Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers

Definitions

• the present invention relates to liposomes designed for enhanced binding to mucosal tissue, and to a drug delivery system and method which uses the liposomes.
• the retention of a solution-form drug on the corneal surface can be enhanced by the use of polymers, such as hydroxyethylcellulose or methylcellulose, which increase the viscosity of the drug solution.
• Polymers such as hydroxyethylcellulose or methylcellulose, which increase the viscosity of the drug solution.
• Polymer containing viscous liquids are used, for example, in the treatment of dry eye, to help keep the corneal surface moist.
• very little of the originally applied liquid is retained for more than about an hour, so frequent dosing is necessary.
• suppositories are a convenient method for releasing medication to the mucosal tissue over an extended period, and for drug release in the stomach, slow release particles that break down at variable rates are commonly used.
• the increased retention is due to the interaction of the liposome surface positive charges with mucin, a negatively charged glycoprotein which is secreted by and present in the environment of mucosal tissue.
• Ocular-retention studies performed in support of the present invention show that at a cholesterol amine concentration of 40 mole percent, liposome retention at the end of an hour increases from about 5% for uncharged liposomes to about 10% of the originally applied liposomes. This small increase in enhancement falls short of the increase in liposome retention which would be needed to provide effective drug release several hours after the liposomes are applied to the mucosal surface.
• Relatively long chain alkyl amines such as stearylamine. have been used to increase retention of liposomes to ocular mucosa (Schaeffer).
• charged amines of this type tend to be toxic at elevated levels (Yashihara) and therefore cannot be used at molar concentrations that give maximal liposome retention properties. This problem is aggravated in part because the single chain molecules of this type can readily dissociate from the liposome bilayer, and because the molecules themselves tend to destabilize the liposome bilayer structure.
• a more specific object is to provide such a composition for use in administering drugs to the eye, at a controlled drug-release rate of over several hours.
• Still another object of the invention is to provide an improved liposome composition for the treatment of dry eye.
• a drug/liposome composition having an enhanced binding affinity for mucosal tissue.
• the outer surfaces of the liposomes contain positive surface charges which are (a) anchored to the lipid outer lipid bilayer structure by vesicle-forming lipids which are relatively tightly associated with the membrane, and (b) spaced by at least about a 3 atom spacer from the polar head regions of such vesicle-forming lipids.
• the concentration of surface positive charges is typically between about 20-50 mole percent.
• the invention includes a liposome composition in which the liposomes have outer lipid bilayer surfaces containing (a) between about 40-80 mole percent of neutral vesicle forming lipid components, and (b) between about 20-60 mole percent of positively-charged vesicle-forming lipid component(s) having (i) 2 aliphatic chains carried on a 3-4 carbon backbone, (ii) a polar atom attached to the backbone at a carbon atom which does not carry an aliphatic chain, (iii) an amine linked to the polar atom through a spacer at least 3 atoms long, and (iv) a net positive charge.
• the liposomes may also include a cholesterol derivative having an amine group linked to the A ring 3 position by a spacer arm at least three atoms long.
• the liposomes preferably have a relatively close-packed lipid structure by virtue of containing between about 20-50 mole percent cholesterol or cholesterol analog or amine derivative, and/or predominantly saturated acyl chain moieties in the phospholipid or diglyceride components.
• One preferred positively charged lipid component is an amine-derivatized phospholipid of the form:
• PE-NH-C-Y-N where PE-NH is phosphatidylethanolamine, and Y is a basic amino acid or peptide containing a basic amino acid.
• the derivated PE is formed by coupling PE with the anhydride of the amino acid or peptide.
• One preferred cholesterol derivative has the form:
• Ch-O-C-Y-N Ch-OH is cholesterol
• Y is a carbon-containing chain at least 2 atoms in length.
• the lipid component is formed by coupling cholesterol with the anhydride of an amino acid or peptide.
• Another preferred cholesterol derivative has the form:
• Ch-NH-Y-N where Ch-NH is cholesterol-3-amine and Y is a
• the component is formed by coupling a diamine with a cholesteryl-3-halide.
• the liposome composition may further be formulated for increased retention near the tissue site (as well as increased retention to the mucosal tissue).
• the formulation may include increased-viscosity polymers.
• the liposomes may be formulated for delayed release in suppositories or slow-release polymer matrices. Aerosolized liposomes for nasal and oral drug delivery, and cream or foam formulations for topical application are also disclosed.
• Also forming part of the invention is an improved method of administering a drug to a mucosal tissue, for sustained drug release at the tissue site over a several hour period. The method utilizes the novel liposome composition described above.
• the invention includes a method of treating dry-eye. by applying to the ocular surface, a preferably optically clear suspension of positively charged liposomes of the type described above.
• the suspension may contain increased-viscosity polymers for greater liposome retention at the ocular site.
• the liposomal lipids contribute to the lubricating properties of the dry-eye composition.
• Figure 1 shows the retention at an ocular tissue of liposomes prepared with increasing concentrations of lysinyl phosphatidylethanolamine, (lysinyl PE), including a neutral liposome control (solid squares), and 10 (open circles), 20 (open triangles). 30 (open squares), and 40 (closed circles) mole percent lysinyl PE;
• lysinyl PE lysinyl phosphatidylethanolamine
• Figure 2 shows the retention on an ocular tissue of liposomes prepared with increasing concentrations of lysine lysinyl PE, including a neutral liposome control (solid squares), and 10 (open circles), 20 (closed triangles), and 30 (open squares), mole percent lysine lysinyl PE;
• Figure 3 shows the retention on an ocular tissue of liposomes prepared with various epi-cholesteryl derivatives, including a cholesterol control (solid squares), cholesterylamine (open circles); and cholesterylpiperazine (open triangles);
• Figure 4 shows the retention on an ocular tissue of liposomes prepared with various cholesterol ester amines, including a cholesterol control (solid squares), and the cholesterol esters of glycine (open circles), ⁇ -alanine (closed triangles), and ⁇ -amino caproic acid (open squares).
• Figure 5 shows the retention on an ocular tissue of liposomes prepared with either 0 (closed symbols) or 40 (open symbols) mole percent cholesterol, and 20 mole percent of either lysine PE (circles) or lysine lysinyl PE (triangles).
