United States Patent 1191
`Radhakrishnan et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,895,719
`Jan. 23, 1990
`
`[54] METHOD AND APPARATUS FOR
`ADMINISTERING DEHYDRATED
`LIPOSOMES BY INHALATION
`
`[75] Inventors: Ramachandran Radhakrishnan; Paul
`J. Mihalko, both of Fremont; Robert
`M. Abra, San Francisco, all of Calif.
`
`[73] Assignee: Liposome Technology, Inc., Menlo
`Park’ Cahf'
`
`[21] APPL No‘ 22,937
`
`22 ' Fil d
`:
`[
`1
`e
`
`M 6 1987
`ar.
`’
`
`[63]
`
`Related U.S. Application Data
`Continuation-impart of Ser- No- 737,221, May 22,
`1935’ abandoned’ and Ser- N°~ 860,528’ May 7» 1936’
`abandoned’ and Set‘ N°' 937’609’ Dec‘ 3’ 1986'
`
`[51] Int. Cl.4 ..................... .. A61K 31/35; A61K 9/14;
`A61K 9/48; A61K 9/68
`[52] U.S. Cl. .................................... .. 424/45; 514/958;
`514/959; 604/ 140
`[58] Field of Search .................. .. 424/45, 46; 514/956,
`514/958, 964, 965
`
`[56]
`
`References Cited
`
`U'S' PATENT DOCUMENTS
`3,551,558 12/1970 Takebe et al. ...................... .. 424/46
`
`3,755,557 8/1973 4,083,953 4/1978
`
`4,232,002 11/1980
`4,462,983 7/1984
`4,752,466 6/1988 Saferstein et al, .............. .. 424/45 X
`FOREIGN PATENT DOCUMENTS
`0158441 10/1986 European Pat. Off. ............ .. 424/45
`4214804 8/1965 Japan ............................... .. 424/46
`woes/01714 3/1986 PCT 1m Appl.
`424/45
`2145107 3/1985 United Kingdom
`424/45
`2166651 5/1'986 United Kingdom ................ .. 424/46
`Primary Examiner--Floycl D. Higel
`Attorney, Agent, or Firm-Peter J. Dehlinger
`[57]
`ABSTRACT
`A system and method for administering a drug, at a
`selected dose, via the respiratory tract. Spray-dried
`liposome particles containing the selected dose of the
`entrapped drug are released into the air in aerosolized
`form, either by entrainment in an air or propellant
`stream, or by release from a pressurized can containing
`3 Suspension of the liposomes in a ?uorchlorocarbon
`solvent.
`
`18 Claims, 1 Drawing Sheet
`
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`

`

`US. Patent
`
`Jan. 23, 1990
`
`V
`
`4,895,719
`
`
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`WATSON LABORATORIES, INC. , IPR2017-01622, Ex. 1024, p. 2 of 13
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`1
`
`METHOD AND APPARATUS FOR
`ADIVIINISTERING DEHYDRATED LIPOSOMES
`BY INHALATION
`
`This application is a continuation-in-part of US. pa
`tent applications for “Liposome Inhalation Method and
`System”, Ser. No. 737,221, ?led May 22, 1985, now
`abandoned “Liposome Concentrate and Method”, Ser.
`No. 860,528, filed May 7, 1986, now abandoned and
`“Liposome Inhalation Method and System”, Ser. No.
`937,609, ?led Dec. 3, 1986 currently pending.
`FIELD OF THE INVENTION
`The present invention relates to drug delivery by
`inhalation, and, in particular, to an improved system
`and method for delivering liposomes containing a me
`tered drug dose via inhalation.
`
`15
`
`25
`
`30
`
`REFERENCES
`20
`The following references are incorporated herein by
`corresponding number:
`1. Hollenbeck, R. G., et al, in “Pharmaceutics and
`Pharmacy Practice” (Banker, G. S., et al, eds), J. P.
`Lippincott, Philadelphia (1982), pp. 344-358.
`2. Szoka, F., Jr., et al, Ann Rev Biophys Bioeng (1980),
`9: 467.
`3. Szoka, F., Jr., et al, Proc Natl Acad Sci (USA)
`(1978) 75: 4194.
