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FIELD OF THE INVENTION
The present inventioin relates to liposomes designed for enhanced binding to mucosal tissue, and to a drug delivery system and method which uses the liposomes.
REFERENCES
Anderson, R.L., et al, Invest. Dermatol. 58:369 (1972).
Chabala, J.C., et al, Carbohydr Res 67:55 (1978).
Doebbert, T.W., et al, J Biol Chem 257(5):2193 )1982).
Doody, M.C., et al, Biochemistry 19:108 (1980).
Heath, T.D., et al, Biochim Biophys Acta 640:66 (1981).
Huang, C.H., et al, Lipids 12:348 (1977).
Kantor, H.L., et al, Biochemistry 17:3592 (1978).
Lawrence, D.J., et al, Ann NY Acad Sci 106:646 (1963).
Lee, V.H.L., et al, Survey of Ophthalmol 29:335 (1985).
Lemp, M.A. et al. Int Ophthalmol Clin 13:185 (1973).
Massari, S., et al. Biochim Biophys Acta 599:188(1980).
Mauk, M.R., et al, Proc Nat Acad Sci USA 77 (8):4430 (1980 )
Nagata, T.et al. Z. Natureforsch 34c:460 (1979).
Papahadjopoulos, D., et al, Biochim Biophys Acta 330:8 (1973).
Papahadjopoulos, D., et al, Biochim Biophys Acta 448:254 (1976).
Ponpipom, M.M., et al, Can J Chem 58:214 (1980).
Ponpipom, M.M. et al, J Med Chem 24:1388 (1980).
Ponipipom, M.M., et al. in Liposome Technology, Vol III, pp 95-115 (1984).
Robbins, J.C., et al, Proc. Nat Acad Sci USA 78(12):7294 (1981).
Schaeffer, H.E., Invest Ophthalmol Vis Sci 21:220-227 (1982).
Sjogren, H., et al, Sury Ophthalmol 16:145 (1971).
Szoka, F., Jr., et al. Ann Rev Biophys Bioeng 9:467 (1890).
Wu, M.S., et al, Biochim Biophys Acta 674:19 (1981 ).
Wu, P.S., et al, Proc Nat Acad Sci USA 78(4):2033 (1981).
Yashihara, E., et al, Biochim Biophys Acta 854:93 (1986).
BACKGROUND
Mucosal body surfaces, such as the corneal surface, and the surface epithelial lining of body cavities, are potentially useful sites for drug administration. For example, many ophthalmic diseases, such as viral and bacterial infections, and
chronic conditions, such as glaucoma, can be treated by topical durg administration to the ocular surface. Other mucosal tissue sites. including the nose. mouth, throat. rectum, vagina, and stomach are also important target areas of direct drug
adiministration.
Currently, many ophthalmic drugs are applied in soulution form to the ocular surface. A majon problem with this approach is limited drug uptake, since the drugs solution is rather quickly washed away by tearing action. Because of the rapid
clearance, an ophthalmic drugs may have to be adiministered several times a day. The frequent doses which are needed reduce patient compliance, and can be quite uncomfortable for the patient, as in the case of common anti-glaucoma drugs which cause
blurred vision for several hours after application.
The retention of a solution-form drugs on the corneal surface can be enhanced by the use of polymers. such as hydroxythylcellulose 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. However, with the increased viscosity, very little of the originally applied liquid is retained for more than about an hour, so frequent dosing is necessary.
For body-cavity sites, 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.
Even though suppositories and slow-release particles may give sustained drug release in the region of the mucosa, only a small percentage of the release drug may be taken up by the mucosa, due to rapid drug "clearance" by the normal cavity fluids.
The concept of using liposomes to enhance the delivery of drugs at a mucosal tissue has been proposed, but this approach has been limited heretofore by relatively poor retention of liposomes on mucosal tissue (Lee). Studies conducted in support
of the present invention, for example, show that retention of conventional liposomes on an ocular surface is less than about 5% after 1 hour. Thus, even though liposomes have the capability of controlled drug release over a several hour period, this
feature has not been exploitable in the past bacause of poor liposome retention at the target site.
Some improvement in liposome retention has been reported for liposome containing charged lipids, such as cholesteryl amine, into liposome. Presumably 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 liposome 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). However, charged amines of this type tend to be toxic at elevated levels (Yashihare) 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.
BACKGROUND OF THE INVENTION
It is therefore a general object of the invention to provide, for administering a drug to a mucosal tissue site, a drug/liposome composition which has significantly enhanced retention on mucosal tissue.
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.
It is yet another object of the invention to provide, for formulation into one of a number of possible liposome vehicles, a drug/liposome composition having an enhanced binding affinity for mucosal tissue.
According to one aspect of the invention, it has been discovered that significantly enhanced liposome binding to mucosal tissue is achieved if 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. In addition to the spacing of positive charges from the liposome surfaces, relatively tight packing in the bilayer membrane has been found to contribute to the enhancement of
liposome retention on mucosal tissue. This packing effect can be achieved either by the presence of cholesterol or a cholesterol derivative, at a concentration of between about 20-50 mole percent, or by the use of phospholipid or diglyceride components
containing predominantly saturated acyl chain moieties. The positive charges can be derivatized to either phospholipid or cholesterol components forming the vesicles.
More specifically, 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 liposome 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:
where PE--NH.sub.2 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 anhydried of the amino acid or peptide.
One preferred cholesterol derivative has the form
where Ch--OH is cholesterol, and 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:
where Ch--NH is cholesterol-3-aine and Y is a carbon-containing chain at least 2 atoms in length. The component is formed by coupling a diamine with a cholesteryl-3-halide.