• Figure 6 shows the retention on an ocular tissue of liposomes prepared with either lysine PE (circles) or lysine lysinyl PE (triangles), in a suspension containing either buffer (closed symbols) or polymers (open symbols); and
• Figure 7 shows the retention on an ocular tissue of liposomes prepared with either lysinyl PE, at 20 (open circles) or 30 (closed circles) mole percent, or lysine lysinyl PE, at 10 (open triangles) or 20 (closed triangles) mole percent, in a suspension containing a polymer additive, and neutral liposomes with (closed squares) or without polymer additive (open squares).
• the positively charged lipid components used in preparing the liposomes of the invention are characterized by: (i) 2 aliphatic chains carried on a 3-4 carbon backbone, (ii) a polar atom attached to the backbone at a carbon atom which does not carry an aliphatic chain, (iii) an amine linked to the polar atom through a spacer at least about 3 atoms long, and (iv) a net positive charge.
• Exemplary lipid components include diglycerides, and amine analogues thereof, in which the polar atom is a hydroxyl oxygen or amine, respectively; glycolipids, in which the polar atom is the acetal oxygen joining the sugar residue to the lipid backbone; and phospholipids, in which the polar atom is a phosphate ester oxygen linking a glycerol backbone to a phosphate polar head group.
• the polar atom is positioned on the outer bilayer surface of the lipid vesicles at a position corresponding approximately to the hydroxyl group of cholesterol.
• the liposomes may also contain positively-charged cholesterol derivatives having an amine group linked to the 6-membered cholesterol A ring by a carbon-containing chain at least 3 atoms in length.
• dialiphatic chain lipids and cholesterol are relatively tightly associated with liposome bilayer structure, and contribute to membrane stability. These properties are in contrast to single acyl chain compounds, such as fatty acids or their derivatives, which readily dissociate from membrane bilayer structures in an aqueous suspension (Doody), and which also promote fusion of lipid bilayers (Kantor) and stimulate phospholipid release (Massari) and intermembrane lipid exchange (Papahadjopoulos).
• Another distinguishing feature of dialiphatic and cholesterol lipids, when compared with single acyl chain components, is their more rigid radial positioning in the lipid planes of the bilayer structure.
• the positively charged amine groups be spaced from the polar head region of the lipid by at a carbon-containing spacer arm at least three atoms long. This spacer is apparently needed to allow the lipid-bound amine groups to interact readily with negatively charged molecules in the mucosal surface environment.
• Evidence for the three-atom spacer requirement comes from a number of studies on the binding of liposomes to ocular and other mucosal tissues which were carried out in support of the invention. Two of these studies, reported in Example X, examine the effect of cholesteryl amines and cholesterol amine esters having various selected spacer chain lengths.
• the glycine derivative in which the amine is spaced from the cholesterol hydroxyl oxygen atom by only two carbon atoms, gives only a slight enhancement .over control liposomes containing underivatized cholesterol (closed squares).
• the cholesterol derivatives of both ⁇ -alanine (three carbon spacer) and ⁇ -aminocaproic acid (6 carbon spacer) gave a severalfold increase in binding retention after 1 hour.
• the spacer chain is a carbon-containing chain having various degrees of saturation and/or heteroatom compositions.
• One preferred type of chain is a simple saturated acyl chain.
• the carbon atoms in the chain may also be partially unsaturated, including either ethylenic or ethynic bonds, and/or may include such heteroatoms as carbon-linked oxygen (O), sulfur (S) or nitrogen (N) atoms, forming ester, ether, thioester. thioether, amide or amine linkages within the chain.
• the chain atoms themselves may be substituted with carbon, hydrogen, O, S, or N atoms, or groups containing these atoms such as short chain acyl groups or the like.
• the chain may contain a glycoside group which carries the amine, and is itself attached to lipid backbone through a suitable spacer arm.
• the positively charged amine may be either a primary, secondary, tertiary, or quaternary amine, with the only requirement that the amine be positively charged at the operative pH.
• primary, secondary, and tertiary amine are positively charged at a pH below about 7.5-10.
• One advantage of quaternary amines is that the species is always positively charged, independent of pH. Structural features and methods of synthesis of selected positively charged lipid components will now be considered.
• dialiphatic lipid is intended to include amphipatic lipids having (i) a 3-4 carbon backbone, (ii) two aliphatic chains carried on the backbone, and (iii) a polar oxygen or nitrogen atom attached to a backbone carbon atom which itself does not carry an aliphatic chain.
• the aliphatic ch ains are attached to the backbone by suitably stable, linkages, including acyl ester, ether, thi ⁇ ether, amine, carbamate, or a dioxolane ring (Nagata) linkages.
• exemplary dialiphatic lipids As indicated above, exemplary dialiphatic lipids.
• aliphatic chain include diglycerides and phospholipids, in which the aliphatic chain are fatty acyl chains attached to a glycerol backbone through acyl linkages and glycolipids, in which one of the acyl chains may be linked to the backbone through an amide linkage.
• the aliphatic chains in the lipid components are preferably at least about 12 atoms in length, and optimally between about 15-20 atoms long.
• the chains are also preferably substantially unsaturated, by which is meant that each chain contains at most one unsaturated bond, and preferably an ethylenic bond.
• the substantially unsaturated aliphatic chains produce better lipid packing in the liposomes, which has been found to increase liposome binding to mucosal surfaces.
• the more unsaturated chains produce greater chemical stability on long term storage, and evidenced by reduced oxidative damage to the lipids.
• diglyceride and diglyceride amine analogues contain an amine attached to the polar oxygen or nitrogen atom through a spacer arm at least three atoms long.
• the diglyceride derivatives may be formed by known coupling methods involving glycerol hydroxyl or amine groups. In general, these methods are similar to used in derivatizing cholesterol or cholesteryl amine, as described below.
• the diglyceride can be reacted with a protected amino acid anhydride, according to above-described methods, to form the diglyceride ester of the amino acid.
• the term phospholipids encompasses phosphatidic acid (PA) and phosphatidyl glycerol (PG).
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidylserine
• plasmalogens and sphingomyelin (SM).
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidylserine
• plasmalogens sphingomyelin
• the polar end region of the phospholipids is defined as the glycerol hydroxy oxygen atom which forms the glycerol/phosphate ester linkage in the phospholipid.
• This oxygen atom occupies roughly the same radial position in a lipid bilayer surface as the hydroxy oxygen atom in diglycerides, and therefore about the same radial position as the 3-hydroxy oxygen atom in cholesterol.
