WO1990006775A1 - Nouvelle composition de liposomes non phospholipidique a liberation soutenue de medicaments - Google Patents

Nouvelle composition de liposomes non phospholipidique a liberation soutenue de medicaments Download PDF

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Publication number
WO1990006775A1
WO1990006775A1 PCT/US1989/005525 US8905525W WO9006775A1 WO 1990006775 A1 WO1990006775 A1 WO 1990006775A1 US 8905525 W US8905525 W US 8905525W WO 9006775 A1 WO9006775 A1 WO 9006775A1
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Prior art keywords
cholesterol
drug
mole
composition
salt
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PCT/US1989/005525
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English (en)
Inventor
Ramachandran Radhakrishnan
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Liposome Technology, Inc.
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Priority claimed from US07/284,216 external-priority patent/US5043165A/en
Priority claimed from US07/284,158 external-priority patent/US4906476A/en
Application filed by Liposome Technology, Inc. filed Critical Liposome Technology, Inc.
Publication of WO1990006775A1 publication Critical patent/WO1990006775A1/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
    • A61K9/0012—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
    • 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
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • the present invention relates to a nove nonphospholipid liposome composition for efficient loadin and sustained release of drugs.
  • the composition i particularly useful in formulating steroids and other drug for inhalation, and targeted systemic, parenteral, oral intrathecal, intraarticular, nasal, ophthalmic and topica administrations for human and veterinary therapeuti applications.
  • dexamethasone is administered systemically by i.v. injection in doses ranging from 0.5 to 9 mg/day. Where, however, dexamethasone is administered via inhalation, th dose may be decreased to approximately 0.084 mg per on inhalation dose.
  • the total dose of inhaled dexamethason daily, even when the inhalation is repeated at the maximu dosing frequency, (12 times a day) corresponds to betwee 0.4 to 1.0 mg of absorbed dexamethasone a day.
  • PDR: 1312 and 1315 (1988) This steroid dose given by inhalatio achieves the same therapeutic effect as systemic dose.
  • Beclomethasone, halogenated synthetic analog of cortiso used in a form of beclomethasone dipropionate (BDP) is ofte used for inhalation for treatment of bronchial asthma an seasonal and perennial rhinitis.
  • BDP beclomethasone dipropionate
  • beclomethason dipropionate is poorly soluble in water, it is currentl formulated as a microcrystalline suspension i chlorofluorocarbons (Freon) propellants.
  • PDR:1003 (1988) The advantages connected with using inhalation rout rather than systemic administration are lessened by th necessity of multiple dosing.
  • ILD interstitial lun diseases
  • ILD International Health Organization
  • steroids in particular corticosteroids or glucocorticoids alone or in combination with other drugs.
  • Most often used therapy for ILD is 40-80 mg/day of prednisone orally for one to two months.
  • a follow-up treatment with lower doses (5-15 mg/day) is needed for weeks, years, or indefinitely.
  • favorable responses to such massive doses of steroids are achieved in only 20-60% of patients.
  • steroids formulated for inhalation seem to be rapidly absorbed in upper respiratory regions with very little, if any, of the steroid ending up in alveoli, a primary area affected by th inflammation leading to ILD.
  • presized liposomes o approximately 0.2 ⁇ or micelles of particle size o approximately 0.02 ⁇ can be used for the generation o aerosol particles that can be deposited in the alveoli i significant amount.
  • th size of aerosol droplet delivering drug into alveoli must b substantially within that size limit, preferably with th majority of single aerosol droplet about or smaller than ⁇ for optimal alveolar deposition.
  • MMAD mass median aerodynamic diameter
  • steroids 7 administration are intraarticular injection of steroid into inflamed joints and intrathecal injection of steroids into the brain and spinal cord during bacterial, inflammatory and viral diseases of the central nervous systems, nasal or oral administration during bacterial, viral or allergic reactions or cold symptoms, topical administration during dermatitis or bacterial infections, various parenteral administrations such as intravenous, intramuscular, intraperitoneal, subcutaneous or percutaneous for treatment of all kinds of infections, inflammations and allergic conditions.
  • agents While in general these agents are considered pharmaceutically acceptable excipients, many of them have undesirable side effects particularly when used in inhalation, parenteral, intraarticular, intrathecal, nasal or topical formulations.
  • the deleterious effect of agents such as PEG in membrane permeabilization and local irritation is well documented.
  • liposomes ar advantageous in that they can provide and improve controlle release of an entrapped drug, reduce side effects b limiting the concentration of free drug in the bloodstream, alter the tissue distribution and uptake of drugs in therapeutically favorable way, and make therapy safer an more convenient by reducing the dose or frequency of dru administration.
  • Decreased toxicity and degradation, use o smaller doses, a targeting the liposomes toward a specifi site, and reducing side effects of a liposome-bound steroi over the use of a free or polymer-bound steroid have bee described in Nature, 271: 372-373 (1978) .
  • the use o liposomes as a solubilizing agent for steroids or othe drugs in aqueous, nebulized inhalation suspension essentially eliminates the use of potentially toxi halogenated hydrocarbon propellants and co-solvents, an prevents irritation caused by drug sedimentation an crystallization often encountered with conventional steroidal suspension.
  • U.S. Patent 4,693,999 discloses new steroid derivatives obtained by modification of corticosteroids with fatty acid esters. These modified steroids incorporated in the lipid portion of liposomes for delivery via inhalation provide prolonged steroid retention in the respiratory tract, however, designing and synthesizing new steroid derivatives is inconvenient, costly, slow, laborious and often changes the drug efficacy.
  • Water-insoluble steroids are generally difficult to load into conventional phospholipid liposomes because these molecules tend to crystallize rather than incorporate into the liposomal membrane. Such drug crystallization causes the same sedimentation problems and free drug toxicity upon administration as do nonliposomal steroidal suspensions.
  • Modified steroids unlike cholesterol which is ubiquitously distributed in biological membranes, in particular seem to be structurally or sterically incompatible with phospholipids in terms of hydrophobic or Van Der Waals interactions and thus crystallize out readily.
  • nonphospholipid liposome composition wherein the poorl water soluble, sedimentation-prone, underivatized o unmodified steroids or other drugs are successfull sequestered within the liposomal lipid vesicles of unifor and controllable particle size, having high encapsulatio values, long-term stability, and effective sustained releas with a controllable potency of the drug.
  • the resultin composition will allow an administration of low doses o steroid or other drugs thus reducing toxicity and systemi side effects and provide pharmacologically bioavailabl doses of the drug .in situ.
  • One aspect of this invention is to provid nonphospholipid liposome based formulation comprisin cholesterol, cholesterol ester salt and underivatized an unmodified steroidal or other drug for therapeutic delivery
  • Another aspect of this invention is to provide formulatio enabling high efficiency liposome entrapment o underivatized steroids and other drugs in the liposom vesicles of uniform and controllable particle size.
  • Yet another aspect of this invention is to provid liposome composition which has lower toxicity, lower sid effects, allows the targeting and release of the drug at site of specific organ, removes need for multiple dosing can be sterilized, and is sufficiently stable in dried for for long-term storage.
  • Another aspect of this invention is to provide controlled, sustained release of the steroidal and other drugs from the n ⁇ nconventional liposome/drug composition.
  • Yet another aspect of this invention is to provide liposome/drug compositions which has lower toxicity, lower side effects, allows the targeting to and release of the drug in a deep lung tissue and removes need for multiple dosing.
  • Yet another aspect of this invention is to provide the method of treatment of interstitial lung diseases by administering the nebulized liposomal drug composition by oral inhalation.
  • Still another aspect is to provide a process for making novel nonconventional liposome composition for controlled sustained release of steroidal or other drugs.
  • Still yet another aspect of this invention is to provide the method of use of the nonphospholipid liposomal drug compositions for delivery by inhalation, intratracheal, peroral, parenteral, such as intravenous, intraperitoneal, intramuscular, or subcutaneous, percutaneous, topical, intraarticular, intraventricular and ocular routes of administration.
  • Figure 1 shows the rat plasma concentration of BDP after intravenous injection of radiolabeled BDP.
  • Figure 2 shows the rat plasma concentration of BDP afte intratracheal instillation of radiolabeled BDP.
  • Figure 3 shows the rat plasma concentration of BDP afte intratracheal instillation of radiolabeled BDP encapsulate in two types of conventional liposomes.
  • Figure 4 shows the plasma kinetics of radiolabeled BD after intravenous administration of free BDP an intratracheal instillation of radiolabeled BDP encapsulate in conventional cholesterol containing liposomes.
  • Figure 5 shows the amount of plasma BDP radioactivit for two hours following the intratracheal instillation o nonconventional liposomal BDP illustrating sustained releas and for three hours following the administration of fre drug.
  • Figure 6 shows the amounts of radiolabeled BDP remainin in the rat lungs following intratracheal instillation o five different liposome-encapsulated BDP formulations an the amount of the radiolabeled BDP in the lungs found afte the intravenous administration of the free BDP.
  • Figure 7 shows the plasma concentration of free BDP an BDP encapsulated in nonconventional liposomes and th sustained release of liposome encapsulated BDP versus total BDP.
  • Figure 8 depicts pulmonary anatomy showing the divisio of one larger bronchus into smaller bronchi, bronchioli terminal bronchioles, respiratory bronchioles, alveola ducts, sacks, and ultimately into individual alveoli.
  • Figure 9 depicts the current concept of pathogenesis clinical symptoms and pathological changes connected wit interstitial lung diseases.
  • Figure 10 is a diagram for nebulization of a steroi liposome suspension and collection of aerosol output o Anderson cascade impactor stages corresponding to the huma respiratory system.
  • Figure 11 depicts Andersen's Sampler as a simulator o a human respiratory system.
  • Figure 12 shows the mass median aerodynamic diameter an aerosol particle size distribution of BECOTIDE ® .
  • Figure 13 shows the mass median aerodynamic diameter an aerosol particle size distribution of liposoma beclomethasone dipropionate.
