposome diameter is greater than about 120 nanometers the liposomes are not released in the bloodstream to any extent. Liposomes with diameters greater than about 120 nm can be used to treat diseases associated with the lymph, such as leukemia. Additionally, these liposomes may be used as a therapeutic compound depot for release of the compound. As can be appreciated the relationship of liposome diameter to the extent of liposome release into the bloodstream can be used for liposome-entrapped compound based therapeutic treatments. For example, to release liposome-entrapped compounds into the bloodstream, the liposome diameter is usually less than 120 nm. About 50% of the total administered liposome dose is delivered to the bloodstream, and peak liposome blood levels are typically about 30% of the injected dose.
  • Fig. 2 shows liposome peak blood levels for 22 different liposome compositions after a single liposome dose was administered.
  • the liposome compositions were prepared as described in Example 1 and liposome blood levels were determined as described in Example 2. These peak blood levels may occur at different times, and typically occurred between twelve to twenty-four hours post-injection.
  • the compositions tested were designated A-V in the order of their peak blood level.
  • the letters in the brackets indicate liposome size and dose.
  • the liposomes were either small (S) , with diameters of 79-93 nm, or big (B) , with diameters of 328-718 nm and were administered at high (H) (10 micromoles/mouse) or low (L) (0.5 micromoles/mouse) doses.
  • H high
  • L low
  • the doses are reported in terms of micromoles phospholipid.
  • compositions corresponded to A: PC40:CH:PEG (SH) ; B: HSPC:CH:PEG (SH) ; C: PC40:PEG (SH) ; D: PC40:CH:PEG (SL) ; E: HSPC:PEG (SH) ; F: HSPC:CH:PEG (SL) ; G: HSPC:CH:PG (SH) ; H: HSPC:PEG (SL) ; I: PC40:CH::PG (SH) ; J: HSPC:CH:PG (SL) ; K: PC40:PG (BL) ; L: PC40:CH:PG (SL) ; M: HSPC:PG (BL) ; N: PC40:PG (SL) ; O: HSPC:CH:PEG (BL) ; P: HSPC:PEG (BL) ; Q: HSPC:CH:PG (BL) ; R: PC40:CH:PG (BL) ; S: PC40:CH:P
  • Fig. 3 compares, as a function of the same liposome compositions (A-V) exemplified in Fig. 2, the percent of injected cpm remaining in the whole animal at 16 days post-injection. This measurement may be used to provide a measure of liposome stability.
  • liposomes containing PEG-DSPE and CH were the most stable over time.
  • Fig. 4A illustrates blood levels of different liposome compositions as a function of time post- injection.
  • the presence of PEG-DSPE resulted in significantly higher blood levels of liposomes, irrespective of whether liposomes were made up of saturated (HSPC) or unsaturated (PC40) liposomes.
  • Fig. 4B shows that the presence of cholesterol in liposomes resulted in higher blood levels of liposomes for HSPC- containing liposomes. The same results are obtained for liposomes containing PC40 (data not shown) .
  • Fig. 4C shows that when large liposomes were injected subcutaneously, blood levels were significantly lower compared to those for smaller liposomes. Higher doses of the liposome composition also resulted in higher blood levels.
  • Table 1 shows blood levels of different liposome compositions over a 48-hour period and their corresponding compound leakage rates.
  • Compound leakage studies were performed by measuring cytarabine (ara-C) release from liposomes in the presence of blood plasma.
  • the compositions that resulted in the highest blood levels were liposomes composed of HSPC:CH:PEG-DSPE and PC40:CH:PEG-DSPE at a molar ratio of 2:1:0.1. These liposome compositions were also the ones that showed the lowest ara-C leakage rates.
  • tissue distribution of PC40:CH:PEG-DSPE liposome at 48 hours after subcutaneous administration is compared to that after intravenous administration in Figs. 5A and 5B.
  • Biodistribution studies were performed as described in Example 2. Tissues tested included blood, liver, spleen, lung, heart, kidney, thyroid, brain, skin, tail, muscle, fat, mesenteric lymph node, cervical lymph node, axillary lymph node (including the brachial lymph node) , limbs, gut, bone marrow and carcass.
  • Subcutaneous administration of the liposomes resulted in higher levels of the liposomes being accumulated in cervical, axillary and brachial lymph nodes compared with intravenous administration.