• the phospholipid derivatives differ from the above cholesterol and diglyceride derivatives in that the polar end region hydroxyl oxygen is itself linked, through a phosphate ester bond, to a carbon containing chain at least three atom long, i.e., the phosphate-ester linked moiety defining the individual class of the phospholipid.
• the positively charged phospholipid other than PA
• phospholipids including PC, and PE, contain chain terminal amines which in the natural phospholipid, balance the negative charge of the phosphate group.
• phospholipids can be converted to the desired positively charged derivatives by acylating the phosphate group, thus neutralizing its charge and imparting a net positive charge (due to the terminal amine) to the derivative.
• Methods for methylating or ethylating ester-linking phosphate groups, to form corresponding methylphosphonate or ethylphosphonate derivatives are known, and would be suitable for use in the present application.
• the phospholipid is derivatived by coupling an amine, such as an amino acid, to a reactive end group in the phosphate-ester link d moiety of the lipid.
• an amine such as an amino acid
• a variety of coupling reactions involving suitable activating agents or reactive species are known, and can be readily adapted for coupling amines to natural phospholipids.
• PE and plasmalogens can be coupled through the terminal primary amine to an amine via an amide linkage, according to known coupling reactions, as will be detailed below.
• PI and a variety of glycolipids, which contain a terminal glycoside group can be coupled to an amine after a prior periodate reaction (Heath) which involves initial aldehyde formation, and proceeds through a Schiff base.
• Heath prior periodate reaction
• PS can be coupled through its terminal acid group, by known amide-forming reactions to suitable amines. It is noted here that the amine coupled to PA must serve to extend the charged amine at least 3 atoms from the phosphate-ester linked oxygen attached to the lipid's glycerol moiety.
• the amine which is coupled to the phospholipid has a total number of charged amine groups which will impart a net positive charge of at least 1 to the derivatized phopholipid.
• the amine must contain at least two amine groups—one to balance the phosphate negative charge, to offset the loss of the terminal amine charge in the phospholipid, and a second to contribute a single net positive charge.
• Preferred double amines for use in derivatizing PE are basic amino acids such as lysine, ornithine, histidine. or arginine, or peptides containing at least one such amino acid.
• a diamine would also be required— one amine to react with the acid group, and a second amine to contribute a single net positive charge.
• the following methods for forming amine-derivatized PE are exemplary.
• Purified or partially purified PE used in preparing the cationic PE derivative is commercially available, or may be prepared by known methods.
• the lipid may be purified and/or modified in acyl chain composition according to known techniques. In certain liposome formulations and applications, to be discussed below, it is desirable to employ PE components having predominantly saturated acyl moieties; for other applications more unsaturated lipid components may be preferred.
• Example I One method for forming the amine derivatized PE component is illustrated in Example I for the preparation of lysinyl and lysine lysinyl PE, and in Example II, for the preparation of arginyl PE.
• the basic amino acid or peptide is N-protected, such as by reaction with di-t-butyldicarbonate.
• the protected amino acid is then reacted with an approximately equimolar amount of a condensing agent, such as dicyclocarbodiimide (DCC), to form the anhydride of the protected amino acid.
• DCC dicyclocarbodiimide
• the reaction conditions described in Example I are generally suitable in the anhydride-forming reaction.
• the anhydride may be used further without removing the dicyclohexylurea which forms as a by-product of the reaction.
• the anhydride is now reacted with PE under anhydrous conditions, to couple the protected amino acid to the PE through an amide linkage.
• the reaction product is deprotected, such as by treatment with trifluoroacetic acid, and may be purified, by chromatography on silica gel.
• the eluate fractions can be monitored conventionally by thin-layer chromatography (TLC), as described in Example I and II.
• TLC thin-layer chromatography
• the purified product may be stored as a dry residue under nitrogen at 4°C for up to several months.
• the protected amine is reacted directly with an N-hydroxysuccinimide in the presence of DCC to form the corresponding N-hydroxysuccinimide ester of the amino acid.
• Typical reaction conditions are similar to those used in forming the amino acid anhydride.
• the material may be employed without further purification for reaction with PE.
• the reaction product is deprotected and purified as above, and as detailed in Example I.
• cholesterol is intended to encompass cholesterol
• the polar end region of cholesterol is defined as the polar atom, such as oxygen or nitrogen, which is directly attached to the 3 position of the 6-membered A ring of the cyclopentanoperhydrophenanthrene nucleus of cholesterol, according to conventional ring and ring position identification.
• the amine-derivatized cholesterol has the general formula: Ch-O-X-N or Ch-NH-X-N, where Ch-O or Ch-N is a cholestene or cholestane structure with a 3 position oxygen or nitrogen, X is a carbon containing chain at least three atoms long, and N is a charged primary, secondary, tertiary, or quaternary amine.
• One exemplary cholesterol derivative is a cholesterol ester of the form:
• Ch-O-C-Y-N where Ch-OH is 3-hydroxy-5,6-cholestene, and Y is a carbon-containing chain at least two atoms long.
• an amino acid of the form CO 2 -Y-N is N-protected, by reaction with di-t-butyldicarbonate, and reacted with a suitable condensing agent, such as DCCI, to form the corresponding anhydride of the protected acid.
• a suitable condensing agent such as DCCI
• the anhydride is reacted with an approximately equimolar amount of cholesterol, forming the derivatized, protected compound, which is then deprotected and purified, for example, by silica gel chromatography.
• the reaction conditions are applicable to other amino acids having one or more free amine groups.
• only those cholesterol ester derivatives in which the free amine is spaced from the cholesterol hydroxyl by three or more atoms produce significant binding enhancement of liposomes to mucosal tissue.
• more than one net positive charge may be derivatized to the cholesterol, either by coupling to a basic amino acid, such as lysine, or by coupling to a peptide containing more than one free amine group.
• a second cholesterol derivative is a cholesteryl amine of the form:
• Ch-NH-X-N where Ch-NH 2 is 3-amino-5,6-cholestene and X and N are as defined as above.
• the derivative is formed by reacting a cholesteryl-3-halide, such as cholesteryl-3-iodide with a diamine of the form NH 2 -Y-N, where N is a primary-quaternary amine, and preferably a primary amine.
• a cholesteryl-3-halide such as cholesteryl-3-iodide
• N is a primary-quaternary amine, and preferably a primary amine.
• a suitable solvent such as dimethylsulfoxide.