  • beclomethasone dipropionate, other steroid in underivatized form and other drugs may be successfull retained in nonconventional liposomes for sustained releas when the liposomes are formulated to contain a mixture of cholesterol and cholesterol ester salt such as for example sodium cholesterol sulfate, also known as cholesteryl sodium sulfate (cholesterol sulfate) .
  • cholesterol and cholesterol ester salts such as for example sodium cholesterol sulfate, also known as cholesteryl sodium sulfate (cholesterol sulfate) .
  • cholesteryl sodium sulfate cholesterol sulfate
  • Sodium cholesterol sulfate or other cholesterol ester salts act as a temporary barrier against drug efflux from the liposomes.
  • the liposome suspension of the invention can be prepared by any of the standard methods for preparing and sizing liposomes. These include hydration of lipid films, solvent injection, reverse-phase evaporation, dehydration rehydration, freeze thaw and other methods, such as those detailed in Am. Rev. Biophvs. Bioencr.. 9:467 (1980).
  • Reverse-phase evaporation vesicles (REV) prepared by the reverse-evaporation phase method is described in U.S. Patent No. 4,235,871, incorporated hereby by reference.
  • the preparation of multi-lamellar vesicles (MLV) by thin-film of a lipid film or by injection technique is described in U.S.
  • Patent 4,737,923 incorporated by reference.
  • a mixture of liposome-forming lipids dissolved in a suitable solvent is evaporated in a vessel to form a thin film, which is covered by an aqueous buffer solution.
  • the lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
  • Either the REV or MLV preparations can be further treated to produce a suspension of smaller, relatively homogeneous-size liposomes, in the 0.1-1.0 micron size range.
  • Advantages of smaller, more homogeneous-size liposomes are, for example the higher density of liposome packing at a mucosal tissue ' surface, the highe concentration of liposome encapsulated drug transported to the target organ or tissue, or the greater optical clarit when applied topically to the eye.
  • One effective sizin method involves extruding an aqueous suspension of th liposomes through a polycarbonate membrane having a selecte uniform pore size, typically 0.2, 0.4, 0.6, 0.8 or l micron Ann. Rev. Biophvs. Bioen ⁇ ..
  • the pore size o the membrane corresponds roughly to the largest sizes o liposomes produced by extrusion through that membrane particularly where the preparation is extruded two or mor times through the same membrane.
  • a more recent metho involves extrusion through an asymmetric ceramic filter The method is detailed in 4,737,323, incorporated hereby b reference.
  • the REV or MLV preparations can b treated to produce small unila ellar vesicles (SUV) , larg unilamellar vesicles (LUV) or oligolamellar vesicles (OLV) .
  • the REVs or MLVs can be treated by sonicatio to produce small unilamellar vesicles (SUVs) which ar characterized by sizes 0.02-0.07 ⁇ . Because of the smal particle sizes, SUVs are particularly suitable for th delivery of steroid to the alveoli.
  • SUVs Another advantage o SUVs is the greater packing density of liposomes at mucosal surface, thus making SUVs preferable for inhalatio for treatment of deep lung diseases such as idiopathi infiltrative pulmonary fibrosis, degenerative interstitial pneumonias and sarcoidosis. Because of the small particl sizes, SUVs suspensions can be optically quite clear, an thus advantageous and preferred for example for ophthalmic applications or for such applications as the delivery of the steroid to the minuscule lung alveoli.
  • SUV is the greater packing density of liposomes at a mucosal surface which can be achieved with smaller liposome particles, thus making SUV preferred for inhalation, for treatment of deep lung diseases such as idiopathic infiltrative pulmonary fibrosis, degenerative interstitial pneumonia or for topical or nasal use.
  • the use of all SUV, LUV, MLV, OLV or mixture thereof is contemplated depending on intended therapeutic application and route of administration.
  • One preferred method for producing SUV is by homogenizing an MLV preparation, using a conventional high pressure homogenizer of the type used commercially for milk homogenization where the MLVs are cycled through the homogenizer, and periodically sampled for particle sizes to determine when the MLV have been substantially converted to SUVs.
  • the drug is encapsulated in the liposomes by using for example the procedure described in U.S. patent 4,752,425, incorporated by reference.
  • the conventional liposomes mean liposomes which contain phospholipids
  • the “nonconventional liposomes” mean liposomes which do not contain phospholipids but are formed solely by cholesterol and cholesterol derivatives or, in alternative by amphipathic lipid components.
  • Both conventional and nonconventional liposomes can be formed by a variety of standard methods from a variety of vesicle-forming lipids.
  • these lipids include dialiphatic chain lipids, such as phospholipids, diglycerides, dialiphatic glycolipids, and cholesterol and derivatives thereof.
  • the various lipid components are present in an amount between about 40-99 mole % preferably 60-90 mole % of the total lipid components in the liposomes, cholesterol or cholesterol derivatives are present in amounts between 0-40 mole %.
  • the cholesterol derivatives are present in amounts between 30-70:20-50:0.01-20 mole % of cholesterol derivative to cholesterol to drug, respectively.
  • phospholipids include phosphatidi acid (PA) , phosphatidylglycerol (PG) , phosphatidylcholin (PC) , egg phosphatidylcholine (EPC) , lysophosphatidylcholin (LPC) , phosphatidylethanolamine (PE) , phosphatidylinosito (PI) , phosphatidylserine (PS) .
  • PA phosphatidi acid
  • PG phosphatidylglycerol
  • PC phosphatidylcholin
  • EPC egg phosphatidylcholine
  • LPC lysophosphatidylcholin
  • PE phosphatidylethanolamine
  • PI phosphatidylinosito
  • PS phosphatidylserine
  • the liposome composition may be formulated t include minor amounts of fatty alcohols, fatty acids, and/o cholesterol esters or any other pharmaceutically acceptabl excipients with the proviso that these minor lipi components do not significantly reduce the binding affinit of the liposomes for mucosal or organ tissue, ar substantially unsaturated, and are not toxic or irritating Preparation of Nonconventional Liposome Composition
  • drugs particularly steroids i underivatized form may be successfully retained in liposome for delayed release when the liposomes are formulated t contain a high percentage of cholesterol ester salt, such a cholesterol sulfate, typically from 30-70 mole %, preferabl 50 mole % in combination with cholesterol, typically fro 20-50 mole %.
  • cholesterol ester salt such as cholesterol sulfate
  • the underivatized drug/cholesterol/sodiu cholesterol sulfate composition of the invention ha improved properties such as lesser toxicity, decreased sid effects, controllable sustained release, improve solubility, high encapsulation, steroid release at th target organ, absence of need for multiple dosing, extende stability in that it can be stored long-term in dried for without significant increase in particle size o rehydration.
  • th current invention combines the lipid components includin cholesterol with cholesterol ester salt, preferably sodiu cholesterol sulfate, providing the hydrophilic group, with the natural or synthetic steroidal drug to be formulated in certain ratio.
  • the composition is engineered to have an increased drug loading and a controllable sustained release rate of the steroid drug. It also provides a means to solubilize the steroids and incorporate them in such liposomal composition without need to modify the drug.
  • the formulation can be easily sterilized and is stable for up to 3 month and thus suitable for long-term storage.
  • Lipid bilayers consisting entirely of cholesterol in their hydrophobic core can be conveniently constructed if a hydrophilic group is built-in as a part of the steroid molecule.
  • Sodium salt preferably sodium cholesterol sulfate, is used to provide such hydrophilic group.
  • equi olar amounts of cholesterol added, initially multilamellar liposomes form which then become unilamellar liposomes on prolonged sonication.
  • the resulting nonconventional nonphospholipid liposomal vesicles are comparable to those of conventional phospholipid vesicles in all aspects except that cholesterol bilayers possess internal barriers that are less easily permeated, thus allowing controllable sustained release of steroid from the core of liposomes.
  • These bilayers can also keep steroidal drugs by hydrophobic and electrostatic interactions in bilayer leaflet thus providing slow release.
  • composition of current invention comprises a lipid component, such as cholesterol, and cholesterol ester salt, and drug in ratio from 20-50:30-70:0.1-20 mole %.
  • lipid component such as cholesterol, and cholesterol ester salt
  • drug in ratio from 20-50:30-70:0.1-20 mole %.
  • the best suited liposomal formulations for sustained release of the steroids were found to be sodium cholesterol sulfate:cholesterol:steroid in mole % ratios of 55:40:10; 50:40:5; 53:37:9, most preferably 50:40:10 mole %.
  • a lipid composition containing sodium cholesterol sulfate: cholesterol:BDP, at a mole ratio of 50:40:10 had the best delayed release of the drug when administered to the experimental animals by way of, for example instillation in the respiratory tract or by inhalation o nebulized composition.
  • All pharmaceutically acceptable cholesterol ester salt and excipients can be used in the formulation.
  • Whil cholesterol sodium sulfate is preferred, the composition i not restricted to this particular salt and any othe suitable cholesterol salt such as cholesterol nitrate maleate, phosphate, acetate, propionate, pyruvate, oxalate malonate, succinate, fumarate, tartarate, citrate, benzoat and such others can be advantageously used.
  • th cholesterol sulfate sodium salt may be converted to othe salts with different cations, which may include potassium lithium, magnesium, and other divalent cations, tris triethanolamine, ethanolamine, heterocycles and such othe salts commonly used and pharmaceutically acceptable i pharmaceutical formulations.
  • Buffer used in the preparation of the nonconventiona liposomes may be any buffer chosen from the group o citrate, carbonate, bicarbonate, acetate, Tris, glycinate cacodylate, maleate, and such other, preferably phosphat buffered saline of pH 7.4.
  • Any organic aqueous solvent such as lower alcohols dimethoxyethane, dioxane, tetrahydrofuran, tetrahydropyran diethylether, acetone, dimethylsulfoxide (DMSO) dimethylformamides (DMF) , and halogenated hydrocarbons, suc as freon, acetonitrile, or mixtures of all those above preferably chloroform/methanol are used in the process o generation of liposomes.