  • the method of the present invention minimizes accumulation of liposomes in the liver or in the bone marrow, sites of the body where a liposome-entrapped drug may have a toxic effect.
  • Fig. 6 shows liposome levels at selected lymph nodes as a function of time post-injection for mice. Injections were made on the back just below the neck. At 24 hours post-injection, the liposomes are located preferentially in the cervical lymph node. At 48 and 72 hours the liposomes are located preferentially in the axillary lymph node.
  • the axillary lymph node as described in this text, includes both axillary and brachial lymph nodes.
  • Figs. 7A-7D provide a comparison of liposome levels in the blood (Fig. 7A) , in the liver (Fig. 7B) , in the spleen (Fig. 7C) , and in the carcass (Fig.
  • Exemplary liposome compositions are discussed in Part A. Studies showing the therapeutic effect of subcutaneous administration of liposome-entrapped compounds is described in Part B below. Different compound administration strategies using the method of the present invention are discussed in Part C below.
  • liposomes are extruded multilamellar vesicles (MLV) composed of HSPC:CH (2:1 molar ratio) containing 5 mole % of PEG-DSPE as a ratio of PEG-DSPE to total phospholipid and cytarabine (ara-C) in liposome-entrapped form.
  • MLV multilamellar vesicles
  • Ara-C belongs to the group of compounds which are water soluble, and have low liposome permeability.
  • Ara-C inhibits DNA synthesis and interferes with replication of DNA viruses and is used in the therapy of non-Hodgkin 's disease and acute myelocytic leukemia.
  • cytarabine After intravenous administration in free form cytarabine is associated with nausea, vomiting, bone marrow depression, neurotoxicity, and hepatic dysfunctions and is rapidly converted to an inactive deaminated form by cytidine deaminases with an initial half-life of 16 to 20 minutes in mice and in humans.
  • liposomes are prepared to contain arg 8 -vasopressin (VP) in liposome-entrapped form.
  • VP belongs to the group of compounds which are water- soluble, and liposome-impermeable. Preparation of liposome-entrapped VP is described in Example 3.
  • Vasopressin (VP) , or antidiuretic hormone, is a peptide hormone released by the posterior pituitary to modulate blood pressure. VP is released to conserve water under conditions of water deprivation. Vasopressin is a nonapeptide with a six amino acid ring and a 3 amino acid side chain. The half life of VP is approximately 20 minutes, with renal and hepatic catabolism via reduction of the disulfide bond and peptide cleavage.
  • VP is utilized for the treatment of transient VP- sensitive diabetes insipidus. Diabetes insipidus results from the failure to secrete sufficient quantities of antidiuretic hormone, and one of the symptoms is dilute urine.
  • liposomes are prepared to contain doxorubicin (DOX) in liposome-entrapped form.
  • DOX belongs to the group of compounds which are lipophilic molecules that tend to partition into the lipid bilayer phase of the liposomes, and which are therefore associated with the liposomes predominantly in a membrane-entrapped form.
  • Doxorubicin is one of the most active antineoplastics identified. It is used to treat acute leukemia, Hodgkin's disease and non-Hodgkin's lymphoma, small cell and non-small cell lung cancer, cancers of the breast, ovaries, stomach, thyroid, bladder, osteogenic and soft tissue sarcomas, and malignant melanoma. Doxorubicin is extensively metabolized to inactive metabolites in the liver. Additionally, doxorubicin causes tissue necrosis if administered intravenously. It would be advantageous to administer these drugs subcutaneously in liposome entrapped form as described by the present invention. Typically, about 1 mg DOX per 10 micromoles phospholipid are incorporated in the liposome.
  • the therapeutic effectiveness of subcutaneously administering a liposome-entrapped compound was tested.
  • the liposomes typically contained a surface layer of polyethylene glycol.
  • single injections of liposome-entrapped ara-C were administered to prolong survival times of mice bearing L1210 leukemia cells given by intravenous or intraperitoneal injection routes.
  • the effect of liposome size, phospholipid bilayer fluidity, and the presence or absence of PEG-DSPE and/or cholesterol were examined. Additionally, the long-term survival of mice bearing L1210 leukemia cells was investigated during subcutaneous administration of ara-C at weekly intervals.