• the product is extracted into a lipophilic solvent, such as toluene, and may be purified by silica gel column chromatography. Reaction details for the synthesis of (5-cholesten-3- ⁇ -N-(3-(4-(3-aminopropyl)piperazino)propy l)amine) from cholesteryl iodide and
• Example IV N,N"-bis-(3-aminopropyl)-piperazine are given in Example IV. Also described in Example IV is the synthesis of cholesterol-3-amine. which was synthesized as a control compound. Methods for derivatizing thiocholesterol through a disulfide linkage have also been reported (Huang. Baldeschweiler).
• the liposomes of the invention are formed of a mixture of neutral and amine-derivatized lipids.
• the neutral lipids which typically constitute between about 40-80 mole percent of the total liposomal lipids, are predominantly phospholipids, such as PC and PE, and/or cholesterol or cholesterol analogues.
• the amine-derivatized lipids preferably make up about 20-60 mole percent of the total lipid components. Studies showing the effect of charge density on liposome retention are presented in Examples VIII, below, and are shown in Figures 1 and 2.
• Figure 1 is a plot of ocular retention against time with liposomes containing increasing amounts of lysinyl PE, from 0 to 40 mole percent.
• enhancement of binding can be achieved at a concentration of charged lipid between about 20-50 mole percent, although higher mole ratios of the charged lipids are permissible.
• the charge concentration can be achieved either by about 20 mole percent of a single charged lipid component, such as lysinyl PE. or 10 mole percent of a doubly charged component, such as lysine lysinyl PE, and so on.
• the liposomes may further include minor amounts of other vesicle-forming lipids, such as fatty acids, negatively charged phospholipids, glycolipids, and the like, with the proviso that these minor lipid components (a) do not significantly reduce the binding affinity of the liposomes for mucosal tissue and (b) are not toxic at the mucosal tissue site.
• the first prescription limits the amount of negatively charged lipid which can be included in the liposomes, and also the amount of lipid which is disruptive of lipid packing in the liposome bilayer.
• phospholipid and sterol components generally apply both to neutral and amine-derivatized components, it being recognized that either the phospholipid or cholesterol components or both may contain at least some amine-derivatized species.
• One consideration in the choice of phospholipid components is the degree of saturation of acyl chain moieties.
• the degree of saturation of phospholipid components can also have a major effect on the rate of release of entrapped drug from the liposomes.
• more saturated lipids prolong the release of entrapped drugs, with a significant increase in release rate being observed near the transition temperature of the lipids.
• the release half life in liposomes with predominantly saturated lipids is several hours to several days, which can mean that over a several-hour period of liposome binding to a mucosal surface, only a small portion of the entrapped drug is actually released from the liposome for uptake by the tissue.
• the presence of high molar amounts of cholesterol does not effect drug release rates substantially. For this reason, it may be advantageous to achieve close packing in the liposomes by the inclusion of cholesterol rather than saturated phospholipid or diglyceride components.
• Lipid peroxidative damage can also be reduced by a combination of a lipophilic free radical scavenger, such as ⁇ -tocopherol ( ⁇ -T), and a water-soluble iron-specific chelator, such as desferrioxamine.
• a lipophilic free radical scavenger such as ⁇ -tocopherol ( ⁇ -T)
• ⁇ -T ⁇ -tocopherol
• desferrioxamine a water-soluble iron-specific chelator
• the lipophilic free radical scavenger used in the composition is preferably ⁇ -T, or a pharmacologically acceptable analog or ester thereof, such as ⁇ -T succinate.
• suitable free radical scavengers include butylated hydroxytoluene (BHT). propyl gallate (Augustin). and their pharmacologically acceptable salts and analogs.
• Additional lipophilic free radical quenchers which are acceptable for parenteral administration in humans, at an effective level in liposomes, may also be used.
• the free radical quencher is typically included in the lipid components used in preparing the liposomes, according to conventional procedures. Preferred concentrations of the protective compound are between about 0.2 and 2 mole percent of the total lipid components making up the liposomes.
• the water soluble iron-specific chelating agent may be selected from the class of natural and synthetic trihydroxamic acids and characterized by a very high binding constant for ferric iron (on the order of 10 30 ) and a relatively low binding constant for ferric iron (on the order of 10 30 )
• 2-valence cations such as calcium and magnesium.
• a variety of trihydroxamic acids of natural origin are known, including compounds in the ferrichrome class. such as ferrichrome. ferrichrome A, and albomycin; compounds in the ferrioxamine class, including the ferrioxamines and ferriomycines; and compounds in the fusaramine class.
• the chelator may be a tetraacetic acid or pentaacetic acid chelator such as EOTA. DPTA, or ED3A.
• the chelating agent is present in the composition at a concentration which is in molar excess of the ferric iron in the liposome suspension.
• the liposomes may be prepared by a variety of techniques, such as those detailed in Szoka et al.
• One preferred method for preparing drug-containing liposomes is the reverse phase evaporation method described in reference 3 and in U.S. Patent No. 4,235,871.
• 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 emulsion.
• the drug to be entrapped is added either to the lipid solution or aqueous medium. After removing the lipid solvent by evaporation, the resulting gel is converted to liposomes, with an encapsulation efficiency, for a water-soluble drug, of up to 50%.
• the reverse phase evaporation vesicles have typical average sizes between about 2-4 microns and are predominantly oligolamellar, that is, contain one or a few lipid bilayer shells.
• the oligolamellar nature of the vesicles may facilitate slow drug efflux and thus contribute to a lower efflux half life for an encapsulated drug.
• One advantage of REVs in the present invention is the high ratio of encapsulated drug to lipid which is possible, allowing greater drug doses to be administered by a surface coating of liposomes. Preparation of REVs is described in Example V.
• a simple lipid-hydration procedure for producing multilamellar vesicles may be preferred where high drug encapsulation efficiency is not desired.
• a mixture of liposome-forming lipids dissolved in a suitable solvent is evaporated in a vessel to form a thin, film, which is then covered by an aqueous solution of the drug.
• the lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.
• the drug to be encapsulated is added either to the initial lipids or to the hydrating medium, depending on its solubility in water.
• the percent of total drug material which can be encapsulated in the MLVs. calculated as the ratio of encapsulated drug to total drug used in vesicle preparation, is typically between about 5-20% for water-soluble drugs.
• MLV preparation is also illustrated in Example V.
• 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: (1) more uniform drug release properties, (2) higher density of liposome packing allowed at a mucosal tissue surface, and (3) greater optical clarity in ophthalmic applications.