  • organic aqueous solvent such as lower alcohols dimethoxyethane, dioxane, tetrahydrofuran, tetrahydropyran diethylether, acetone, dimethylsulfoxide (DMSO) dimethylformamides (DMF) , and halogenated hydrocarbons, suc as freon, acetonitrile, or mixtures of all those above preferably chloroform/methanol
  • cholesterol, cholesterol ester salt, preferably sodium cholesterol sulfate, and steroidal drug i dry form in amounts from 20-50 mole % of cholesterol, 30 70 mole % of cholesterol salt and 0.1-20 mole% of steroid, preferably 40 mole % of cholesterol, 50 mole % of sodiu cholesterol sulfate and 10 mole % of a drug;
  • dissolving the mixture in 5-30 ml of an organic solvent, preferably in 10 ml of methanol:chloroform (2:1 v/v) ;
  • composition of the invention are not limited to those named above, but all methods of liposome preparation such as solvent injection, thin film hydration, dehydration-rehydration, and reverse evaporation are equally suitable.
  • Drug encapsulation means the amount of the drug incorporated, loaded, associated, bound or otherwise attached to the liposomes or their bilayers. In general, the ability of liposomes to encapsulate drug is expressed in % of the starting amount. Thus, the optimal encapsulation of 100% is achieved where all drug is encapsulated in liposomes. Technically, however, it is often difficult to achieve 100% encapsulation because the encapsulation depends on the lipid properties, on the drug properties and on the encapsulating method used.
  • nonconventional liposomes demonstrate higher drug loading with encapsulatio values of 100%, when 10 mole % dose is used (total lipi concentration of 40 umol/ml) compared with conventiona phospholipid liposomes. These liposomes generally allo only about 1 mole percent drug encapsulation at a tota lipid concentration of 40 umol/ml. For example, unsaturate conventional liposomes without cholesterol have th flexibility of accommodating only 1 mole percent o steroidal drug and their encapsulation value is therefor very small.
  • Stability problems are also overcome in a current nonconventional liposome formulation, in terms of the sedimentation and crystallization problems encountered with nonliposomal or conventional liposome suspensions. Because of the unique cholesterol sulfate formulations which accommodate the drug by steric fit, and because of their high encapsulation and high retention values, drug crystallization does not occur outside or inside the liposomes, nor does sedimentation occur from the suspension. Such nonconventional liposomes are stable at 4°C for up to 3 months and do not form the drug crystals.
  • the nonconventional liposome composition may be prepared and stored as suspension, dry powder, dehydrated liposomes and as liposome paste.
  • These liposome formulations provide the following advantages: relatively good stability on storage, a high drug capacity, a high ratio of liposome-entrapped to free drug, and very high viscosity for enhanced retention to the mucosal and ocular surface.
  • the concentrate is preferably formed by ultrafiltration with continued recycling of the liposome suspension material. These concentrates have equilibrium maximal loading of steroidal drugs and stable for storage for at least three months at 4°C.
  • the dried particle (dry powder) liposome formulation can be prepared either by lyophilization of liposomes or spray drying. In the former method, the small-particle suspension is quick frozen and lyophilized or subjected to slow process lyophilization at a shelf temperature of preferably -20°C or less.
  • the particle suspension is dried in a conventional apparatus in which the particles to be dried are sprayed in aerosolized suspension form into a stream of heated air or inert gas, and the aerosolized droplets are dried in the gas stream as they are carried toward a dry powder collector where the dried liposomes are collected.
  • An exemplary spray dry apparatus is a Buchi 190 Mini Spray Dryer. BBA 897:331-334 (1987).
  • the drying temperature is at least about 25°C, preferably between about 30-200°C.
  • the temperature of the collection chamber is generally lower than that of the heated air, and typically about 30 ⁇ C.
  • the dried particle are collected and stored as a powder in dehydrated form under an inert atmosphere in the presence of a desiccant Such powders are storable under these indications for a least a year at ambient temperature.
  • Dry powder liposome can be used as injectable materials after reconstitution o suspended in appropriate dilutants or freon propellants fo aerosol administration or formulated to topical, nasal o oral dosage forms.
  • steroids may be solubilized in surfactan micelles and nebulized into small aerosol particles by usin appropriate nebulizers.
  • Typical mixed micellar formulation of steroid contain an appropriate surfactant detergent suc as sodium methyl cocoyl taurate (Tauranol ® WS) obtained fro
  • BASF Wyandotte Corp. N.J. in amount from 1-100 mg per ml, preferably between 40-60 mg/ral, mixed with steroid drug i amounts from 0.1-20 mg/ml, preferably in amount 0.2-1 mg/ml.
  • the weight ratio of surfactant to drug is from 100-200:0.2
  • the mixture is let stand unde stirring for 2-48 hours, preferably overnight at temperatur between 16-40°C, preferably at ambient temperature.
  • the the mixture is filtered over filter with pore sizes smalle than steroid crystals, usually using 0.1-l ⁇ filter. Filter, on which the undissolved drug is deposited, is discarded and the micelle filtrate is used for nebulization as described below.
  • Micelle is the term used to describe the suspension of surfactant in water.
  • drug is intercalated between two layers of surfactant with polar group being situated on outside.
  • pH of micelles varies and maybe from around 4.25 to preferably around 7.4- 7.8.
  • other additives such as saline, mono or dibasic sodium phosphate may be added in amount to reach and/or maintain osmolality of the mixed micelles between 200-500, preferably around 300 mOsm/kg.
  • the micelles are prepared in deionized distilled water to make up volume wherein per each ml there is present surfactant, steroidal drug, saline or other salt in amount to fall within ratios given above, preferably about 60 mg/ml surfactant; 0.4 mg/ml of drug and 9 mg/ml of saline.
  • micelles as particle aerosol useful for treatment of interstitial lung diseases
  • the loading of drug into micelles and the sustained release of drug are limited.
  • interstitial lung diseases are primarily diseases of the deep lung
  • the delivery of corticosteroids and other drugs used for treatment of alveolar inflammation to the site of the inflammation is of primary interest.
  • Focused administration of steroids or other drugs to the lung parenchyma via oral inhalation represents an attractive alternative to the oral route for the treatment of ILD and offers the potential to concentrate the drug at a site where it is needed while minimizing systemic absorption and accompanying side effects.
  • Solubilization of steroids in an aqueous formulation and subsequent generation of small aerosol droplets by nebulization are important prerequisites toward achieving this goal.
  • Several inhalation dosage forms of steroid drugs have been previously developed for the treatment of bronchial asthma.
  • steroid preparations could only be formulated as propellant suspensions, such as for example Freon 11-clathrate suspended in Freon 12/114 mixture or as aqueous suspensions with surfactants.
  • propellant suspensions such as for example Freon 11-clathrate suspended in Freon 12/114 mixture or as aqueous suspensions with surfactants.
  • steroids may be advantageously formulate in nonconventional i.e., nonphospholipid liposomes
  • steroids may be formulated in surfactant micella solutions. Steroids solubilized in either of these entitie are able to be nebulized using appropriate nebulizers t form small particles with good drug output as describe above.
  • Nonconventional liposomes offer several advantage including greater loading efficiencies and safety. Fo example, nonconventional cholesterol sulfate liposome ar able to incorporate around 2 mg or more of drug per ml o solution used for nebulization, generating aerosol droplet with a mass median diameter between 0.4-0.9 ⁇ .
  • the aerosol droplets generated by th method described below are able to be deposited, upo inhalation, in the deep lung of alveolar tissue.
  • compositions of this invention ar suspensions of nonconventional liposomes or micelle containing steroid, preferably beclomethasone dipropionat in as large amounts as can be possibly formulated.
  • Fo nonphospholipid liposomes these amounts are from 0.1 mg/m to about 2 mg/ml of suspension.
  • the suspende amount of steroid in surfactant preferably Tauranol WS, i about 0.4 mg/ml.
  • Liposomes or micelles are prepared as describe above. Liposomes are presized to contain substantially homogeneou liposome population with a mean particle size of 0.2 ⁇ . Th liposomal or micellar suspension is placed in the nebulize and, as illustrated in Figure 10, the air compressor i attached to the lower part of the nebulizer at point B. B the pressured air generated from the compressor, th solution in the nebulizer is agitated into a mist o aerosolized particles droplets of sizes predominantl between 0.02-3 ⁇ m with an MMAD not exceeding 2.1 ⁇ m. Thes particles are then moved to the connecting tubing havin inserted one-way valve with filter. The aerosol particles move toward the mouthpiece to be used for a patients' inhalation. Larger particles fall back to nebulizer and again undergo aerolization. In the real life situation, expired air carrying very small particles may be trapped in the air filter provided.
  • the nonconventional liposome steroidal suspension or micellar solution prefor ulated in the concentration and amount as described above (or the formulation may be sufficiently diluted with sterile saline or a suitable diluent to known concentration of active ingredient) is poured into the nebulizer, the nebulizer is connected to the air compressor, and the patient inhales via a mouth piece the aerosolized suspension.
  • Figure 10 represents a model for studying a nebulization of steroid suspension on the Anderson cascade impactor stages.
  • the principle of the model is that the impactor is divided into Stages 0-7, having segments separated from each other by the stages with pores 10 ⁇ and above-preseparator stage, 9-10 ⁇ - Stage 0; 5.8-9 ⁇ - Stage 1; 4.7 - 5.8 ⁇ -
  • Stage 7 A suitable filter is placed at the end to collect any submicronic droplets.
  • Stages 5, 6, 7 and filter correspond to droplets of 0.4 to about 2.1 ⁇ (MMAD) reaching alveoli. Consequently, only aerosol particles which pass Stage 4 into Stages 5, 6, 7 and submicronic filter are useful for delivering drugs into alveoli. Aerosolization of nonconventional liposomal suspension or micelles produces droplets containing the expected amount of steroid, i.e., around 1.7-2 mg/ml of aerosolized solution for liposomes and 0.4-0.5 mg/ml of aerosolized micellar solution with a mass median aerosol diameter of 0.4 - 0.9 ⁇ .