  • Liposomes containing entrapped ara-C were prepared as described in Example 2 and used in experiments as described in Example 5 to determine their therapeutic efficacy. As seen in Table 2, ara-C, administered at doses of 25 mg liposome-entrapped ara-C and 25 mg free ara-C per kg body weight, were effective to increase the mean survival time of mice bearing L1210 leukemia cells by between about 133 to 200%. Long-term mice survivors were obtained during subcutaneous administration of ara-C at weekly intervals, 2 out of 5 experimental animals survived.
  • vasopressin because it has a short plasma half-life unless incorporated in liposomes. Bioactivity experiments can be performed easily by measuring urine production in Brattleboro rats which have a hereditary deficiency in vasopressin production, but are not deficient in the number of vasopressin receptors (Valtin) . Antidiuresis, one of the biological responses to vasopressin, is easily measured and was used to demonstrate that vasopressin administered in this manner can be therapeutically useful.
  • Fig. 8 shows dose response curves for vasopressin administered subcutaneously to Brattleboro rats in liposome-entrapped form.
  • liposomes containing HEPC cholesterol and PEG-DSPE at doses of 25, 100 or 400 micrograms/kg body weight
  • reduction of urine production lasted up to 30 days at a dose of 400 micrograms/kg body weight.
  • vasopressin bioactivity was maintained for about one month after subcutaneous administration of vasopressin in liposome-entrapped form and the liposomes contained a surface layer of polyethylene glycol.
  • Vasopressin entrapped in conventional liposomes lacking polyethylene glycol was active for about two weeks, and free vasopressin was active for even less time.
  • Fig. 9 shows the effect on diuresis of vasopressin administered at 0.18 mg/kg in different preparations.
  • VP When VP is administered in an aqueous solution, the antidiuretic effect of vasopressin is observed up to about 7 days.
  • VP When VP is administered in liposomes composed of HEPC, cholesterol and phosphatidylserine, the antidiuretic effect of vasopressin is apparent until about day 20.
  • VP is administered in liposomes composed of HEPC, cholesterol and PEG-DSPE, the antidiuretic effect of vasopressin at the 0.18 mg/kg dose is extended up to 30 days.
  • Fig. 10 and 11 show detectable vasopressin plasma concentrations in Brattleboro rats when vasopressin is administered in an aqueous solution or in liposomes that contain HEPC, cholesterol and either PEG-DSPE or phosphatidylserine. The results establish that there are detectable vasopressin blood levels after subcutaneous administration of liposome-entrapped vasopressin.
  • detectable vasopressin blood levels are higher for liposomes containing PEG-DSPE than for liposomes containing PS instead of PEG-DSPE, or for aqueous VP solutions.
  • the present method can be used in the treatment of diseases associated with the bloodstream or the lymph.
  • liposomes are sized to a diameter less than about 120 nanometers, preferably between 50 and 120 nanometers, and the liposome-entrapped compounds, such as asparaginase or superoxide dismutase, have a specified therapeutic effect in the blood stream.
  • the compounds may be immune response effectors, such as interleukins, interferons and colony stimulating factors, or antiproliferative effectors, such as bleomycin, doxorubicin, daunomycin.
  • the method of the present invention reduces accumulation of a liposome-entrapped compound in tissues, such as the liver, spleen, lungs and bone marrow, and in the bloodstream, while increasing liposome-entrapped compound accumulation in the lymph nodes.
  • Subcutaneous administration of the liposome- entrapped compound may therefore minimize toxic side effects of the compound when it is administered by another method.
  • subcutaneous administration of the compound in liposome-entrapped form may ensure that the drug is not rapidly degraded to an inactive form in tissues, such as the liver, and may help extend the lifetime of the therapeutically active compound.
  • two compounds may be administered simultaneously.
  • This treatment will result in high blood levels of the first compound during the first 6 hours after compound administration. Peak blood levels of the second compound will be obtained typically 6 to 24 hours after drug administration.
  • drug treatment protocols may be simplified, since both drugs are administered simultaneously to obtain effective levels of the drugs in the bloodstream sequentially.
  • first and second drugs may be two different drugs, such as recombinant interleukin-2 and d acarbazine or cyclophosphamide. Both dacarbazine and cyclophosphamide are used in the treatment of metastatic melanoma (Stoter, Mitchell) .
  • First and second compounds are administered to obtain high levels of first and second compounds sequentially, in order to avoid toxic side effects resulting from co-administering both compounds in the bloodstream.
  • the first compound is administered for a desired therapeutic effect in the bloodstream of a subject, and the second compound cancels the therapeutic effect of the first compound.