• One effective sizing method involves extruding an aqueous suspension of the liposomes through a polycarbonate membrane having a selected uniform pore size, typically 0.2. 0.4. 0.6, 0.8 or 1 microns (reference 3).
• the pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane.
• a more recent method involves extrusion through an asymmetric ceramic filter. The method is detailed in co-owned U.S. patent application for Liposome Extrusion Method. Serial # 829.710, filed February 13, 1986.
• the REV or MLV preparations can be treated to produce small unilamellar vesicles which are characterized by sizes in the 0.04-0.08 micron range.
• SUV suspensions can be optically quite clear, and thus advantageous for ophthalmic applications.
• Another advantage of SUVs, as suggested above, is the greater packing density of liposomes on a mucosal surface which can be achieved with smaller liposome particles. This feature is valuable, for example, in one of the novel uses of the invention, where the liposomes are used as an ocular lubricant in the treatment of dry eye.
• One preferred method for producing SUVs is by homogenizing an MLV preparation, using a conventional high pressure homogenizer of the type used commercially for milk homogenization.
• the MLV preparation is cycled through the homogenizer, with periodic sampling of particle sizes to determine when the MLVs have been substantially converted to SUVs.
• Example V which describes the conversion of MLVs to SUVs with both moderate-pressure and high-pressure homogenization.
• SUVs containing either 30 mole percent lysinyl PE or 30 mole percent cholesterol ester of ⁇ -alanine were examined for stability, as evidenced by the leakage of encapsulated radiolabeled sucrose from the liposomes.
• REVs and MLVs would be expected to be even more stable for encapsulation, since leakage effects related to membrane curvature strain and unequal charge distribution across the membrane would be less.
• the liposomes can be treated, if necessary, to remove free (non-entrapped) drug. Conventional separation techniques, such as centrifugation, diafiltration. and molecular-sieve chromatography are suitable. The following examples illustrate specific methods for synthesizing amine-derivatized lipid components, and for producing liposomes with selected mucosal retention properties.
• Example XII The studies reported in Example XII verify that a significantly enhanced liposome retention is seen with a variety of other tissue types, including trachea, esophagus, stomach, small intestine, and rectum.
• the study compared the binding to the tissue of uncharged liposomes with liposomes containing either 20 mole percent stearylamine or 20 mole percent lysine lysinyl PE.
• both positively charged liposome preparations showed enhanced adhesion to most of the mucosal tissue type compared to the adhesion of the neutral liposomes.
• Lys-lys-PE liposomes showed twice the percent adhesion to the trachea, esophagus and small intestine and a lesser but significant enhanced adhesion to the stomach and rectum relative to the stearylamine containing liposomes.
• aqueous liposome suspensions prepared as above are suited to ophthalmic uses in which the liposomes are applied in droplet form to the eye.
• the liposomes are preferably sized, relatively small REVs or MLVs, or SUVs. typically at lipid concentrations of between about 5 and 50 umole lipid/ml.
• the retention of liposomes on mucosal tissue can be enhanced by including in the suspension, high molecular weight polymers which act to increase suspension viscosity.
• Typical polymers for use in ophthalmic formulations are methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and polyvinylalcohol. The effect of these polymers on ocular retention was examined in the studies reported in Example XI. In a first study. SUVs prepared with either 20 mole percent lysinyl PE or lysine lysinyl PE were formulated in a dilute suspension of buffer alone or buffer containing 0.8% hydroxyethylcellulose and 0.2% polyvinylalcohol, and ocular retention over a 1 hour period was measured. As may be seen, addition of polymers (solid symbols) significantly increased the level of liposome retention after 1 hour in both lysinyl PE SUVs (circles) and in lysine lysinyl PE SUVs (triangles).
• Neo-TearsTM polymer a product of Barnes Hind.
• the liposomal formulation is used to produce a sustained drug release at the ocular tissue site.
• Ophthalmic drugs suitable for delivery in liposomal-entrapped form, for sustained release over a several-hour period include: antiviral agents, such as fluorouracil, iodouridine. trifluorouridine. vidarabine, azidothymidine, ribavirin, phosphonoformate, phosphonoacetate. and acyclovir; anti-allergic agents such as cromolyn, cemetidine, naphazoline, lodoxamide. and phenylepinephrine; anti-inflammatory agents such as predisolone, dexamethasone, and supraphen; and anti-glaucoma agents which act by lowering intraocular pressure, such as carbacol, N-demethylcarbacol.
• antiviral agents such as fluorouracil, iodouridine. trifluorouridine. vidarabine, azidothymidine, ribavirin, phosphonoformate, phosphonoacetate. and acyclovir
• anti-glaucoma agents which act as cholinesterase inhibitors, such as isoflurophate, exothioiodate, and demecarium bromide
• anti-glaucoma agents which act as ⁇ -blockers, such as timolol, depaxolol, meti-pranalol, levobunalol, and celiprolol.
• the amount of drug which is delivered in drop form may be substantially higher than that allowable in free solution form, since undesired side effects related to high free drug concentrations are reduced.
• the liposomes suspensions are also useful in ophthalmics for treating dry eye. This condition, which is characterized by poor moisture retention on the eye, has a number of distinct etiologies, including poor water-secretion by the lacrimal gland (Sjögren), , poor mucin secretion by goblet cells (Lemp), vitamin A deficiency (Lawrence), and alteration of film-forming lipids as a result of chronic blepharitis. These are primarily long-chain alcohols and acids and cholesterol esters, which are required for forming a stable preocular tear film (Anderson).
• One preferred lipid composition contains lipid components designed to contribute to film forming on the eye surface.
• the phospholipid components can be selected to yield optimal long chain fatty acids after hydrolysis. Small amounts of long chain alcohols and fatty acids can also be included without destabilizing the liposomes appreciably.
• Oxidative damage can be reduced, as discussed above in Section IIA, by using predominantly saturated phopholipids and cholesterol analogs (e.g., cholestanol), and actively inhibiting free-radical reactions by the combined action of a lipophilic free-radical scavenger, such as ⁇ -to ⁇ opherol and a water-soluble iron-specific chelator, such as desferrioxamine.
• a lipophilic free-radical scavenger such as ⁇ -to ⁇ opherol
• a water-soluble iron-specific chelator such as desferrioxamine.