`4. Hollenbeck, R. G., op cit. pp. 382-391.
`BACKGROUND AND SUMMARY
`Inhalation provides an effective means for delivering
`a variety of drugs, including nasal decongestants, drugs
`useful in the treatment of asthma and other bronchial
`35
`and pulmonary conditions (1). One advantage of inhala
`tion in treating nasal, bronchial, and pulmonary condi
`tions is the ability to deliver the drug directly to the site
`of drug action. A related advantage is the rapid onset of
`the therapeutic effect, compared with other routes of
`40
`administration, such as intramuscular and oral routes.
`For drugs which are susceptible to breakdown in the
`gastrointestinal. tract, or which otherwise cannot be
`administered orally, inhalation may be preferred for a
`variety of reasons over intravenous or intramuscular
`45
`injection. Other drugs, such as nitroglycerin, whose
`primary drug action is systemic, can also be delivered
`ef?ciently by inhalation.
`Several methods for delivering drugs via inhalation
`are known. In one, the drug is dissolved in a suitable
`solvent which can be aerosolized to form a small-parti
`cle mist. The drug solution may be aerosolized by pneu
`matic or ultrasonic nebulizer, or, more conveniently, by
`means of a self-contained nebulizer containing a pres
`surized, ?uorocarbon propellant. Inhalation of the aero
`sol mist, i.e., drawing the mist from the mouth or nose
`into the respiratory tract, acts to deposit the drug-con
`taining aerosol particles on various sites of the respira
`tory tract, including the upper nasopharyngeal region,
`the tracheobronchial region, and the pulmonary region.
`In the latter region, the drug has the opportunity for
`rapid absorption into the bloodstream for systemic ac
`tion.
`Also well known in the prior art are inhalation sys
`tems in which a drug is administered in particulate form,
`either as a dry powder or as a micronized suspension in
`a suitable carrier solvent system. Typically the drug is a
`water-soluble compound which is suspended in micron
`
`4,895,719
`2
`ized form in a ?uorocarbon-type propellant solvent.
`Following aerosolization, most of the propellant sol
`vent is lost through ?ash evaporation and replaced by
`moisture in the respiratory tract, leading to the deposi
`tion of hydrated micronized particles.
`Both types of inhalation systems mentioned above are
`based on delivery of the drug in a free form to sites in
`the respiratory tract. As such, the drug is rapidly uti
`lized and, in the case of pulmonary deposition, taken up
`systemically at the site of deposition. Because of this
`rapid drug uptake and utilization, the drug effect may
`be relatively short-lived, requiring frequent dosing. A
`related problem is the limited amount of drug that can
`be administered safely at each dosing, particularly
`where the drug has unwanted systemic side effects. This
`problem is illustrated by a number of Bg-adrenergic
`agonist type brochodilators which also produce marked
`tachycardia. Even at relatively low doses of these
`drugs, the stimulatory effect of the drug on the heart
`and other side effects, such as dizziness and insomnia,
`are a nuisance to the patient. Additionally, micronized
`particles may irritate the respiratory tract.
`More recently, liposome inhalation systems for ad
`ministering a drug to the respiratory tract in liposome
`entrapped form have been proposed. UK patent appli
`cation GB 2,l45,l07A describes an aerosol device
`which brings aqueous and organic-solvent phase solu
`tions together under pressure, and passes the mixture
`through a nozzle to form aerosolized liposomes. EPO
`patent application 0,158,441 discloses liposome forma
`tion, in aerosol form, from a water/lipid/ethanol mix
`ture. In PCT application WO 86/01714, it is proposed
`to spray lipid droplets in a volatile liquid carrier, with
`liposome formation occurring upon contact of the drop
`lets with a moist aqueous surface. UK patent application
`GB 2,170,815 describes a system in which an aqueous
`solution is emulsified in a lipid-containing propellant
`solvent, then sprayed through an atomizing nozzle to
`form lipid-coated droplets which can form liposomes
`upon contact with a moist surface. All of these ap
`proaches are characterized by “in situ” liposome forma
`tion, i.e., liposome formation at the spray valve or on
`contact with the moist surface of the lungs. As such, the
`concentration and size of the liposomes formed, and the
`percentage of drug entrapment in the liposomes, will
`vary from one dose delivery to another, depending
`upon temperature and humidity conditions, the extent
`of solvent mixing, and the total and relative amounts of
`solvent components present in the system. Thus each of
`these systems would be difficult to adapt for metered
`dose delivery, in which a reproducible amount of lipo
`some-encapsulated drug is needed.