  • the first compound is heparin to enhance anticoagulation in the bloodstream
  • the second compound is protamine sulfate that binds to heparin and inhibits the anticoagulation action of heparin.
  • the method is of use in imaging a target site in a subject, wherein the first compound is an imaging agent, such as a chelated form of technetium-99, and the second compound is one effective to reduce the concentration of the imaging agent in the bloodstream, such as an antibody.
  • the method is of use in targeting an agent to a selected site in the subject, wherein the first compound is a targeting agent effective to localize at a site, and said second compound is one effective to reduce the concentration of the targeting agent in the bloodstream.
  • the second compound in this embodiment, is effective to form a multivalent complex with the first compound, to accelerate clearance of the first compound from the bloodstream.
  • the method is of use in the administration of an anesthetic, wherein the first compound is an anesthetic, such as an opioid, and the second compound, an opioid antagonist, is effective to block the action of the anesthetic in the subject.
  • an anesthetic such as an opioid
  • the second compound an opioid antagonist
  • the method is of use in the administration of a therapeutically active compound, wherein the first compound is an inactive compound which is converted, by interaction with the second compound, to a therapeutically active compound.
  • liposomes prepared as described in Example 1, with a selected liposome diameter of at least 120 nanometers results in liposomes that are minimally released in the blood stream as shown in Fig. 1.
  • These liposomes are effective to treat diseases associated with the lymphatic system or are effective to serve as a depot for the release of drug from a subcutaneous site.
  • liposomes with an average size of about 166 nm serve as depots for the localized release of the liposome-entrapped compound. These liposomes were most effective in extending the mean survival time of mice injected with leukemic cells after subcutaneous administration. Therefore, subcutaneous administration of large liposomes causes retention of these liposomes at the site of subcutaneous administration and permits controlled compound release from the injection site to the lymphatic system.
  • the compounds are preferably effective for treatment of a disease localized in the lymph, such as leukemias, lymphomas and metastasizing cancers.
  • liposomes with average diameters greater than 120 nm can be employed as therapeutic compound depots for release of the compound at the site of subcutaneous administration.
  • the compounds can be released from the subcutaneous site in a pulsatile manner by treating the skin surface with heat or other agents that may assist in destabilizing the liposome composition.
  • Cholesterol (CH) , N-tris(hydroxymethyl)methyl-2- aminoethanesulfonic acid (TES) , vasopressin, and ara-C were purchased from Sigma Chemicals (St. Louis, MO) and [5- 3 H] ⁇ ytosine- ⁇ -D-arabinoside ( 3 H-ara-C, 0.55-1.1 TBq/mmol) was purchased from Amersham (Oakville, O ntario) .
  • Hydrogenated soy phosphatidylcholine (HSPC) , partially hydrogenated soy phosphatidylcholine, iodine number 40, (PC40) , hydrogenated egg phosphatidylcholine (HEPC) were purchased from Asahi Chemicals, Japan.
  • Liposome Preparation Incorporating Radiolabelled Tyraminylinulin Liposomes were extruded multilamellar vesicles (MLV) composed of HSPC:CH (2:1 molar ratio) or PC40:CH (2:1 molar ratio) containing 5 mole % of either PEG-DSPE or PG as a ratio of these lipids to total phospholipid content. PG was substituted for PEG-DSPE in control liposome preparations in order to form in liposomes with the same overall net charge.
  • MMV multilamellar vesicles
  • Liposomes were prepared by vortexing dried lipid films in 10 mM TES-buffered saline, 154 mM NaCl, pH 7.4, containing radiolabelled 125 I-Tyraminylinulin (TI) as an aqueous space label. Liposomes were sized by extruding through two stacked Nucleopore (Nucleopore Corp. , Pleasanton, CA) filters from 1.0 to 0.05 micrometers in pore diameter (Olson, Mayer) . The resulting liposomes sizes were determined by dynamic light scattering using a Brookhaven BI-90 particle sizer (Brookhaven Instruments, Holtsville, NY) .
  • Liposome size ranged from 79-93 nm for vesicles extruded through 50 nm filters and 328-718 nm for vesicles extruded through 1 micron filters. Free tyraminylinulin was removed from entrapped label by chromatography over an Ultragel AcA34 column (IBF
  • Example 2 Liposome Preparation Containing Ara-C Liposomes were prepared by the extruded multilamellar vesicle (MLV) method (Mayer) . Briefly, dried lipid films were hydrated with an aqueous solution of 247 ⁇ M ara-C (60 mg/ml, 290 mOsm) containing 'H-ara-C such that the specific activity of the final ara-C solution was 37 kBq/ml.