  • MMAD 2.1 ⁇
  • Figures 12 and 13 compare the alternative aqueous steroida suspension of BECOTIDE ® ( Figure 12) to a liposoma beclomethasone dipropionate Formulation ( Figure 13) .
  • Figure 12 shows the liquid aerosol particle siz distribution of BECOTIDE ® generated using an ultraven nebulizer with pulmoaide compressor pump mass distributio being done by QCM impactor with an isokinetic flow divider.
  • MMAD effective mass media aerodynamic diameter
  • MMAD is Stokes Diamete described in An Introduction to Experimental Aerobiology, p. 447, Wiley (1966) and is an equivalent mean diameter. Whe in the same experimental set-up, the liposomes containing 2 mg/ml of beclomethasone are aerosolized, 50% of al particles have MMAD around 0.4 ⁇ . Only 15% are larger tha 2 ⁇ , with 50% equal or smaller than 0.4 ⁇ .
  • Andersen cascade impactor is obtained from Andersen Ai Sampler Inc., Atlanta, GA; QCM Cascade impactor is obtaine from California Measurements, Sierra Madre, CA.
  • Single-us ultravent nebulizer is obtained from Mallinckrot, St. Louis, MO, and Respigard II nebulizer is obtained from Marquest, Englewood, CO.
  • Parameters followed for aerosolization were percent of drug recovery, nebulization or aerolization rate, MMAD, percent alveolar deposition relative to total nebulizer volume and analyses of fractions in nebulizer, throat, Y- joint, stages and down stream submicronic filter.
  • FIG. 5 shows the plasm radioactivity of 14C-BDP following intratrachea instillation of free C-BDP and intratracheal instillatio of 14C BDP encapsulated i.n nonconventi.onal liposomes. Whil the free BDP is quickly removed from the lungs into plasm and metabolically eliminated, the rate of release of th liposomal BDP into the plasma is much slower. Th concentration of 14C-BDP i.n plasma i.ni.ti.ally increases probably due to presence of some percentage of free BDP
  • BDP BDP; and 50:40:10 mole % with 0.035 mg/kg of BDP wa compared with the free BDP administered intravenously an with one formulation of conventional liposomes containin sodium cholesterol sulphate/egg phosphatidylcholine/ 14C-BD in ratio of 30:60:1.2 mole % with 0.007 mg/kg of BDP (Figur
  • the amount of drug present in the lungs at time zero (T 0 ) varied among formulations and was 90 - 48% for these nonconventional liposomes, although in vitro measurements by membrane exchange assay did not detect any free drug in the formulations. This would indicate that there are rapidly and slowly released pools of drug within each liposomal formulation.
  • the absorption kinetics was determined by measuring of percentage of C BDP remaining in the lungs following the intratracheal instillation of the above described five liposome formulations and one intravenous administration of free drug. In less than thirty minutes, 99.7% of free C-BDP was removed from the lungs and 98.8% of the BDP encapsulated in conventional liposomes. In contrast, only 20% of radioactivity of BDP encapsulated in the best nonconventional liposomes wa removed from the lungs with 23% of radioactivity still bein present at 180 minutes. The other three nonconventiona liposome formulations also should sustain release of th steroid for the same time.
  • the plasm concentration curve of Figure 7 reflects the sum o concentrations due to "free” and "encapsulated” drug. Th concentration time curve for "free” drug was estimated b assuming 27% of the dose was immediately absorbed an followed the kinetics observed for i.v. administration o free BDP. This curve was subtracted from the experimentall observed data to give an estimate of the plasm concentration due to liposomal sustained-release BDP (Figur 7) . It is clear that the plasma concentration versus tim curve for the cholesterol/cholesterol sulfate formulation differ substantially from those observed following i.v. an conventional EPC liposome administration of BDP ( Figure 4) .
  • the cholesterol ester salt and cholesterol are mandatory components of the nonconventional liposome formulation and are not interchangeable with a phospholipids, normally used in conventional liposome compositions.
  • the cholesterol is primarily responsible for, and greatly affects the sustained release, but the in vivo drug-release half life depends on the relative amount of cholesterol sulfate and on the absolute presence of cholesterol in the composition. Drug release half life can be varied accordingly.
  • liposome composition containing egg phosphatidyl choline: cholesterol sulfate:BDP (60:30:10) has a drug-release half life in vivo only slightly lower than the conventional liposomes without cholesterol sulfate salt or the free drug, but it has pronounced drug retention in vitro compared to compositions without cholesterol sulfate (Example V and Table II) .
  • nonconventional liposome compositions containing sodium cholesterol sulfate:cholesterol: BDP, (50:40:10; 55:40:5; 53:37:9 mole %) gave markedly delayed release in vivo of the drug when instilled in the respiratory tract of an experimental animal together with having much prolonged drug retention as compared to the retention of the free drug and conventional liposomes ( Figure 6) .
  • th nonphospholipid liposomes Therapeutic applications and advantages of th nonphospholipid liposomes are numerous. Sustained releas of the steroid from the nonconventional liposomes prolong a therapeutic activity after each administration, reduce the frequency of administration, further improves the rati of localized-to-systemic effects, and provides increased an extended local therapeutic effect in the lungs, deep lung joints, brain, spinal cord, blood, muscles, skin, mucosa tissue, eye, and other organs or antineoplastic effect o tumors.
  • a sustained release formulation will reduc the amount of drug absorbed by the oral ucosa (due to th salivary action which would more likely clear aqueou liposome suspension into G.I.) thus reducing the incidenc of oral or mucosal tissue infection following inhalation, nasal or peroral therapy.
  • the formulation will be delivered into the airways from which it can be slowly and continuously released to the bloodstream and can treat the inflammation of the airways, bronchitis, pneumonia, or tracheal allergic or infectious or inflammatory infections and conditions.
  • the formulation will deliver the steroidal drug into the brain, to the central nervous system and spinal cord. Such delivery is important for treatment of acute, subacute, or recurrent meningitis, encephalitis, aseptic meningitis, brain abscesses or spinal cord neoplasms.
  • Intraarticularly the formulation will be injected into joints for treatment of arthritis, gout, Lyme arthritis, osteoarthritis where the systemic cortiscosteroidal treatment is specifically contraindicated but intraarticular injections are beneficial albeit unpleasant and uncomfortable.
  • the antiarticular injection of the nonconventional liposome steroidal formulation will avoid repeated injections because the formulation will be able to release the needed amount of steroid from the liposome for prolonged period of time.
  • spray dried or lyophilized liposomes containing steroid are diluted with 0.9% sterile saline and the'suspension placed, after mixing, in a Mallinckrot Ultravent nebulizer and the aerosol is breathed until there is no more liquid in the nebulizer.
  • a typical volume of nebulized solution, deliverable over 10- 30 minutes time period is 1-2 ml. Consequently, the ideal aerosolized liposome-steroid suspension contains from 0.2-2 mg of steroid per ml of the nebulized solution.
  • one inhalation dosage daily is sufficient to provide a daily needed dosage of steroid for treatment of interstitial diseases of lung.
  • the dosage with the same, larger or smaller amounts of the drug may be administered to a patient according to a treatment regimen prescribed by a physician.
  • the examples for providing the data and evaluating th novel composition in this application use primarily th antiinflammatory steroid beclomethasone dipropionate, cortisone or hydrocortisone with inhalation, intravenous intrathecal, intraarticular or topical routes o administration.
  • the scope of the invention is not limited to BDP, cortisone or hydrocortisone as a steroid, nor th described routes of administration, but are intended t include all other steroids named below and all other route of administration.
  • the invention is applicable, more broadly, to al steroids such as dexamethasone, aldosterone, betamethasone, cloprednol, cortisone, cortivazol, deoxycortone, desonide, dexamethasone, estrogenes, difluorocortolone, fluclorolone, fluorocortisone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluorocortolone, fluorometholone, flurandrenolone, halcinonide, hydrocortisone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, triamicinolone, testosterone or their respectiv pharmaceutically acceptable salts or esters.
  • al steroids such as dexamethasone, aldosterone, betamethasone, cloprednol, cortisone, cortivazol, deoxycortone, desonide, de
  • salts refer to salts such a chloride, bromide, iodide, sulfate, phosphate, nitrate, acetate, propianate, glycolate, pyruvate, oxalate, malate, maleate, malonate, succinate, cimamate, mendelate, salicilate, sulfonate, and the like.
  • esters such as for example metyl ester, etyl ester, butyl ester, bexyl ester, octyl ester or dodecyl and the like.
  • bronchodilators such as metaproterenol sulfate, aminophylline, terbutaline, albuterol, theophyline, ephedrine, isoproterenol, bitolterol, pirbuterol, adrenaline, norepinephrine, procaterol, and salmeterol
  • antiinflammatory steroids such as BDP, dexamethasone, prednisolone, hydrocortisone, fluoromethasone, medrysone, fluticasone, triamcinolone, and flunisolide
  • anticholinergics such as atropine methyl nitrate, ipratropium bromide, (4) mast cell stabilizers.
  • cardiovascular compounds including cromolyn sodium and nedocromil, (5) cardiovascular compounds, (6) oncology drugs for treatment of lung cancer such as, bleomycine, azathioprine, doxorubicin, daunorubicin, cy ⁇ lophosphomide, vincristine, etoposide, lomustine, cisplatin, procarbazine, methotrexate, mitomycin, vindesine, ifosfa ide and altretamine, (7) antiviral drugs, including acyclovir, azidothymidine, ganciclovir, enviroxime, ribavarin, rimantadine and amantadine; (8) antibiotics including penicillin, erythromycin, tetracyclin, cephalothin, cefotaxime, carbenicillin, vancomycin, gentamycin, tobramycin, piperacillin, moxalactam, cefazolin, cefadroxil, ce
  • the increased retention of the drug in the liposomes can be exploited in any type of delivery systems, such as inhalation, parenteral, intravenous or topical steroid administration and devices in solid, liquid, aerosol, nebulized, cream or spray form.