`SUMMARY OF THE INVENTION
`Co-pending patent application for “Liposorne Inhala
`tion Method and System” Ser. No. 737,221, filed May
`22, 1985, now abandoned, discloses a lipsome-based
`aerosol system for delivering a drug, at a controlled
`release rate, via the respiratory tract. The invention is
`based on two discoveries: First, that rapid systemic
`uptake of drugs from the site of administration in the
`respiratory tract can be eliminated or greatly reduced
`by administering the drug in a predominantly liposome
`encapsulated form. Secondly, it was found that the rate
`of release of a water-soluble drug from a drug/liposome
`composition delivered to the respiratory tract can be
`modulated according to the acyl-chain composition of
`the phospholipids making up the liposomes. As a rule,
`
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`20
`
`4,895,719
`3
`slower drug release rates correlate with longer in vitro
`drug efflux half lives in serum. The liposome aerosol
`compositions used in these studies were prepared under
`conditions in which the drug was predominantly in
`liposome-encapsulated form, and the liposome suspen
`sions were delivered in metered dose form from a ?xed
`volume nebulizer.
`Co-owned patent application for “Liposome Concen
`trate and Method”, Ser. No. 860,528, ?led Apr. 22,
`1986, now abandoned addresses another aspect of effec
`tive drug delivery in a liposome-based inhalation sys
`tem: that of delivering a water-soluble, liposome-perme
`able drug in predominantly encapsulated form, from a
`dilute aqueous liposome suspension. The method of the
`invention involves preparing and storing a liposome/
`drug suspension initially in paste form, then diluting the
`paste to a concentration suitable for aerosolizing.
`Co-owned US. patent application Ser. No. 937,607,
`?led Dec. 3, 1986, currently pending additionally
`showed that administration of the ?z-agonist meta
`proteranol sulfate (MPS) in liposomal form via inhala
`tion reduced initial plasma levels of the drug more than
`about 8 fold with respect to free drug, and that plasma
`levels remained substantially constant over a two hour
`period, compared with a rapid drop in plasma levels of
`25
`the drug administered by inhalation in free form. At the
`levels of MP8 which were studied, the percent protec—
`tion against bronchoconstriction provided by the drug
`was about the same for both free drug and liposomal
`entrapped drug.
`It was further discovered, according to the teaching
`of co-owned patent application for “Liposome Bron
`chodilator System and Method”, Ser. No. 022,669, ?led
`Mar. 6, 1987, that Bz-adrenoreceptor agonists, when
`administered in liposome-entrapped form at a therapeu
`tic drug dose (i.e., minimum dose required for optimal
`or near-optimal short-term therapeutic effect), produce
`signi?cantly greater bronchodilation, over an extended
`time period, than is produced by the the same amount of
`B2-agonist delivered to the respiratory tract in a free
`40
`drug aerosol form.
`The inventions mentioned above show that liposome
`drug delivery by inhalation provides advantages of (a)
`reduced side effects due to rapid systemic drug uptake,
`(b) improved therapeutic action over an extended per
`iod, and (c) the ability to modulate rate of drug release
`from the target site.
`The present invention is concerned with a self-con
`tained apparatus, or system and method for delivering a
`selected amount of drug, ef?ciently and reproducibly,
`in liposome encapsulated form. The apparatus of the
`invention includes liposome particles which have been
`formed by spray drying a dilute aqueous suspension of
`the liposomes. The particles formed (a) have a ?ne
`particle size, (b) retain the majority of their originally
`encapsulated material, and (c) are stable, in a preferred
`formulation, when suspended in a fluorochlorocarbon
`solvent. The particles are preferably formed, according
`to one method of the invention, by forming the lipo
`somes from partially or totally saturated phospholipid
`60
`components and drying the liposomes in a stream of
`heated gas whose temperature does not degrade the
`lipid components or structural integrity of the lipo
`somes.