  • MLV multilamellar vesicle
  • the liposomes were then extruded (Extruder, Lipex Bicm ⁇ mbranes, Vancouver, B.C.) multiple times through 0.05 ⁇ m or 0.2 ⁇ m Nucleopore polycarbonate filters at 75°C, and sized by quasielastic light scattering with a BI-90 particle sizer (Brookhaven Instruments, Holtsville, NJ) .
  • Liposome-entrapped ara-C was separated from free ara-C by centrifugation at 370,000 x g at 10°C for 3 hours. The supernatant containing free ara-C was removed, but the pellet was not washed. The pellet was resuspended in buffer to the desired drug concentration.
  • the total amount of free ara-C was equivalent to the total amount of liposomal ara-C, as determined by chromatography of the samples over Sephadex G-50.
  • the concentration of ara-C in the preparations was calculated from the specific activity of 3 H-ara-C. Phospholipid concentration was determined using the method of Bartlett ( Bartlett) .
  • the average mean diameter of the liposomes was approximately 85 nm and 166 nm for the 0.05 and 0.2 ⁇ m extruded MLV, respectively.
  • Example 3 Liposome Preparation Containing Vasopressin
  • Liposomes containing vasopressin were prepared by hydration of lipid films with an aqueous VP solution followed by extrusion through polycarbonate filters with defined pores.
  • Appropriate amounts of fully hydrogenated egg phosphatidylcholine (HEPC) , phosphatidylserine (PS) , cholesterol (CH) and polyethylene glycol derivatives of distearoylphophatidylethanolamine (PEG-DSPE) were mixed in various combinations.
  • Lipid mixtures sufficient for a 10 ml final volume of liposomes with a phospholipid concentration of 100 micromole/ml were dissolved in chloroform and evaporated to drynes ⁇ under reduced pressure.
  • mice in the weight range of 23-30 grams were obtained from Charles River Canada (St. Constant, QUE) , and maintained in standard housing. Mice (three per group) were given a single bolus injection of 0.2 ml liposomes containing approximately 10 6 125 I-TI cpm and either 0.5 or 10 micromole phospholipid. Some groups of mice received injection of free radiolabelled TI. Injections were either subcutaneous (on the back just below the neck) , intravenous (via the tail vein) or intraperitoneal. After specified periods of time, animals were anaeasthized with halothane (M.T.C. Pharmaceutical, Ontario) and sacrificed by cervical dislocation.
  • halothane M.T.C. Pharmaceutical, Ontario
  • liposomes containing 125 I- tyraminylinulin as an aqueous space marker were administered subcutaneously into the back of the neck in B6D2F1/J female mice (3/group) .
  • the liposomes were labelled with 125 I-tyraminylinulin which is metabolically inert.
  • Data is presented as % of in vivo cpm, which represents the % of counts remaining in the body at a given time point. This corrects for leakage of the label from the liposomes and represents intact liposomes remaining in the body.
  • compositions that resulted in the highest blood levels were liposomes composed of HSPC:CH:PEG-DSPE and PC40:CH:PEG-DSPE at a molar ratio of 2:1:0.1.
  • Table 1 liposomes with a diameter of about 80 to 90 nanometers and the 2:1:0.1 lipid ratio showed the highest blood levels after liposome subcutaneous administration.
  • the liposome blood levels reported as % of in vivo cpm in the blood, increased from about 10% at six hours to about 30% at 24 hours post-injection. The fluidity of these liposome compositions did not appear to markedly affect the blood levels. Liposomes containing either HSPC or PC40 appeared to have similar % of in vivo cpm in blood.
  • Liposomes with a diameter of 155 nanometer are released to the bloodstream at very low levels as indicated in Table 1.
  • a maximum level in the blood of 2% of in vivo cpm in blood is obtained at 24 hours post- injection.
  • Liposomes lacking either cholesterol or the PEG derivatized phospholipid had intermediate blood levels with liposomes containing PEG-DSPE having peak blood levels at 12 hours post-injection of 15%, and liposomes containing cholesterol having peak blood levels at 24 hours post-injection of 4.2%.