• liposome/polymer suspensions In addition to liposome size, which is discussed above, the liposomes and polymers must be stable in terms of aggregative effects, such that if aggregation occurs, the liposome/polymer complexes can be readily dispersed by shaking. For both of the hydroxyethylcellulose/polyvinylalcohol, and NEO-TEARSTM polymers used in formulating ophthalmic liposomes, good optical clarity after two months storage at room temperature, and clouding which was observed could be cleared by moderate shaking.
• Aerosolized liposomes. or liposome sprays are a convenient vehicle for applying the liposomes to the nasal or oral mucosa.
• the liposomes are formulated as a dilute aqueous suspension, and sprayed from a conventional pump or squeeze spray bottle.
• the liposomes are formulated for use with fluorocarbon propellant solvents in a pressurized cannister system.
• the liposomes may be suspended in the propellant in powdered or aqueous paste form, or combined in paste or powdered form with the propellant during propellant release from the pressurized cannister.
• Exemplary drugs for delivery to the nasal mucosa in liposomal form include anti-allergens, anti-histimines, such as benadryl. diphenhydramine HCl, clemastine fumarate, promethazine HCl. and triprolidine HCl; vasoconstrictors, such as metaraminolbitartrate, epinephrine, norepinephrine, phenylephrine HCl, and ephedrine; and peptide hormones, such as insulin. calcitonin, growth hormone, epidermal growth factor, atrial natriuretic peptide, vasopressin, and oxytocin.
• anti-allergens such as benadryl. diphenhydramine HCl, clemastine fumarate, promethazine HCl. and triprolidine HCl
• vasoconstrictors such as metaraminolbitartrate, epinephrine, norepineph
• Exemplary drugs for delivery to the oral mucosa include anesthetics, such as benzocaine, lidocaine HCl, dyclonine HCl; and antiviral or antibacterial agents, such as amantadine HCl, fluorouracil, iodouridine, gentamicin, erythromycin, cephalosporin, and tetracycline.
• anesthetics such as benzocaine, lidocaine HCl, dyclonine HCl
• antiviral or antibacterial agents such as amantadine HCl, fluorouracil, iodouridine, gentamicin, erythromycin, cephalosporin, and tetracycline.
• Paste or foam formulations of the liposomes provide advantages of (1) relatively good stability on storage, (2) high drug capacity and (3) a high ratio of liposome-entrapped to free drug, particularly for water-soluble, liposome-permeable drugs.
• Liposome pastes or foams are suitable for application to burned or broken skin, ocular tissue, and in body cavities, where the high viscosity of the material helps maintain the material at the site of application.
• Methods for generating liposome pastes with up to 70% encapsulated aqueous volume have been described in co-owned patent application for "Liposome Concentrate and Method", filed May 7, 1986.
• the concentrate is preferably formed by ultrafiltration with continued recycling of the liposome suspension material.
• Liposome foams can be prepared using conventional two-chamber propellant devices, such as are used for cosmetic foams, such as shaving cream.
• a heavy liposome suspension contained in one chamber is mixed with propellant gas contained in a second chamber, and the gassified mixture or foam is expelled under the propellant release pressure through a discharge nozzle.
• U.S. Patent #3.326,416 describes a two-chamber propellant foam device which could be adapted readily for use in liposome foam generation.
• the high-retention liposomes of the invention can be embedded or encapsulated within several types of solid matrix supports, either to protect the liposomes from rapid clearance or breakdown and/or to provide slow release the liposomes from the matrix into the region of tissue mucosa.
• One type of matrix is a suppository designed either to be melted or dissolved in a body cavity, to release the embedded liposomes. Conventional materials and preparation methods for suppositories would be suitable, to the extent the liposomes are not exposed to transient temperatures above about 60 C.
• Biocompatible polymers such as collagen, polylysine, polylactic acid, polymethyacrylate, polyurethanes, polyglycolic acid, hydroxypropylcellulose, agar and agarose, are also suitable bulk carriers for the liposomes of the invention.
• Methods for preparing these polymers in cross-linked and/or gel form are well known, and the methods can be readily adapted to incorporate liposomes, again with the proviso that transient temperatures above about 60 C are avoided.
• Many of the polymers, such as agar, collagen, and polyurethanes can be formulated in permeable cross-linked structures which allow liposome movement through and out of the matrices at a selected rate.
• Matrices of this type are suitable for drug delivery in body cavities, where the matrix can be held in place over an extended period, or for ocular use, where the implant can take the form of a clear lens or the like.
• Other polymer compositions like polylactate, can be formulated as a biodegradable solid which release the entrapped slowly over an extended polymer degradation period Such matrices are suitable for liposome release in the mouth or stomach.
• Some of the polymer compositions, such as polylysine can be polymerized in a liposome suspension to form a polymer shell about individual liposomes, to form a coating which, for example, would protect the liposomes from rapid breakdown in the stomach.
• the liposomes of the invention give significantly enhanced binding to mucosal surface, by virtue of a high concentration of spaced positive charges on the liposomes surfaces. Additional enhancement is achieved in a close-packed lipid arrangement in the liposomes.
• the phospholipid. diglyceride, and cholesterol lipid moieties which anchor the positive charges to the liposome membrane are tightly bound and contribute generally to close-packing in the lipid bilayer.
• the derivatized components of the present invention do not show toxicity effects at the relatively high concentrations needed to enhance binding to mucosal tissue.
• the liposomes can be prepared to include a broad range of drugs or other pharmaceutical agents, such as vitamins, peptides, enzymes or enzyme cofactors and the like, which are usefully administered to mucosal tissue in sustained release form.
• drugs or other pharmaceutical agents such as vitamins, peptides, enzymes or enzyme cofactors and the like
• the combination of liposomes. which provide slow release of the entrapped drugs, and long-term liposomal retention at the mucosal tissue site, allow an entrapped drug to be released in a sustained fashion over a several hour period, and at the same time, allow relatively high drug doses with reduced side effects associated with a high concentration of free drug.
• the enhanced-retention liposomes provide several advantages over polymer solutions for treating dry eye.
• the liposomes can be formulated in a variety of drug-delivery vehicles, including suspensions, aerosols, pastes, and solid matrix vehicles for delivering the liposomes to mucosal tissue in an optimal manner.