`The apparatus further includes a self-contained deliv
`ery device for producing an airborne suspension of the
`liposome particles containing a metered dose of drug, in
`liposome-entrapped form. In one embodiment, the lipo
`
`4
`somes are contained in a suspension of a pressurized
`?uorochlorocarbon solvent in a metered-dose spray
`device designed to release a selected volume’ of the
`suspension in aerosolized form.
`In a second embodiment, the liposomes and a metered
`amount of the liposome-entrapped drug are contained in
`individual packets. The delivery device may be a pro
`pellant spray device designed to release a stream of
`aerosolized propellant particles through the packet, to
`entrain the liposomes in the stream. Alternatively, the
`delivery device may be a ?ow-through air chamber
`designed to support a liposome packet such that the
`liposomes are entrained in a stream of air drawn
`through the chamber.
`These and other objects and features of the present
`invention will be more fully understood when the fol
`lowing detailed description of the invention is read in
`conjunction with the accompanying drawings.
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIGS. 1A and 1B are renderings of light micro graphs
`of spray dried liposomes which have been suspended in
`a propellant solvent and sprayed onto a dry (1A) or
`moist (1B) slide;
`FIGS. 2A and 2B are renderings of light micrographs
`of liposomes, as in FIGS. 1A and 1B, respectively, but
`where the liposomes were spray dried from a 5% lac
`tose solution;
`FIG. 3 is a side cutaway view of a liposome delivery
`apparatus according to one embodiment of the inven
`tion, in which spray-dried liposomes are delivered from
`a suspension in a pressurized propellant;
`FIG. 4 is a side cutaway view of a liposome delivery
`apparatus constructed according to another embodi
`ment of the invention, in which spray-dried liposomes
`are contained in a cylindrical packet, for entrainment in
`an aerosol stream produced by a self-contained propel
`lant device;
`.
`FIG. 5 is a side cutaway view of a liposome delivery
`apparatus like the one shown in FIG. 4, except where
`the liposomes are supported loosely on a porous matrix
`in a packet, for entrainment in an aerosol stream pro
`duced by a self-contained propellant device; and
`FIG. 6 is a side cutaway view of a liposome delivery
`apparatus constructed according to a third embodiment
`of the invention, in which spray-dried liposomes are
`contained in a capsule-like packet, and the packet is
`supported in the air passageway of a delivery device,
`for entrainment of the liposomes in a stream of air
`drawn through the passageway.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`I. Spray-Dried Liposome Particles
`This section discusses methods for preparing spray
`dried liposomes having desired properties in the inhala
`tion system of the invention. The most important of
`these properties are (a) high drug entrapment, (b) se
`lected pharmacokinetic behavior when delivered to the
`respiratory tract, (0) ability to form fine particle sizes on
`spray drying, and (d) stability on storage in dehydrated
`or propellant suspension form. Section IA below dis
`cusses lipid composition factors which are important to
`drug release rates, most compatible with spray drying,
`and give greatest storage stability. Sections IB describes
`several methods for forming lipsomes containing en
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`4,895,719
`6
`5
`trapped water-soluble or lipid-soluble drugs. Spray
`and in more rigid membranes, where sterol have a fluid
`izing effect, drug release half lives were decreased.
`drying methods are discussed in Section IC.
`Another important consideration in the choice of
`A. Liposome Components
`lipid components is the phase transition temperature of
`The effect of liposome lipid components on the rate 5 the lipids, which may be greater than the temperature of
`of drug release in the respiratory tract has been reported
`heated gases used in drying the liposomes such that the
`in the above-cited co-pending patent applications.
`drug is retained in liposomes on drying. Most efficient
`Brie?y, studies on in vitro drug release rates, as a func-
`drying temperatures are at least about 37° C., and pref
`tion of lipid composition in the liposomes, showed that
`erably between about 40°~50° C., although higher tem
`liposomes whose phospholipid components contain 10 peratures are also possible. Table 1 below gives the
`longer and/or more saturated acyl chain moieties have
`transition temperatures and acyl chain composition for
`longer drug-release half lives. The drug-release rates
`a number of different liposome lipid components.