  • composition (molar cpm in vivo leakage of ratio, diameter) 6 hrs 12 hrs 24 hrs 48 hrs ⁇ 48 hrs ara-C (hrs)
  • PC40:CH:PEG-DSPE 0.5 ⁇ 0.5 1.0 ⁇ 0.8 2.0 ⁇ 0.7 0.1 ⁇ 0.1 58.5 ⁇ 6.2 ND (2:1 :0.1 , 155 nm)
  • the area under the blood-time concentration curve (AUC) for sc injection was about half the size of the AUC obtained by the iv injection method.
  • the AUC after sc administration provides a measurement of bioavailability in the bloodstream.
  • the true bioavailability of the drug after sc administration is probably much higher since a large fraction of the liposomes never is released in the bloodstream.
  • the time necessary to achieve peak liposome levels in the bloodstream was about 0.6 hours for ip and 9 hours for sc injection. Liposomes containing saturated phospholipids tended to be released more slowly into the bloodstream than liposomes containing unsaturated phospholipids. The rate of liposome removal is not significantly different after the liposomes have been localized in the bloodstream.
  • Drug leakage from the different liposome compositions were determined by measuring the rate of leakage of ⁇ -ara-C from liposomes at 37°C in 25% human plasma, by measuring the free and entrapped ara-C peaks following chromatography of samples over Sephadex G-50 columns.
  • Half-times for leakage were calculated using Graphpad software (ISI, Philadelphia, PA) (Allen, 1992) .
  • liposomes containing PEG- DSPE, cholesterol and less fluid vesicle-forming lipids, such as HSPC have the greatest half-times for ara-C leakage (463 hours) .
  • mice In vivo Experiments with Ara-C Survival experiments were performed in female B6D2F1/J hybrid mice. Groups of 5 female mice (2-4 months, 20-23 gm) were injected by either the ip or the iv (tail vein) route with 10° L1210/C2 cells. Twenty- four hours after implantation of the leukemic cells, treatment began. Treatment consisted of single or multiple injections of a combination of equal amounts of both free and liposome-entrapped drug, by either the iv or the sc route. For example, mice received 25 mg/kg of liposome-entrapped ara-C in combination with 25 mg/kg free ara-C.
  • Table 2 shows the therapeutic effect of liposome- entrapped ara-C given by the intravenous (iv) or subcutaneous (sc) routes of administration.
  • the table shows the route of injection of the leukemic cells, the route of administration of ara-C, the mean survival times (MST) of mice in the various treatment groups. MST values were compared by analysis of variance (ANOVA) . Results were also expressed as % ILS, which represents the % increase in the MST of test mice as compared to control untreated mice. The number of 70-day survivors in the different treatment groups is also indicated.
  • the drug treatments included administering half of the drug in free form and the other half in liposome-entrapped form. Drug treatments employing various liposome compositions were compared.
  • Liposome compositions varied in terms of their lipid components, the mole percentages of the different lipid components, and the average diameter of the liposomes.
  • the liposome compositions were administered as a single injection or as three weekly iv or sc injections.
  • a liposome composition tested contained PC40:cholesterol (CH) :PEG-DSPE in a 2:1:0.1 molar ratio with a 89 nm average size.
  • a second liposome composition tested contained PC40:CH:PEG-DSPE in the same molar ratio but with an average diameter of 166 nm.
  • Liposome compositions lacking cholesterol enhanced the MST of leukemic mice by only about 50%. Mice treated with these liposome compositions subcutaneously fared better than mice treated with the same liposomes intravenously.
  • ara-C was administered in liposome compositions containing hydrogenated soy phosphatidylcholine (HSPC) , instead of PC40.
  • HSPC hydrogenated soy phosphatidylcholine
  • Liposomes containing HSPC:CH:PEG-DSPE in the same molar ratio and an average diameter of 88 nm were tested. Iv and sc administration of the drug were comparable for increasing the MST.
  • drug was administered at weekly intervals. The dose was 25 g free and 25 mg in liposome-entrapped form/kg body weight.
  • the %ILS was increased by about 300 to 500% depending on the method of drug administration and the method of leukemic cell injection. Seventy-day survivors were obtained by both intravenous and subcutaneous administration methods. TABLE 2
  • vasopressin vasopressin
  • vasopressin bioactivity was maintained for about one month after subcutaneous administration of 400 micrograms vasopressin in liposome-entrapped form when the liposomes contained a surface layer of polyethylene glycol.