• L-lysine, L-arginine, L-histidine L-ornithine, L-lysinyl-lysine, and L-lysinyl-lysinyl-lysine were obtained in monohydrochloride form from Sigma Chem Co. (St. Louis. MO); di-tert-butyldicarbonate, from
• N,N'-bis-(3-aminopropyl)-piperazine from Aldrich Chemical Co. (Milwaukee, WI); potassium phthalimide, from Aldrich Cem. Co (Milwaukee, WI); trinitrobenzenesulfonic acid (TNBS) from Aldrich Chemical Co. (Milwaukee, WI); silica gel TLC plates, from J.T. Baker Chem. Co. (Phillipsburg. NJ); Kieselgel 60. 70-230 mesh, from E.M. Science Company, (San Francisco, CA); alumina. 80-200 mesh, from Fisher Scientific (Springfield, NJ); aluminum-hydroxide coated TLC plates, from E. Merck (Daumstadt. Germany); celite 545.
• the suspension was filtered with suction to remove the dicyclohexylurea. and the filter cake was washed with a few ml of chloroform. The washings were combined with the filtrate and used immediately in the preparation of the PE amide of tBOC-lysine.
• the entire chloroform solution (filtrate), containing no more than 1.5 mmole of the anhydride of tBOC-lysine. was added to 900 mg (1.21 mmole) of egg PE and 200 ⁇ l (1.43 mmole) of triethylamine. The solution was permitted to stand 18 hours at room temperature and then evaporated to constant weight under vacuum. The residue was redissolved in 10 ml chloroform and used directly in the preparation of the PE amide of lysine.
• the crude L-lysine PE was dissolved in 10 ml chloroform and placed at the top of a 21 mm x 270 mm chromatographic adsorption column packed with Kieselgel 60, 70-230 mesh. Development was with 100 ml 5% methanol in chloroform, followed by 100 ml 10% methanol in chloroform, followed by 200 ml 20% methanol in chloroform, followed by 400 ml 100% methanol. Separate 50 ml aliquots of column effluent were saved and analyzed by TLC, using CHCl 3 , CH 3 OH, H 2 O; 65:25:4.
• the chloroform solution of the egg PE amide of tBOC-arginine from IIB was diluted to 2.0 ml with chloroform and 0.7 ml trifluoracetic acid and 18 ⁇ l of water (1 mmole) and let stand at room temperature for 1 hour.
• the combined chloroform extracts were dried over anhydrous sodium sulfate and placed at the top of a 10 mm x 240 mm chromatographic absorption column packed with Kieselgel 60, 70-230 mesh.
• the column was developed by passing through it, in sequence. 50 ml 100% chloroform. 50 ml 10% methanol in chloroform, 50 ml 20% methanol in chloroform, 50 ml 40% methanol in chloroform, and 100 ml 100% methanol. Twenty ml portions of effluent were saved and analyzed by TLC on silica gel coated plates, using CHCl 3 , CH 3 OH, H 2 O, 65:25:4 (v/v) as developer. Those fractions which contained a phosphate positive and ninhydrin positive spot with R f about 0.17 were combined. Evaporation to dryness yielded about 9 mg of colorless waxy material.
• esters are formed by the steps of (A) N-protecting the corresponding amino acid, (B) forming the anhydride of the protected amino acid, (C) reacting the hydride with cholesterol, to form the corresponding cholesterol ester, and (D) deprotecting the ester to form the final product.
• the amino acid protection reaction follows the general preparing tert-butoxycarbonyl amino acids described by Moroder, et al. 4.45 gm (50mmole) of ⁇ -alanine were dissolved in a mixture of 50 ml 1 N sodium hydroxide, 50 ml water, and 100 ml/dioxane. To the resulting solution cooled in an ice bath was added with stirring, at 10°C. 12.0 gm (55 mmole) of di-tert-butyl dicarbonate. When the reaction mixture temperature had fallen to 5°C, the ice bath was removed and stirring at room temperature was continued for another hour.
• Table III gives the corresponding yields, R f values on silica gel developed with 1:n y cyclohexane: ethyl acetate 1:1, and color when heated in the presence of sulfuric acid.
• the syrup was redissolved in 100 ml chloroform and shaken with enough 10% aqueous sodium carbonate to raise the ph to 10.
• the resulting concentrate was suction-filtered through a filter coated with celite 545 to remove most of the suspended solid.
• the chloroform was separated from the aqueous phase of the filtrate and the aqueous phase reextracted with 100 ml chloroform.
• the chloroform phases were combined, washed with 20 ml 10% Na 2 CO 3 , and evaporated to dryness to obtain 15.2 gm of a white paste.
• the chromatogram was developed successively with 50 ml methylene chloride, followed by 50 ml 5% methanol, followed by 50 ml 10% methanol in methylene chloride. Separate 20 ml portions of effluent were collected. Evaporation of the second portion of effluent collected yielded 10 mg of material still impure by TLC, on silica gel plates developed with 50% ethylacetate/50% cyclohexane.
• Re-chromotography was developed successively with 50 ml methylene chloride, followed by 50 ml 5% methanol followed by 50 ml 10% methanol in methylene chloride. Separate 20 ml portions of effluent were collected. Evaporation of the second portion of effluent collected yielded 10 mg of material still impure by TLC, on silica gel plates developed with 50% ethylacetate - 50% cyclohexane. Re-chromotography on Al 2 O 3 , using 50 ml
• Example V Preparation of REVs, MLVs and SUVs
• This example describes the preparation of reverse phase evaporation vesicles (REVs), multilamellar vesicles (MLVs) and small unilamellar vesicles (SUVs) with a representative lipid composition containing 30 mole percent egg PC, 40 mole percent cholesterol, and 30 mole percent egg PE Amide of Lysine (Example I)
• REVs A total of 8 mg of the above lipid composition, containing 1 mole percent of ⁇ -tocopherol, was dissolved in 1 ml of diethyl ether. An aqueous buffer containing 13 mM phosphate, 140 mM NaCl, pH 7.4 was added to the organic solvent to a final volume of 1.3 ml. and the mixture was emulsified by sonication for 1 minute, maintaining the temperature of the solution at or below room temperature. The ether solvent was removed under reduced pressure at room temperature, and the resulting gel was taken up in 1 ml of the above buffer, and shaken vogorously.
• the resulting REV suspension had particle sizes, as determined by microscopic examination, of between about 0.1 to 20 microns, and the was composed predominantly of relatively large (greater than 1 micron) vesicles having one or only a few bilayer lamellae.
• Profiles of lipid phosphorous eluting from the column reflect the transformation of large, multilamellar vesicles which are contained in the void volume, to small unilamellar vesicles which are included in the column and elute as a broad peak between about 0.08 and 0.04 microns.