`ranged from less than about 0.5 hour for soy phos-
`As can be appreciated from the table, liposomes can
`phatidylcholine (SPC) to nearly ten days for a mixture
`be formulated with mixtures of relatively low and rela
`of distearoyl PC/distearoyl phosphatidyl glycerol 15 tively high phase transition temperature components, to
`(DSPC/DSPG). The most signi?cant increases in drug _ achieve an overall phase transition temperature in the
`release rates were observed when the liposomes con-
`37° to 50° C. range. Alternatively, partially hydroge
`tained a signi?cant proportion of lipids whose transition
`nated lipid components, such as PHPC, are suitable
`temperature (T6) are above the temperature at which
`when used in combination with other components. The
`the ef?ux half lives are measured, e.g., 37+ C.
`20 transition temperature of liposomes formed with the
`Studies on the effect of lipid charge on drug release
`selected lipid components can be determined conven
`rates indicated that the addition of a negatively charged
`tionally by differential scanning calorimetry (2).
`TABLE 1
`Some Properties of Phospholipids Used in Liposomes
`Ref.
`Abbrev.
`Charge Tc (°C.)
`EPC
`0
`— 15 to —7 109
`DLPC
`0
`— 1.8
`122
`DMPC
`0
`23
`109
`DPPC
`0
`41
`109
`DSPC
`0
`55
`109.1
`MPPC
`0
`27
`97
`
`'
`
`Lipid
`Egg phosphatidylcholine
`Dilaurylolylphosphatidylcholine (Cl2:0)
`Dimyristoylphosphatidylcholine (c14=0)
`Dipalmitoylphosphatidylcholine (C1620)
`Distearoylphosphatidylcholine (C18z0)
`l-Myristoyl-2-palmitoylphosphatidyl-
`choline (c14:0.16=0)
`l-Palmitoyl~2-myristoyl phosphatidyl-
`choline (C16:O.14:0)
`l-Palmitoyl-Z-stearoyl phosphatidyl-
`choline (Cl6:0.l8:0)
`l-Stearoyl-Z-paln?toyl phosphatidyl-
`choline (C18:0.16:0)
`Dioleoylphosphatidylcholine (Cl8:l)
`Dilaurylolylphosphatidylglycerol
`Dimyristoylphosphatidylglycerol
`Dipalmitoylphosphatidylglycerol
`Distearoylphosphatidylglycerol
`Dioleoylphosphatidylglycerol
`Dimyristoyl phosphatidic acid
`Dimyristoyl phosphatidic acid
`Dipalinitoyl phcsphatidic acid
`Dipalmitoyl phosphatidic acid
`Dimyristoyl phosphatidylethanolarnine
`Dipalmitoyl phosphatidylethanolamine
`Dimyristoyl phosphatidylserine
`Dipalmitoyl phosphatidylserine
`Brain phosphatidylserine
`Brain sphingomyelin
`Dipalmitoyl sphingomyelin
`Distearoyl sphingomyelin
`
`PMPC
`
`PSPC
`
`SPPC
`
`0
`
`0
`
`0
`
`0
`DOPC
`— 1
`DLPG
`DMPG — l
`DPPG
`— l
`DSPG
`— 1
`DSPG
`— l
`DMPA —1
`DMPA —2
`DPPA
`—1
`DPPA
`-2
`DMPE —
`DPPE
`—
`DMPS
`—
`DPPS
`—
`PS
`—
`BSP
`0
`DPSP
`0
`DSSP
`0
`
`35
`
`44
`
`47
`
`—22
`4
`23
`41
`55
`— 18
`51
`45
`67
`58
`50
`60
`38
`5 l
`6-8“
`32
`41
`57
`
`97
`
`97
`
`97
`
`109
`46
`158
`86
`153
`46
`86
`153
`153
`153
`153
`203
`203
`120
`86
`191
`18
`42
`
`lipid, such as phosphatidyl glycerol (PG), at a mole
`ratio of about 10%, produces a slight to moderate in
`crease in ef?ux half life. The lipid-charge effect is de
`pendent somewhat on the degree of saturation and
`chain length in the charged and uncharged lipids used in
`forming the liposomes, with the charge effect produc
`ing a greater increase in drug efflux half life where the
`liposomes are formed of predominantly shorter and/or
`unsaturated lipids, and producing less effect in the case
`of longer-chain and/or saturated lipids. The effect of
`sterol components, such as cholesterol, was relatively
`minor, and generally followed the known ?uidizing
`effect of sterol on liposomal membranes. That is, in
`relatively ?uid membranes, where sterol increase mem
`brane rigidity, drug-release half lives were increased,
`
`60
`
`65
`
`Other lipid components, including PG, and cholesterol,
`can be included. In general, cholesterol has a ?uidizing
`effect in liposomes containing predominantly saturated
`phospholipid components, and thus is expected to lower
`phase transition temperature slightly. The effect of
`added PG or other phospholipid components will, of
`course, depend largely on the acyl chain composition of
`the lipids.