  • Vasopressin entrapped in conventional liposomes lacking polyethylene glycol was active for about two weeks, and free vasopressin was active for even less time.
  • Plasma concentration of VP over time was determined in the same strain of rat by obtaining serial blood samples at various time points following a single subcutaneous dose of 200 micrograms VP/kg body weight. Blood seunples from 0-30 hour post dose were obtained via an indwelling femoral artery cannula surgically implanted 1-2 days prior to dosing. All samples were placed on ice immediately until separation. Plasma samples were stored frozen until vasopressin analysis (Hazelton Labs America, Washington, D.C.) was complete.

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Abstract

L'invention concerne un procédé permettant d'obtenir un apport de longue durée d'un composé thérapeutique dans le sang. Le composé est administré de manière sous-cutanée dans une composition de liposomes présentant des dimensions inférieures à environ 120 nm, un enrobage de surface de polyéthylèneglycol, et le composé sous une forme piégée. L'invention se rapporte également à un procédé d'administration d'une paire de composés médicamenteux destinés à apparaître séquentiellement dans le sang.
PCT/US1994/005072 1993-05-07 1994-05-06 Procede d'apport sous-cutane de liposomes WO1994026251A1 (fr)

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Cited By (7)

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EP0818989A1 (fr) * 1995-04-05 1998-01-21 Imarx Pharmaceutical Corp. Compositions nouvelles de lipides et de materiaux de stabilisation
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5827533A (en) * 1997-02-06 1998-10-27 Duke University Liposomes containing active agents aggregated with lipid surfactants
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US6673364B1 (en) 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
WO2014144842A3 (fr) * 2013-03-15 2015-04-09 Rhythm Metabolic, Inc. Compositions pharmaceutiques

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DE2747378A1 (de) * 1976-10-23 1978-04-27 Choay Sa Liposomen, verfahren zu ihrer herstellung und sie enthaltende arzneimittel
EP0300806A1 (fr) * 1987-07-24 1989-01-25 Vestar, Inc. Système de libération liposomique d'opoides analgésiques
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DE2747378A1 (de) * 1976-10-23 1978-04-27 Choay Sa Liposomen, verfahren zu ihrer herstellung und sie enthaltende arzneimittel
EP0300806A1 (fr) * 1987-07-24 1989-01-25 Vestar, Inc. Système de libération liposomique d'opoides analgésiques
WO1991005545A1 (fr) * 1989-10-20 1991-05-02 Liposome Technology, Inc. Procede et composition a microreservoir de liposomes

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5820873A (en) * 1994-09-30 1998-10-13 The University Of British Columbia Polyethylene glycol modified ceramide lipids and liposome uses thereof
US5885613A (en) * 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
EP0818989A4 (fr) * 1995-04-05 2000-09-13 Imarx Pharmaceutical Corp Compositions nouvelles de lipides et de materiaux de stabilisation
EP0818989A1 (fr) * 1995-04-05 1998-01-21 Imarx Pharmaceutical Corp. Compositions nouvelles de lipides et de materiaux de stabilisation
US6673364B1 (en) 1995-06-07 2004-01-06 The University Of British Columbia Liposome having an exchangeable component
US6296870B1 (en) 1997-02-06 2001-10-02 Duke University Liposomes containing active agents
US5882679A (en) * 1997-02-06 1999-03-16 Duke University Liposomes containing active agents aggregated with lipid surfactants
US5827533A (en) * 1997-02-06 1998-10-27 Duke University Liposomes containing active agents aggregated with lipid surfactants
US6734171B1 (en) 1997-10-10 2004-05-11 Inex Pharmaceuticals Corp. Methods for encapsulating nucleic acids in lipid bilayers
WO2014144842A3 (fr) * 2013-03-15 2015-04-09 Rhythm Metabolic, Inc. Compositions pharmaceutiques
AU2014228460B2 (en) * 2013-03-15 2018-11-01 Rhythm Pharmaceuticals, Inc. Pharmaceutical compositions
AU2019200101B2 (en) * 2013-03-15 2021-02-04 Rhythm Pharmaceuticals, Inc. Pharmaceutical Compositions
US11129869B2 (en) 2013-03-15 2021-09-28 Rhythm Pharmaceuticals, Inc. Pharmaceutical compositions

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