  • the liposomal composition of the invention can be prepared and delivered in a number of ways.
  • the delivery is achieved by (a) aerosolization of a dilute aqueous suspension by means of a pneumatic or ultrasonic nebulizer, (b) spraying from a self-contained atomizer using a propellant solvent with suspended, dried liposomes in a powder, (c) spraying dried particles int the lungs with a propellant, or (d) delivering drie liposomes as a powder aerosol using a suitable device.
  • the improved retention of the drug in the liposomes ca be advantageously exploited in intravenous or topica delivery systems in devices, intravenous infusions injections, capsules, cremes, drops, lotions, ointments.
  • I can be used for treatment of infection, inflammation, injury, or diseased conditions involving eye or skin wounds, rheumatoid arthritis, joint inflammation, allergi reactions, hormonal disturbances, asthma, emphysema, intestinal fibrosis, respiratory disease syndrome, cardiovascular disorders, infections, and other inflammator conditions and allergic conditions.
  • the composition of the current invention shows a grea stability, thus increasing a shelf-life of the drug fo extended period of time for up to one year.
  • liposome delivery system An added benefit to the liposome delivery system is tha it can be used for combination therapy. For instance, i certain asthmatic conditions, a steroid is used fo antiinflammation, or as antiallergenic agent while bronchodilator is needed to relax the bronchial muscle an expand the bronchial air passages. Both can be incorporate in the liposomes for slow release. Antibiotics, antivirals, antiallergens, vitamins, nutrients, or any other water- soluble compound can be used when dual therapy is needed to counteract the immunosuppressive characteristics of steroids.
  • Liposomes were formed by modified thin film hydration method according to BBA, 691:227 (1982). Unlabeled BDP
  • the MLVs formed were heterogeneous in size between about 0.05 to 20 microns, and a predominance of multilayered structures. These liposomes were extruded through a 0.4 or a 0.2 micron polycarbonate membrane by using a stainless steel extrusion cell (Lipex Biomembrane, Inc., Vancouver, British Columbia, Canada) to produce uniform homogeneous size distribution and to remove free drug crystals.
  • a stainless steel extrusion cell Lipex Biomembrane, Inc., Vancouver, British Columbia, Canada
  • formulation C Using the procedure of Section A, 10 mole % o BDP, 60 mole % of egg phosphatidylcholine and 30 mole % o cholesterol sulfate was formulated as formulation C.
  • Table I illustrates the encapsulation values an efficiency of various conventional and nonconventiona liposome formulations.
  • EPC:EPG:BDP (96:3:1.3)
  • B. EPC:BDP (98:2)
  • C. EPC:BDP (95:5)
  • PHEPC:BDP (99:1)
  • EPC:LEPC * :BDP (90:8:2)
  • EPC:CHS0 4 :BDP 60:30:10)
  • CHS0 4 :CH:BDP (50:40:10)
  • CHS0 4 :CH:BDP (55:40:5)
  • CHS0 4 :CH:BDP (50:40:10)
  • DLPC and DLPG refer to dilauroyl phosphatides.
  • LEPC refers to lyso egg phosphatidylcholine.
  • Initial drug/lipid ratio refers to percent mol fraction of the drug used in the formulation.
  • the final drug/lipid ratio means mole % fraction of drug in liposomes after formulation and removal of free drug not associated with liposomes.
  • the encapsulation efficiency shows the amount of the steroidal drug which can be encapsulated in various nonconventional (I-L) or conventional (A-H) liposomes.
  • I-L nonconventional
  • A-H conventional liposomes.
  • the conventional phospholipid containing liposomes can have rather high encapsulation efficacy with respect to limited amount of drug used in the formulation.
  • final drug/lipid ratio shows that only 2 mole % of drug could be incorporated into these liposomes at total lipid concentration of 40 umole/ml.
  • Nonconventional liposome formulations prepared as described in Example III below show high encapsulation efficiency at high drug concentration.
  • the overall encapsulation of steroid in nonconventional liposomes was around 100% even when 10 mole % drug was used in the formulation with requirement for the amount of lipid approximately 10 times lower than for conventional liposomes.
  • Beclomethasone dipropionate phospholipid liposome formulations were tested for their release behavior in an in vitro and in vivo exchange with membrane systems as described in Examples V and VI.
  • a mixture of partially hydrogenated egg phosphatidylcholine (PHEPC IV-40, 1.98 mmol), and steroid (BDP, 0.02 mmol), in the mole ratio of 99:1 was spiked with radioactive label as in Example I.A. and dissolved in 100 ml of Freon 11 containing 1.0 ml of ethanol.
  • Liposomal BDP dispersion was formed by slowly injecting the lipid/drug/freon solution into 50 ml of the phosphate buffered saline pH 7.4 under the following conditions: Injection rate: 1.25 ml/min; Vacuum: 400 mm Hg; Temperature: 20°C; Mixer rate: 1000 rpm.
  • U C-BDP used as a marker in formulations was obtaine by conversion of C sodium propionate (1 mCi, Sp. Act. 5 mCi/mmol) to propionic anydride which was used to acylat nonlabeled beclomethasone in the presence of acylatio catalyst dimethylaminopyridine.
  • Steroidal drug BDP (10 mole%) and lipid cholesterol sulfate (50 mole%) and cholesterol (40 mole%) i amounts (40 u mole/ml per liposomal formulation) were dissolved in 10 ml methanol:chloroform (2:1), added to screw-cap test tube and dried under nitrogen. The proceedur was repeated three times and the dried film was lyophilize for half an hour at room temperature.
  • the residue was resuspended i about 2 to 5 ml of phosphate buffered saline (pH 7.4, mOsm 295, originally preserved with sodium azide) and sonicate with a bath sonicator (Model G112SP1T, 600 volts, 80 KC, .0 Amps) for half an hour to prepare multilamellar vesicle (MLVs) .
  • An aliquot of the sonicated, pre-extruded MLVs sample was saved and volume of preparation recorded for determination of baseline values.
  • Liposomes were then extruded with a stainless steel Cullis high pressure extrusion cell one time through a 8.0 um Nucleopore polycarbonate membrane and two times through a 0.4 um Nucleopore polycarbonate membrane at ⁇ 500 psi using the extrusion method described in U.S. Patent 4,737,323.
  • a post-extrusion sample was saved to determine the amount of drug or lipid lost in the sizing process. Post- extrusion volume was noted. Free drug, if any, was removed by repeated washing with phosphate buffered saline and centrifugation. Liposomes were centrifuged three times on the Beckman L8-70M Ultracentrifuge at temperature of 4°C, at 47,600 rpm, for 1 hour, using 50 Ti rotor. The supernatant was discarded and the pellet resuspended in a volume equal to the post-extrusion volume after each centrifugation. The cleaned sample obtained by resuspending the pellet after the third centrifugation was labeled as T 0 sample. This sample was saved to determine percent encapsulation.
  • dexamethasone, hydrocortisone, prednisolone, fluoromethasone, medrysone, and all other steroids are similarly formulated in nonconventional liposomes.
  • EXAMPLE IV Encapsulation Efficiency and Stability This example illustrates lipid compositions screened by varying the level of drug BDP, by determining the amount of the drug incorporated into the liposomes i.e. drug encapsulation, and by monitoring the stability of drug that remains associated with liposomes over time (Table I) .
  • Multilamellar vesicles were formed containing
  • the samples were washed and centrifuge several times to remove the free drug that is not associate with the liposomes according to Examples 1-3.
  • the vesicles were visually examined under a ligh microscope to detect the presence of drug crystals. N crystals were observed after encapsulation of steroidal dru BDP into nonconventional liposomes. Conventional liposome had to be washed to remove the excess of the drug befor they were microscopically clear of crystals. In additio BDP incorporation was low.
  • the level of incorporation of the drug in th liposomes was determined based on radioactive counts an expressed as encapsulation efficiency as shown in Table I.
  • the stability of the incorporated steroidal drug i the liposomes was followed for several days to severa months.
  • liposome sample obtained above were further diluted with PBS at pH 7.4 (1:5 v/v) and incubated at ambient temperature. Time aliquots were withdrawn and pelleted by centrifugation (19,000 rp , 4°C, 30 min) . The supernatant and pellets were monitored fo the presence of lipid and drug.
  • the amount of drug remaining in the liposomes after three days to three months was determined to assess the stability of the incorporation. Very little, if any, of the steroid leaked out of the nonconventional liposomes after three days indicating that the incorporation was very stable at ambient temperature.
  • Nonconventional liposomes also showed no crystals after three months of storage at 4°C by light microscopy.
  • Conventional liposomes although appearing stable for 3 days at ambient temperature in buffer solutions, lost readily their drug content during the longer period of storage and/or in the presence of an acceptor membrane.
  • Conventional liposomes such as A-G (Table I) even though they showed no crystals after 3 months at 4°C, readily lost the drug content both in vitro in the presence of a membrane reservoir (Table II) and in vivo.
  • EXAMPLE V In Vitro Membrane Exchange Assay This example illustrates the sustained release from the nonconventional liposome formulations prepared according to the current invention.
  • BDP as a steroid poorly soluble in water, and is primarily entrapped in the lipid bilayer rather than in the aqueous core of liposomes. Thus, very little of the drug can be released into a surrounding aqueous environment unless a huge volume of buffer is used based on partitioning characteristics of the drug. Since BDP has good solubility in phospholipid membranes, liposomal BDP may be rapidly exchanged from the bilayer of liposomes to surrounding cell membranes in the lung. To mimic the cell membranes in the lung, in vitro system was set up using small unilamelar vesicles (SUVs) .