`.
`The liposomes may also be formulated to include
`various types of drug-protective or lipid-protective
`agents, such as the antioxidant a-tocopherol, which is
`typically included at a mole ratio of between about
`0.1-2 mole percent.
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`7
`B. Liposome Preparation
`The liposomes are prepared initially as a dilute aque
`ous suspension which is suitable for spray drying. The
`suspension may be prepared by a variety of techniques,
`such as those detailed in reference 2. The choice of the
`liposome preparation method will depend, in part, on
`the nature of the drug to be entrapped. As de?ned
`herein, “drug” is intended to include any pharmacologi
`cally active agent which has a site of action in the respi
`ratory tract or is therapeutically active when it is taken
`up systemically from the respiratory tract. Such drugs
`may include antibiotics, peptide hormones, enzymes,
`enzyme inhibitors, anti-tumor agents, bronchodilators,
`allergens, antihistamines, and biogenic compounds such
`as steroids and prostaglandins.
`For purposes of discussion, two general classes of
`drugs will be considered. The ?rst class are predomi
`nantly water-soluble drugs which tend to partition into
`the aqueous phase of a water/oil two-phase system.
`More particularly, this class of drugs tend to partition
`preferentially in the aqueous interior phase of lipo
`somes, rather than in the lipophilic bilayer phase. Drugs
`in this class include relatively small, liposome-permea
`ble drugs such as albuterol (salbutamol) sulfate, ephi
`drine sulfate, ephidrine bitartrate, isoetharine hydro
`chloride, isoetharine mesylate, isoproteranol hydro
`chloride, isoproteranol sulfate, metaproteranol sulfate,
`terbutaline sulfate, procaterol, and bitolterol mesylate,
`atropine methyl nitrate, cromolyn sodium, propranalol,
`?uoroisolide, ibupro?n, gentamycin, tobermycin, pent
`amidine, penicillin, theophylline, bleomycin, etoposide,
`captopril, n-acetyl cysteine, and verapamil; and rela
`tively large, liposome-impermeable drugs, such as pep
`tide hormones, enzymes, enzyme inhibitors, apolipo
`proteins, and higher molecular weight carbohydrates,
`as exempli?ed by calcitonin, atriopeptin, a-l antirypsin
`(protease inhibitor), interferon, oxytocin, vasopressin,
`insulin, interleukin-2, superoxide dismutase, tissue plas
`minogen activator (T PA), plasma factor 8, epidermal
`growth factor, tumor necrosis factor, lung surfactant
`protein, and lipocortin.
`The second general class of drugs are lipophilic drugs
`which partition preferentially in the oil phase of a
`water/oil system, and more particularly, preferentially
`in the bilayer region of liposomes. Representative drugs
`in this class include prostaglandins, amphotericin B,
`progesterone, isosorbide dinitrate, testosterone, nitro
`glycerin, estradiol, doxorubicin, beclomethasone and
`esters, vitamin E, cortisone, dexamethasone and esters,
`DPPC/DPPG or any other lipid/surfactant (to relieve
`RDS), and betamethasone valerete.