• the mean particle size of the vesicles progressively decreased. Based on the column elution profiles, about 60%, 75%, 87%, and 90% of the MLVs were converted to SUVs (less than about 0.06 microns) after 10, 20, 30, and 50 passages, respectively.
• the final preparation showed good optical clarity
• Example VI Sucrose Release from Positively Charged Liposomes REVs containing 30 mole percent PC, 40 mole percent cholesterol and 30 mole percent lysinyl PE (composition 1) or 70 mole percent. PC and 30 mole percent cholesteryl B-alanine were prepared as in
• Example IV The aqueous medium used in liposome preparation contained 14 C sucrose, yielding REVs with encapsulated 14 C sucrose. The vesicles were freed of free 14 C sucrose by washing with centrifugation.
• the washed liposomes containing the entrapped sucrose were resuspended in a physiological- saline-buffer, and incubated over a four hour period at room temperature. The amount of released sucrose was measured each hour over the incubation period. For both liposome compositions, the amount of free sucrose measured after 4 hour was about 10% of the total entrapped sucrose, indicating that the liposomes are quite stable for encapsulation of small molecular weight molecules.
• Example VIII Ocular Retention- Effect of Surface Charge Concentration
• Liposomes containing 40 mole percent cholesterol, either 10. 20, 30, or 40 mole percent lysinyl egg PE (from Example II), and remainder mole percent egg PC were prepared as in Example V.
• Control SUVs contained cholesterol and egg PC in a 40:60 mole ratio. All of the preparation contained about 10 5 counts per minute (CPMs) of1 125 I-PE per 100 nmole lipid. The final concentration of the liposome preparations was about 10 umole lipids/ml.
• PE were applied to the rabbit eye. Retention was assessed with the gamma probe positioned over the eye.
• the percent retention was measured at 2, 5, 10, 15, 30, 30, 45, and 60 minutes. Retention times of the five SUV preparations are shown in Figure 1. All values represent the mean of four rabbit eye measurements. As seen from the figure PC SUVs (solid squares) are poorly retained, falling to less than about 5% within one hour.
• the PE lysinyl SUVs show increasing levels of lysinyl PE, on progressing from 10 mole percent (open circles), to 20 mole percent (open triangles), to 30 mole percent (open squares) to 40 mole percent (closed circles), where 1 hour retention is about 45 % of the originally applied CPMs.
• the retention of the 40 mole percent lysinyl PE liposomes was similarly measured at every hour over a five hour period. The data show a gradual drop in liposome counts to about 20 percent within three hours, and thereafter, a very gradual loss to slightly below 20 % between hours 3 and 5.
• the four SUV preparation studies contained 40 mole percent cholesteryl, 10, 20, or 30 mole percent lysinyl-lysinyl PE, and remainder amounts of egg PC.
• SUV preparations containing either lysinyl PE with or without cholesterol or lysine lysinyl PE with or without cholesterol were tested for ocular retention.
• the compositions were: (1) egg PC:lynsinyl PE, 80:20, (2) egg PC:lysinyl PE:cholesterol 40:20:40, (3) egg PC:lysine lynsinyl PE, 80:20, and (4) egg PC: lysine lysinyl PE: cholesterol 40:20:40.
• the SUVs were prepared as in Example V, and tested for ocular retention as in Example VIII. The results are shown in Figure 3.
• Example X Effect of Cholesterol/Amine Spacer Arm Length
• SUV preparations containing 30 mole percent egg PC, 40 mole percent egg PE and 30 mole percent of (a) cholesteryl ester of glycine, (b) cholesteryl ester of B-alanine, (c) cholesteryl ester of ⁇ -amino caproic acid, and (d) cholesterol (control) were prepared.
• compositions (a), (b), and (c) have n values 1. 2, and >3, respectively.
• the compositions were prepared as in Example V and tested for ocular retention as in Example VIII.
• Example XI Ocular Retention- Effect of Polymer Additives In a first study, SUVs containing egg PC: lysinyl PE, 80:20 were mixed with equal volumes of either phosphate buffer (control) or a polymer solution containing 0.8% hydroxyethylcellulose and 0.2% polyvinylalcohol. Both compositions showed about 20% retention of labelled lipid counts after 1 hour, showing that the polymers produce very little improvement in ocular retention in non-cholesterol SUVs.
• Neo-Tears TM a commercial ocular polymer solution on ocular retention of SUVs containing either lysinyl PE (20 or 30 mole percent) or lysine lysinyl PE (10 or 20 mole percent).
• a control preparation contained 20 mole percent PE.
• the SUVs all contained 40 mole percent cholesterol and remainder egg PC.
• the polymer solution was mixed with an equal volume of each SUV preparation.
• the ocular retention over a 1 hour period of the polymer/SUV preparations is shown in Figure 6.
• the two low-retention curves are for control SUVs with (solid squares) and without (open squares) the polymer.
• the polymer provides some improvement, even in the absence of charge effects.
• the two lysinyl PE SUVs gave the retention plots indicated by the open and closed circles (20 and 30 mole percent lysinyl PE, respectively).
• the two lysine lysinyl PE SUVs gave the retention plots indicated by the open and closed triangles (10 and 20 mole percent lysine lysinyl PE, respectively).
• the three preparations each contained about the same specific activity of 125 -I PE, as a radioactive marker.
• the tissue pieces were placed on gauze pads and 20 ⁇ l of1 125 l-PE-liposomes samples were spread onto the lumen side such that none of the sample was in contact with the back side of the tissue. After one minute to several minutes, the samples were siphoned off and BME medium was used to rinse the mucosal tissue once. The wash medium was removed by siphon and the tissue was gently picked up by fine forceps and washed 3 times in 6 ml each of BME supplemented as described above. The tissue piece was then counted for 125 I.
• tissue piece was blotted dry and weighed.
• the 125 l cIounts were then normalized to 100 mg tissue weight for comparison within each tissue type. At least 3 pieces of similar tissues were used to determine the adhesivity.
• Both positively charged liposome preparations showed enhanced adhesion to most of the mucosal tissue types compared to the adhesion of the neutral liposomes.
• Lys-lys-PE liposomes showed twice the percent adhesion to the trachea, esophagus and small intestine and a significantly less enhanced adhesion to the stomach and rectum relative to the stearylamine containing liposomes.

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