  • SUVs small unilamelar vesicles
  • the trachea was cannulated with a 4 cm long section of Teflon tubing (1.2 mm I.D.), inserte at the level of the fifth tracheal ring below the thyroi cartilage and tied in place with a suture. Excess fluid i the trachea was aspirated through tubing attached to syringe. A 0.5 ml glass syringe with a blunt needle an short length of polyethylene tubing attached was used t administer the formulations. The tubing was inserted to th level of the bronchial bifurcation and the dose (100 to 40 ul) rapidly administered during an inhalation. Animals were supported head up on a tilted dorsal support (approximatel 70°) during the instillation process.
  • BDP was carried out by liquid scintillation counting. The actual dose administered in each study was determined by measurement of duplicate dose control samples of the formulation which were delivered by the same apparatus used in dosing the animals.
  • the pharmacokineti ⁇ parameters of free BDP were determined following intravenous administration of 14C-BDP (0.008 mg/kg in 50% aqueous ethanol) to a group of 12 rats. Plasma and lung levels of radiolabel were measured as previously described. The decrease in plasma concentration versus time following free drug administration was biphasic ( Figure 4) . These data were subjected to analysis by a non- linear least squares curve fitting program (RSTRIP, MicroMath, Salt Lake City, UT) and the resulting exponential slopes and intercepts interpreted according to a two compartment open pharmacokinetic model.
  • RSTRIP non- linear least squares curve fitting program
  • EPC/cholesterol sulfate liposomes were virtually identica to those observed following the i.v. administration of similar dose of free drug (Figure 4).
  • the amount o radiolabel remaining in the lungs after 35 minutes was onl
  • the present study shows that the lipophilic steroid beclomethasone dipropionate can be successfully incorporated into a nonconventional liposomal formulation that provides sustained in vivo release of the drug following intratracheal instillation.
  • Table IV illustrates the in vitro and the in vivo exchange of conventional and nonconventional liposomes.
  • Example VII Preformulation Studies This example determines the localization of the steroid in the liposomal structure and illustrates the steroid's water insolubility.
  • Beclomethasone dipropionate is a lipophilic drug.
  • the solubility of the drug in different solvents is listed below in Table V: Table V Solvent Solubility
  • the partition coefficient for beclomethasone dipropionate between octanol and phosphate buffer saline was determined at pH 7.4. Nearly all (95%) of the BDP was associated with the octanol. This indicates that the drug will most likely reside in the membrane core of the bilayer.
  • EXAMPLE VIII Intrathecal Administration of Liposomal Steroids This example illustrates the intrathecal administration of the steroids formulated in nonconventional liposomes.
  • the treatment is useful for boosting the effect of antibiotic or other treatments in severe sepses, blood poisoning, meningitis, brain inflammations and infections or other conditions when the immediate and prolonged administration of the steroid is indicated.
  • the rats are divided into one experimental and one control group.
  • the experimental group is injected with 50 ul of liposomal cortisone composition of 50 mole % of cholesterol sulfate, 40 mole % of cholesterol and 10 mole % of cortisone (spiked with radioactive cortisone) prepared according to procedure of Example III.
  • the control group is injected with 50 ul of the free cortisone 20 mg/ml suspended in 0.9% NaCl. Injection is done over 25 minutes using a syringe infusion pump. At the end of the injection, the needle is removed and the skin defect is closed with a surgical staple.
  • the brain in the cranial cavity is lifted out with a spatula and the cranial cavity is washed out thoroughly with a 0.9% NaCl solution to collect all drug remaining in the cranial compartment.
  • the spinal cord is extruded forward into the cranial vault by inserting in the rostral direction a 19 gauge hypodermic needle in the low lumbar spinal canal at a point 2.5 cm rostral to the origin of the tail and then pushing 0.9% NaCl solution into the canal at high pressure.
  • the empty spinal canal is then washed thoroughly with 0.9% NaCl solution to collect all the drug in the spinal canal.
  • the brain compartment specimen is collected separately from the spinal specimens.
  • the specimens are homogenized on ice with distilled water using a Dounce manual tissue grinder, sonicated to disrupt intact liposomes and filtered through the ultrafiltration membrane (YMT membrane) .
  • the ultrafiltrates are analyzed with HPLC.
  • the amount of the drug is measured in cranial and spinal compartments and in cerebral fluid.
  • the liposomal steroid remained in the cranial or spinal compartments for as long as 24 days while the free drug almost completely disappears within the first 2.8 hours.
  • the free drug appears immediately in the high concentration and is quickly eliminated from the cerebrospinal fluid within the 2.8 hours.
  • the liposomal cortisone on the other hand remains in the cerebrospinal fluid for as long as 16 days.
  • This example illustrates the use of the nonconventional steroidal liposomes for treatment of arthritis, rheumatoid arthritis, tendonitis and other inflammatory diseases of the joints by injection of liposomal steroid into joints.
  • New Zealand rabbits of 2.5 to 3.5 kg are shaved around the joints of both hind legs. Between 8 and 9 a.m. the right joints received an intra-articular injection of 0.5 ml of freshly prepared nonconventional liposomes with encapsulated hydrocortisone (3 mg) the left joints are injected with 0.5 ml physiological saline as control. Blood samples are collected from the ear veins at timed intervals.
  • the rabbits are kept in metabolism cages.
  • the rabbits are anesthetized 24 or 48 hours after the i.a. injection and the joints rinsed with 2 ml of physiological saline ("synovial fluid") . Then they receive 1 ml of Disulphine BlueTM 6.2 per cent s.c, between the toes of both hind paws to stain the lymph nodes.
  • synovial fluid physiological saline
  • Disulphine BlueTM 6.2 per cent s.c between the toes of both hind paws to stain the lymph nodes.
  • At death total synovectomy of both joints is carried out, the heads of the femur, fibula and tibia and the popliteal lymph nodes excised.
  • the tissues were grouped as follows: (1) menisci, tendons, ligaments and cartilage scraped off from femur, fibula, tibia, patella and fabellas were put together and named as “menisci etc.”, (2) “synoviu “, (3) “patella and fabellas”, (4) "femur, fibula and tibia-heads", (5) "bone marrow” which was scraped out from femur, fibula and tibia, (6) popliteal "lymph nodes”. Menisci etc.
  • synovium and bone marrow are digested with Packard Soluene 350, the bones with concentrated HC10 4 /H 2 O 2 (l:2 v/v) .
  • Aliquots of whole blood, plasma, synovial fluid and after centrifugation and the digested materials are subjected to liquid scintillation counting. In the fresh synovia the number of cells is counted and cell differentiation is carried out.
  • Urine and faeces are collected daily, aliquots of urine are subjected to liquid scintillation counting directly, aliquots of faeces after digest with Packard Soluene 350.
  • Nonconventional preparations give indistinguishably low plasma levels of radiolabel for 48 hour post i.a. injection. The levels range from 0.001 to 0.3 per cent of the dose in the whole rabbit blood.
  • the liposomes have shown the long term 150 hours retention of radiolabel in synovial fluid and therefore in the whole joint.
  • a bilateral arthritis is induced by the intraarticular injection of a preformed insoluble complex of poly-D-lysine and hyaluronic acid into both knee joints (Shaw et al., 1979) .
  • Liposomes (0.5 ml) containing the cortisol (3 mg) encapsulated in nonconventional liposomes prepared according to Example III and conventional liposomes prepared according to Example I, are injected into one knee joint 4 days after the induction of the arthritis. The temperatures and diameters of injected and contralateral joints is monitored for 3-5 days after treatment. In the treatment of an acute inflammatory "flare" superimposed on a chronic arthritis, liposomes are injected 4 days after induction of the acute episode.
  • Liposome Steroid Composition This example illustrates the use of the nonconventional steroid liposomes for intravenous treatment of various diseases requiring steroidal treatment.
  • the nonconventional steroidal liposome composition prepared according to Example III was administered intravenously and the ability of mouse organs to bind and/or take up the radiolabel was studied. Liposome formulation was concentrated, if necessary, by adding one volume of 50%
  • Example III were administered to a mouse via a tail vein.
  • mice were lightly anesthesized with diethyl ether and a 1 ml blood sample was rapidly removed from the jugular vein with a heparinized syringe. Livers and spleens were subsequently removed, weighed and set aside for analysis together with the remaining carcass. An attempt was made to remove and discard the bladder and its content from each carcass prior to storage. Three types of studies were performed as follows.
  • mice Three groups of three experimental mice were each given 0.2 ml phosphate-buffered saline containing free radioactive spike beclomethasone intravenously via a tail vein. Mice receiving imperfect injections were discarded. Groups of three mice were killed at 1, 5 and 24 hours after injection and their organs sampled as described above in order to determine the clearance of free beclomethasone.
  • Plasma concentration of free and encapsulated beclomethasone in nonconventional liposomes was determined. The results are shown in Figure 7. Free beclomethasone disappears rapidly from the plasma, while the beclomethasone encapsulated in nonconventional liposomes remains circulating in the plasma as liposome plasma reservoir from which the amount of steroid is slowly released to the circulation.
  • EXAMPLE XII Intraperitoneal Administration of Nonconventional Liposome Steroid Composition This example illustrates the use of nonconventional steroid liposomes for intraperitoneal treatment of internal inflammatory diseases.
  • mice Male Sprague-Dawley rats weighing 250-300 grams are used. Each rat is given an oral dose of soybean oil (4.0) mL/kg) and 1 hour later anesthetized with an injection of urethane (1.2 g/kg sc) .
  • a polyethylene cannula (PE 10, Clay Adams) is inserted into the thoracic duct proximal to the juglosubclavian junction according to the method of Saldeen and Linder, Acta. Path.. 49:433(1960).
  • Another cannula (PE- 50) is put into the left femoral artery, and a third is used to cannulate the urinary bladder. The anesthesia is maintained for the duration of the study.
  • the rat is placed on a plate and kept at 37°C in a supine position. Fluid balance is maintained with a 4 mL/h/kg infusion of saline containing 2.5 U/mL of herparin via the arterial cannula.
  • the test liposome steroid formulation according to Example III or free drug suspension (5 mM in phosphate-buffered saline, 2 mL/kg) is administered intraperitoneally 30 minutes after surgery. Lymph and urine are collected continuously. Blood is sampled periodically over the 5-h study period.