`One preferred method for preparing drug-containing
`liposomes is the reverse phase evaporation method de
`scribed in reference 3 and in US. Pat. No. 4,235,871. In
`this method, a solution of liposome-forming lipids is
`mixed with a smaller volume of an aqueous medium,
`and the mixture is dispersed to form a water-in-oil emul
`sion. The drug to be entrapped is added either to the
`lipid solution, if a lipophilic drug, or in the aqueous
`medium, if- a water soluble drug. After removing the
`lipid solvent by evaporation, the resulting gel is con
`verted to liposomes, with an encapsulation ef?ciency,
`for a water-soluble drug, of up to 50%. The reverse
`phase evaporation vesicles (REVs) have typical aver
`age sizes between about 1-5 microns and are predomi
`nantly oligolamellar, that is, contain one or a few lipid
`bilayer shells. The oligolamellar nature of the vesicles
`
`25
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`30
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`35
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`40
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`45
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`50
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`60
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`65
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`4,895,719
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`10
`
`15
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`8
`may facilitate drug ef?ux and thus contribute to a lower
`efilux half live for an encapsulated drug.
`A simple lipid-hydration procedure for producing
`multilamellar vesicles (MLVs) may be preferred where
`high drug encapsulation ef?ciency is not desired. In this
`procedure, a mixture of liposome-forming lipids dis
`solved in a suitable solvent is evaporated in a vessel to
`form a thin ?lm, which is then covered by an aqueous
`solution of the drug. The lipid ?lm hydrates to form
`MLVs, typically with sizes between about 0.1 to 10
`microns. As in the REV method, the drug to be encap
`sulated is added either to the initial lipids or to the hy
`drating medium, depending on its solubility in water.
`The percent of total drug material which can be encap
`sulated in the MLVs, calculated as the ratio of encapsu
`lated drug to total drug used in vesicle preparation, is
`typically between about 5-20% for water-soluble
`drugs.
`It is mentioned here, for the case of lipid-soluble
`drugs, and where spray-dried liposomes are suspended
`in a ?uorochlorocarbon solvent which is itself a solvent
`for the drug, that the drug may be included initially in
`the propellant solvent rather than in the liposomes. In
`this embodiment, the spray dried liposomes are formed
`without added drug, with the drug being incorporated
`into the lipid vesicles at the time of contact with and
`evaporation of the solvent in an aerosol. This system
`will be described in Section II below.
`The liposome preparation methods described above
`produce heterogeneous liposomes sizes, typically rang
`ing from less than about 0.1 micron to up to about 20
`microns in size. Optimal liposome size, after spray dry
`ing, is preferably less than about 5 microns, although
`respirable particles as large as 5-8 microns are suitable.
`The size of the liposomal particle formed on dyring the
`liposomes in suspension will depend both on the initial
`size of the aqueous-suspension liposomes and on the
`degree of liposome aggregation which occurs on dry
`ing. As will be seen below, liposomes having an initial
`suspension size of about 1.5 microns produce particles
`ranging from about 1.5 to 4 microns on drying. In gen
`eral, therefore, it is desirable to process liposomes
`formed by either of the above two methods to reduce
`the largest liposomes to less than about 2 and preferably
`1.5 microns. Liposome sizing can be achieved by ex
`truding an aqueous suspension of the liposomes through
`a polycarbonate membrane having a selected uniform
`pore size, typically 1 micron or less (reference 3). A
`more recent method for sizing liposomes involves extru
`sion through an asymmetric ceramic ?lter. The method
`is detailed in co-owned US. patent application for Lip
`osome Extrusion Method, Ser. No. 829,710, ?led Feb.
`13, 1986, now US. Pat. No. 4,737,323.
`In the case of an encapsulated water-soluble drug, the
`liposomes may be further treated to remove non-encap
`sulated drug from the suspension. Conventional separa
`tion techniques, such as centrifugation, dia?ltration, and
`molecular-sieve chromatography are suitable. The sepa
`ration step should be carried out after sizing, since the
`sizing procedure itself can lead to liposome rupture and
`release of encapsulated drug. It will be appreciated that
`in the case of a water-soluble liposome-permeable drug
`which can rapidly equilibrate between encapsulated and
`bulk-phase aqueous spaces in the liposomal suspension,
`drug removal needs to be carried shortly before spray
`drying, i.e., before appreciable leakage of the drug from
`the liposomes.