  • rats are sacrificed and the peritoneal cavity is rinsed with at least 60 mL of saline and 20 mL of 1% Triton X-100 to recover unabsorbed liposomes and marker.
  • saline aline
  • Triton X-100 1% Triton X-100
  • the molecular weight cut-off of the peritoneal- vascular permeability barrier is determined using sucrose, inulin, and fluorescein isothiocyanate (FITC) dextrans of molecular weights 20,000, 70,000, and 150,000; these are dosed intraperitoneally as phosphate-buffered saline solutions (sucrose and inulin at 5 mM; FITC dextrans at 20 mg/mL with 2 mL/kg injected) .
  • sucrose, inulin, and fluorescein isothiocyanate (FITC) dextrans of molecular weights 20,000, 70,000, and 150,000; these are dosed intraperitoneally as phosphate-buffered saline solutions (sucrose and inulin at 5 mM; FITC dextrans at 20 mg/mL with 2 mL/kg injected) .
  • FITC fluorescein isothiocyanate
  • This example illustrates the use of the nonconventional steroidal liposomes for topical treatments.
  • the nonconventional steroid liposome radiolabeled composition according to Example III was used.
  • the hamster flank organ test was carried out according to the method described in Endocrinology. 92:1216- 1222 (1983).
  • the female hamsters are separated into six groups of five animals and treated according to the following scheme.
  • Group I 4 ug fluocinolone (dissolved in acetone) ;
  • Group II 20 ug of free fluocinolone (dissolved in acetone) ;
  • Group III 40 ug fluocinolone (dissolved in acetone)
  • Group IV 4 ug fluocinolone (encapsulated in nonconventional liposomes) ;
  • Group V 20 ug fluocinolone (encapsulated in nonconventional liposomes) ;
  • Group VI 40 ug fluocinolone (encapsulated in liposomes)
  • Group VII 40 ug fluocinolone (encapsulated in liposomes) .
  • the animals are treated once daily (five days a week) . After 28 days of treatment the flank organs are excised for morphometrical and histochemical examination according to the method of Goos et al.. Arch. Derm. Res..
  • This example illustrates preparation of mixed micelles containing steroid beclomethasone dipropionate.
  • the filtrate is then poured into nebulizer vessel and nebulized to generate microaerosol particles according to Example XV.
  • the same procedure is used but the initial amount of
  • Tauranol is A. 750 mg (15 mg/ml) or B. 1.5 g (30 mg/ml), with 450 mg (9 mg/ml) of sodium chloride. The amount of drug is the same and the volume is made up to 50 ml.
  • N.D. means not determined
  • BDP and Tween 20 was prepared in amounts and with recover amount shown in Table VII.
  • micelle solution of BDP and poloxamer was prepared in amount as shown in Table VIII.
  • This example illustrates in vitro testing of aerosolized liposome BDP formulation.
  • Example 4 ml of presized (0.02 ⁇ ) nonphospholipid liposome (or adequate volumes of the liposome formulation to provide known amounts of drug diluted to a total volume of 4 ml with sterile saline or suitable diluent) obtained in Example 4 or mixed micelles of Example 8 containing BDP were placed in single-use Mallinckrot Ultravent nebulizer and the compressor pump was attached according to Figure 10.
  • the compressed air generated by the compressor is introduce into the nebulizer and agitates the liposomal or micella solution into mist of aerosolized particle droplets. These droplets enter via the valve into Andersen cascade impactor. The flow velocity of air through the impactor is adjusted to 28 L/minute.
  • the aerosol mist is separated into preseparator chamber and into segments 0-7.
  • the preseparator, and each stage of segments 0-7 are separated from each other by stages with decreasing sizes of pores.
  • the aerosolized particles are then forced by the compressor to enter the impactor and are selectively deposited on the glass slides place on the stages when their sizes are bigger than the pores of that stage or pass through the to the next stage until they are deposited on the glass slide.
  • the sizes of stages corresponding to various segments of the lungs are shown in Figure 11.
  • Equipment (Andersen Cascade impactor) is set-up as shown in Figure 10 after ascertaining that all sections of the equipment are clean.
  • a blank trial is run with 4 ml of deionized water placed in the nebulizer to determine how long it takes to nebulize 1 ml.
  • the flow meter is adjusted to allow the air flow rate of 28 1/min.
  • 4 ml of liposome or micelle suspension (or diluted formulations in appropriate cases) is introduced into the nebulizer and nebulized to deliver approximately 1 ml of the material.
  • Y-side arm, throat including mouthpiece, and all glass slides from stages of the impactor and submicronic aerosol filter are disconnected and removed.
  • BDP concentrations were determined spectrophotometrically for the nebulizer solutions before and after aerosolization and on the aerosol output deposited in the stages and Y-side arm.
  • the amounts of drug (BDP) in each location was calculated and the material balance was verified as follows:
  • D j Drug initially introduced into the nebulizer in ⁇ g or mg.
  • V j Initial volume of solution in nebulizer
  • D F Drug present in the nebulizer residue
  • V p Final volume of solution in nebulizer
  • D s ⁇ - 8 Drug deposited on the seven stages and the fine or submicronic aerosol particle filter (S 8 ) D p could be very high, implying that only water was aerosolized preferentially and "crystalline" drug remained in residue. This may very well be the case with BECOTIDE ® .
  • Phospholipid concentrations are determined similarly to verify that there is a comparable material balance with lipid as well.
  • the drug/lipid ratio in the initial and final solutions in the nebulizer with liposomal formulations is checked.
  • Ratio should remain constant. Any deviation points to drug crystallization and therefore unavailability fo aerosolization.
  • Liposome particle size of the nebulization solutions ar measured before and after aerosolization experiments usin the NiComp laser particle sizer. Liposome particle sizes i the collected aerosol are also measured.
  • Typical results obtained with Cascade Impactor Analysis of commercial Becotide ® formulation and the liposomal BD formulation are summarized in Table XI.
  • a graphic plot of this data by standard methods gives the mass median aerodynamic diameter (MMAD) of the aerosol droplets (Figure 12, 13).
  • Drug/phospholipid deposition on stages and throat is used to extrapolate how much of the nebulizer output will reach "alveoli” assuming that aerosol particles of MMAD 0.02-2.1 ⁇ can be deposited in deep lung.
  • the Becotide suspension generates larger particles and more significantly only 3.2% of the aerosolized dose reaches stages 5, 6, 7 (Table IX) corresponding to alveolar region.
  • liposomal aerosol droplets have very small size (MMAD 0.4 ⁇ ) and deliver 28% of aerosolized dose in stages 5, 6, 7 (Table IX) corresponding to alveolar region. Results are summarized in Table IX.
  • This example illustrates the clinical protocol and results of treatment of patients suffering from ILD.
  • T lymphocytes of the inflamed lung are the sources of the pathology in their release of interleukin (IL2) and T cell growth fact or and their ability to continuously proliferate making lung an immune organ (Spencer, H. Pathology of the Lung r (1985) . New Engl. J. Med.. 308 793 (1983), Rev. Respir. Pis.. 128:634 (1983)).
  • Active pulmonary sarcoidosis therefore, simulates a relevant model to evaluate, in humans, the in vitro findings that corticosteroids can suppress the level of the ly phokine IL2 and T lymphocyte proliferation.
  • bronchoalveolar lavage to sample activated T lymphocyte population, it is possible to monitor the effects of corsticosteroid formulations of the present invention in suppressing IL-2 levels (either by protein monitor using antibody or at genetic level using mRNA probes) and their ability to reduce cell proliferation in cell culture.
  • the following experimental protocol was designed to monitor the disease course in patients with control population of normal volunteers.
  • volume recovered ranges from 55-70 ml.
  • the lavage fluid was passed through 2 layers of sterile gauze and cells were pelleted at 250 g for 5 minutes and washed twice in RPM 1640 (M.A. Bioproducts, Walkersville, M.D.) and then resuspended to a concentration of 10 7 cells/ml before use.
  • Monoclonal antibodies were all obtained from Becton Dickinson, Sunnyvale, CA. T cells were identified by the monoclonal antibody Leu-4 (CD 3 ) . Helper inducer T lymphocyte was recognized by monoclonal Leu-3. (CD4) and Suppressor cytotoxic T subtype was identified by Leu-2 (CD8) .
  • Antibodies were FITC labeled. Unstained preparations were used as control to assess auto fluorescence and non relevant mouse IgG subclasses were used as controls for nonspecific binding. Stainings were done in microtiter plates by standard methods. Because alveolar macrophages tend to clump, each sample was diluted in 400 ⁇ l of staining buffer prior to analysis. Forward light scattering was used to analyze macrophages from lymphocytes first. Limiting gates were set on lymphocyte peak.
  • a fraction of lung mononuclear cells at a concentration of 10 6 /ml was cultured in RPM11640 with 1% fetal Calf Serum for 48 hours. After this time period, supernatants were obtained by centrifugation and stored at -20°. Supernatants were assayed for the biochemical marker IL2 by their ability to stimulate H 3 thymidine incorporation in murine IL2 dependent CT-6 cells. Results are expressed as IL2 units by comparison with a standard. Quantitation uses profit analysis.
  • Lung lymphocyte replication was assessed by autoradiography. Lung mononuclear cells were incubated (10 6 cells/ml) in flat-bottom microliter wells in RPM I 1640 containing 10% heat-inactivated autologous serum and H 3 thymidine (0.5 ⁇ ci, 2 ci/mmol, Amersham) for 24 hours. At this time, cytocentrifuge slide preparations were made of nonadherent cells. The cells were then fixed and extracted in methanol:acetic acid (3:l;v/v), developed for autoradiography with a ten day exposure and then stained with Wright-Giemsa.
  • Lymphocyte labeling index labeled lymphocyte x 100 total number of lymphocyte
  • Diagnosis of pulmonary sarcoidosis based on lung measurements including lung biopsy or mediastinal lymph node biopsy;
  • Active lymphocytic alveolitis (BAL data showing % lymphocytes > 30% (normal + 6%) ; Leu 3+ : Leu + > 2.7 (normal 2+ 0.3); spontaneous IL2 level >_ 5U/10 6 lung mononuclear cells (normal OU) ; spontaneous proliferation of lymphocytes in 24 hour > 4% (normal - 1%) ] .
  • 21 patients included in the study ten were treated with steroidal formulations and all individuals were monitored every month up to six months. All were monitored for pulmonary function and the biochemical markers underlying the disease namely IL2 level and spontaneous proliferation of lymphocytes collected from patients.

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Abstract

Nouvelle formulation de liposomes non phospholipidique permettant une libération et un acheminement soutenus de stéroïdes. La formulation assure une libération prolongée du médicament, un rapport thérapeutique amélioré, une toxicité plus faible, des effets secondaires systémiques réduits, et est stable pendant trois mois. Ladite formulation est adaptée pour un acheminement soutenu de stéroïde par inhalation, administration parentérale, intrarachidienne, intraarticulaire, locale, ophtalmique et orale, et convient pour un traitement des maladies inflammatoires, arthritiques, rhumatoïdes, topiques, pulmonaires et pulmonaires interstitielles.
PCT/US1989/005525 1988-12-14 1989-12-06 Nouvelle composition de liposomes non phospholipidique a liberation soutenue de medicaments WO1990006775A1 (fr)

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US284,158 1988-12-14
US07/284,216 US5043165A (en) 1988-12-14 1988-12-14 Novel liposome composition for sustained release of steroidal drugs
US07/284,158 US4906476A (en) 1988-12-14 1988-12-14 Novel liposome composition for sustained release of steroidal drugs in lungs
US284,216 1988-12-14
US444,738 1989-12-01

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0416925A2 (fr) * 1989-09-07 1991-03-13 Glaxo Group Limited Utilisation du 4-hydroxy-alpha 1-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol ou ses sels dans le traitement de l'allergie et de l'inflammation
WO1996014849A1 (fr) * 1994-11-11 1996-05-23 HAIDER, Angelika Utilisation des aminoglycosides pour le diagnostic des desordres formant barriere sang-air
WO1997048381A1 (fr) * 1996-06-15 1997-12-24 Boehringer Ingelheim Pharma Kg Preparation pharmaceutique sous forme de liposomes
WO1999027940A1 (fr) * 1997-12-01 1999-06-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Preparation pour traitement local des infections de la peau
WO2002003998A2 (fr) * 2000-07-10 2002-01-17 Chiron Corporation Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
WO2004028545A1 (fr) * 2002-09-25 2004-04-08 Astrazeneca Ab Combinaison d'un agoniste $g(b)2 a action prolongee et d'un glucocorticosteroide dans le traitement de maladies fibrotiques
EP1419803A2 (fr) * 1995-02-06 2004-05-19 Medivir Ab Formulation pharmaceutique pour l'application topique contenant une substance antvirale et un glucocorticoide
WO2006027787A1 (fr) * 2004-09-09 2006-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Compositions liposomales a base de glucocorticoides et de derives de glucocorticoides
USRE40045E1 (en) 1989-09-08 2008-02-05 Glaxo Group Limited Medicaments
US7473433B2 (en) 2000-12-21 2009-01-06 Nektar Therapeutics Pulmonary delivery of polyene antifungal agents
JP2009067771A (ja) * 2008-05-27 2009-04-02 Pacira Pharmaceuticals Inc 神経学的疾患の治療方法
WO2009094641A2 (fr) 2008-01-24 2009-07-30 Dor Biopharma, Inc. Stéroïdes actifs par voie topique destinés à être utilisés dans le cas d'une fibrose pulmonaire interstitielle
WO2014128233A1 (fr) * 2013-02-21 2014-08-28 Sigmoid Pharma Limited Formulations de stéroïdes administrées par voie orale destinées à être utilisées pour le traitement d'une fibrose intestinale
US9155700B2 (en) 2003-10-22 2015-10-13 Chiesi Farmaceutici S.P.A. Process for the preparation of pharmaceutical suspensions for inhalation
US9844513B2 (en) 2007-04-04 2017-12-19 Sigmoid Pharma Limited Oral pharmaceutical composition

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US4235871A (en) * 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes
US4693999A (en) * 1984-07-30 1987-09-15 Aktiebolaget Draco Liposomes containing steroid esters
US4780455A (en) * 1985-04-12 1988-10-25 The Trustees Of Columbia University In The City Of New York Lipophilic complexes of pharmacologically active organic compounds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) * 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4515736A (en) * 1983-05-12 1985-05-07 The Regents Of The University Of California Method for encapsulating materials into liposomes
US4693999A (en) * 1984-07-30 1987-09-15 Aktiebolaget Draco Liposomes containing steroid esters
US4780455A (en) * 1985-04-12 1988-10-25 The Trustees Of Columbia University In The City Of New York Lipophilic complexes of pharmacologically active organic compounds

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0416925A3 (en) * 1989-09-07 1991-09-25 Glaxo Group Limited Use of 4-hydroxy-alpha 1-(((6-(4-phenylbutoxy)hexyl)amino)methyl)-1,3-benzenedimethanol or its salts in the treatment of allergy and inflammation
EP0416925A2 (fr) * 1989-09-07 1991-03-13 Glaxo Group Limited Utilisation du 4-hydroxy-alpha 1-[[[6-(4-phenylbutoxy)hexyl]amino]methyl]-1,3-benzenedimethanol ou ses sels dans le traitement de l'allergie et de l'inflammation
EP1078629A2 (fr) * 1989-09-07 2001-02-28 Glaxo Group Limited Utilisation du Salmeterol ou ses sels dans le traitement de l'allergie et de l'inflammation
EP1078629A3 (fr) * 1989-09-07 2001-05-23 Glaxo Group Limited Utilisation du Salmeterol ou ses sels dans le traitement de l'allergie et de l'inflammation
USRE40045E1 (en) 1989-09-08 2008-02-05 Glaxo Group Limited Medicaments
WO1996014849A1 (fr) * 1994-11-11 1996-05-23 HAIDER, Angelika Utilisation des aminoglycosides pour le diagnostic des desordres formant barriere sang-air
EP1419803A2 (fr) * 1995-02-06 2004-05-19 Medivir Ab Formulation pharmaceutique pour l'application topique contenant une substance antvirale et un glucocorticoide
EP1419803A3 (fr) * 1995-02-06 2004-06-09 Medivir Ab Formulation pharmaceutique pour l'application topique contenant une substance antvirale et un glucocorticoide
WO1997048381A1 (fr) * 1996-06-15 1997-12-24 Boehringer Ingelheim Pharma Kg Preparation pharmaceutique sous forme de liposomes
WO1999027940A1 (fr) * 1997-12-01 1999-06-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Preparation pour traitement local des infections de la peau
WO2002003998A3 (fr) * 2000-07-10 2002-06-13 Chiron Corp Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
WO2002003998A2 (fr) * 2000-07-10 2002-01-17 Chiron Corporation Formulations a base de macrolides destines a l'inhalation et procede de traitement des infections endobronchiales
US7473433B2 (en) 2000-12-21 2009-01-06 Nektar Therapeutics Pulmonary delivery of polyene antifungal agents
WO2004028545A1 (fr) * 2002-09-25 2004-04-08 Astrazeneca Ab Combinaison d'un agoniste $g(b)2 a action prolongee et d'un glucocorticosteroide dans le traitement de maladies fibrotiques
US9155700B2 (en) 2003-10-22 2015-10-13 Chiesi Farmaceutici S.P.A. Process for the preparation of pharmaceutical suspensions for inhalation
WO2006027786A3 (fr) * 2004-09-09 2006-04-27 Yissum Res Dev Co Utilisation de glucocorticoides pour le traitement d'etats inflammatoires
AU2005281352B2 (en) * 2004-09-09 2011-01-27 Yissum Research Development Company Of The Hebrew University Of Jerusalem Liposomal compositions of glucocorticoid and glucocorticoid derivatives
WO2006027786A2 (fr) * 2004-09-09 2006-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Utilisation de glucocorticoides pour le traitement d'etats inflammatoires
US8932627B2 (en) 2004-09-09 2015-01-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Liposomal compositions of glucocorticoid and glucocorticoid derivatives
WO2006027787A1 (fr) * 2004-09-09 2006-03-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Compositions liposomales a base de glucocorticoides et de derives de glucocorticoides
US9844513B2 (en) 2007-04-04 2017-12-19 Sigmoid Pharma Limited Oral pharmaceutical composition
WO2009094641A2 (fr) 2008-01-24 2009-07-30 Dor Biopharma, Inc. Stéroïdes actifs par voie topique destinés à être utilisés dans le cas d'une fibrose pulmonaire interstitielle
EP2242477A2 (fr) * 2008-01-24 2010-10-27 Soligenix, Inc. Stéroïdes actifs par voie topique destinés à être utilisés dans le cas d'une fibrose pulmonaire interstitielle
EP2242477A4 (fr) * 2008-01-24 2011-04-27 Soligenix Inc Stéroïdes actifs par voie topique destinés à être utilisés dans le cas d'une fibrose pulmonaire interstitielle
JP2009067771A (ja) * 2008-05-27 2009-04-02 Pacira Pharmaceuticals Inc 神経学的疾患の治療方法
WO2014128233A1 (fr) * 2013-02-21 2014-08-28 Sigmoid Pharma Limited Formulations de stéroïdes administrées par voie orale destinées à être utilisées pour le traitement d'une fibrose intestinale
US9320746B2 (en) 2013-02-21 2016-04-26 Sigmoid Pharma Limited Method for treating intestinal fibrosis
CN105073127A (zh) * 2013-02-21 2015-11-18 希格默伊德药业有限公司 用于治疗肠纤维化的口服类固醇制剂
US9980902B2 (en) 2013-02-21 2018-05-29 Sigmoid Pharma Limited Method for treating intestinal fibrosis

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