`
`WATSON LABORATORIES, INC. , IPR2017-01622, Ex. 1024, p. 6 of 13
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`

`15
`
`20
`
`25
`
`4,895,719
`10
`9
`It is also possible, using recently-developed liposome
`tose or other sugar. The bulking agent, which is typi
`cally added between about 2-7 percent by weight, may
`preparation methods (detailed in co-owned U.S. patent
`applications for “High-Concentration Liposome Pro
`also facilitate spray drying, both by drawing water from
`the liposomes in the terminal stages of drying, and by
`cessing System and Method" Ser. No. 909,122, ?led
`reducing the tendency of liposomes to aggregate during
`Sept. 18, 1986, now U.S. Pat. No. 4,781,781, issued
`the drying procedure.
`November 1988 and “High-Encapsulation Liposome
`Processing System and Method” Ser. No. 908,765, ?led
`The liposome suspension can be spray dried in a con
`ventional drying apparatus in which the material to be
`Sept. 18, 1986), now U.S. Pat. No. 4,752,425, issued
`June 21, 1988, to prepare a liposome suspension which
`dried is sprayed in aerosolized form into a stream of
`heated air or inert gas, and the aerosolized liposome
`(a) has maximum sizes of about 1.5 microns or less and
`(b) contains at least about 50%, and typically 70% or
`particles are dried in the gas stream as they are carried
`more, of the drug in liposome-entrapped form. Thus,
`toward a plate collector where the dried liposomes are
`collected. An exemplary spray dry apparatus which is
`liposomes produced by these methods do not need to be
`suitable for use in the present invention is a Buchi 190
`further sized or treated to remove free drug. In this
`Mini Spray Dryer.
`method, a solution of lipids (and lipid-soluble drug, if
`As noted above, the drying temperature is at least
`such is used) is injected in an organic solution into an
`about 37° C., and preferably higher than 40° C. More
`aqueous buffer (and a water-soluble drug, if such is
`particularly, the drying temperature is such that the
`used) under selected temperature and pressure condi
`liposomes remain in a rigid, unmelted state during the
`tions, with liposome formation in the aqueous medium.
`Lipid injection into the liposome suspension is contin
`heating process. The temperature of the collection
`chamber is generally lower than that of the heated air,
`ued until a ?nal lipid concentration of at least about 250
`and typically about 37° C.
`mM and preferably between about 300-500 mM is
`By way of examples, liposomes formed from
`reached. At this lipid concentration, the liposomes are
`PHPCzcholesterolzPc (5:4:1) and containing 1 mole
`predominantly in the size range below about 1.5 mi
`percent a-tocopherol were formed by a solvent injec
`crons, and up to 70% or more of the drug is associated
`with the liposomes, either in encapsulated form in the
`tion into an aqueous metaproteranol sulfate (MPS) solu
`tion (100 mg/ml), to a ?nal liposome concentration of
`case of a water-soluble drug, or in membrane-entrapped
`about 400 umole/ml and a final encapsulation ef?ciency
`form in the case of a lipid-soluble drug. The ?nal sus
`pension may have a paste-like consistency. The paste
`of about 70%. The liposomes, which had maximum
`sizes of about 1.5 microns, were diluted to 3 percent by
`may be diluted to a selected concentration, as described
`weight in low osmolarity phosphate buffer, pH 7.4, and
`below, immediately before spray drying.
`spray dried at about 48° C. The size and stability charac
`C. Spray Drying
`teristics of the liposomes are discussed in Section II
`A liposome suspension prepared as above is diluted, if
`below.
`The dried liposomes are collected and stored in dehy
`necessary, to a ?nal dilute suspension which is suitable
`for spray drying. The suspension has the following
`drated form, at lower temperature (typically 4° C.).
`preferred characteristics:
`II. Delivery Devices
`a. a liposome concentration of between about 2-10
`and preferably 3-6 weight percent;
`This section describes three self-contained delivery
`devices designed for producing an airborne suspension
`b. More than half and preferably about 70% or more
`40
`of the total drug in liposome-entrapped form,