The liposome composition may further be formulated for increase retention near the tissue site (as well as increased retention to the mucosal tissue). For ophthalmic uses, the formulation may include increase-viscosity polymers. For uses in
body cavities, the liposome may be formulated for delayed release in suppositories or slow-release polymer matrices. Aerosolized liposomes for nasal and oral drug deliverly, 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.
In still another aspect, 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.
These and other object and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 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:
FIG. 2 shows the retention on an ocular tissue of liposomes prepared with increasing concentrations of lysine lysinyl PE, including a neutral lipsome control (solid squares), and 10 (open circles), 20 (closed triangles), and 30 (open squares),
mole percent lysine lysinyl PE:
FIG. 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):
FIG. 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), .beta.-alanine (closed triangles),
and .epsilon.-amino caproic acid (open squares).
FIG. 5 shows the retention on an ocular tissue of liposomes prepared with either O (closed symbols) or 40 (open symbols) mole percent cholesterol, and 20 mole percent of either lysine PE (circles) or lysine lysinyl PE (triangles).
FIG. 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
FIG. 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).
DETAILED DESCRIPTION OF THE INVENTION
I. Preparing Amine-Derivatized Lipid Components
A. Structural Requirement
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 suger 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. In all of these lipid types, 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.
Both the 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.
According to an important aspect of the invention, it has been found that good enhancement of liposome retention requires that 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 ester
having various selected spacer chain lengths. The data on cholesteryl amines is summarized in FIG. 3, which shows that liposomes containing epi-cholesteryl amine (open circles) are comparable to uncharged liposome (closed squares) in liposome retention. By contrast, a epi-cholesteryl piperazine derivative, with a several atom chain (open triangle), shows about 50% retention after 1 hour.
A more systematic study on chain length, also reported in Example X, compares the ocular retention of liposome containing 40 mole percent of the cholesterol ester of glycine (open circles), .beta.-alanine (open squares) and .epsilon.-aminocaproic
acid (closed triangles). As seen in the FIG. 4, 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 liposome containing underivatized
cholesterol (closed squares). By contrast the cholesterol derivatives of both .beta.-alanine (three carbon spacer) and .epsilon.-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. Further, 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. In general 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 Derivatives
As defined herein, the term 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 chains are attached to the backbone by suitably stable linkage, including acyl ester, ether, thioether, amine, carbamate, or a dioxolane ring (Nagata) linkages. As indicated above,
exemplary dialiphatic lipids 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. In addition, the more
unsaturated chains produce greater chemical stability on ling term storage, and evidenced by reduced oxidative damage to the lipids.
In derivatized form, 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. For example, 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.
As defined herein, the term phospholipids encompasses phosphatidic acid (PA) and phosphatidyl glycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), Phosphatidylserine (PS), plasmalogens, and
sphingomyelin (SM).
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 (other than PA derivatives) differ from the above cholesterol and diglyceride derivatives in that the polar end region hydroxy 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. Thus it is only necessary, in forming the positively charged phospholipid (other than PA) for use in the invention, to place a net positive
charge at or near the end of the phosphate-ester linked moiety in the phospholipid. Several phospholipids, including PC, and PE, contain chain terminal amines which in the natural phospholipid, balance the negative charge of the phosphate group. These
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.
In another general approach, the phospholipid is derivated by coupling an amine, such as an amino acid, to a reactive end group in the phosphate-ester linked moiety of the lipid. 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. For example, 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. 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 phospholipid. Thus, where the amine is joined to PE or a plasmalogen through an
amide linkage, 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. As a further illustration, in derivatizing PS through an amide linkage involving the terminal
acid group, 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 by prepared by known methods. The lipid may be purified and/or modified in acyl chain composition according to known techniques. In
certain liposome formuations 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.
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. As a first step, 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.
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 in 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. The purified product may be stored as a dry residue under nitrogen at 4.degree. C. for up to several months.
In a second method for forming the amine PE derivative, 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.
C. Cholesterol Derivatives
As defined herein, the term cholesterol is intended to encompass cholesterol, (3-hydroxy-5,6-cholestene), and related analogs, such as 3-amino-5,6-cholestene, and 5,6-cholestene and cholestane and related analogs, such as 3-hydroxy-cholestane.
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.
To form the cholesterol ester, an amino acid of the form CO.sub.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. 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. Example III illustrates these reaction
methods for forming the cholesterol esters of the five amino acids CO.sub.2 (CH.sub.2).sub.n NH.sub.2, where n-1-5. The reaction conditions are applicable to other amino acids having one or more free amine groups. As noted above, only those cholesterol
ester derivatives in which the free amine is spaced from the cholesterol hydroxyl by three or more atoms (n greater than or equal to 2 in the Example III compounds) produce significant binding enhancement of liposomes to mucosal tissue.
It will be appreciated that 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.sub.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.sub.2 --Y--N, where N is a primary-quaternary amine, and preferably a primary amine. Typically, about a tenfold molar excess
of the diamine is reacted with the cholesteryl halide in 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-.alpha.-N-(-3-(4-(3-aminopropyl)piperazino)propyl)amine) from cholesteryl iodide and 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).
In addition, a variety of cholesterol amine and amino glycoside compounds having the requisite cholesterol/spacer arm/amine structure have been reported. The compounds were prepared, along with a variety of different uncharged thio-linked
glycoside cholesterol analogues, to examine the feasibility of targeting liposomes containing a selected surface glycoside to specific tissues, based on liposome interactions with glycoside-specific tissue receptors. (Ponpipom, 1980, 1981, 1984;
Chabala; Wu, M; Wu, P.; Robbins; Mauk; and Doebbert). Liposomes incorporating several of these cholesterol derivatives show a variety of glycoside-specific effects, including enhanced uptake by macrophages, increased retention at subcutaneous injection
site, increased stability in vivo, and tissue-specific liposome distribution. Comparative studies with a variety of uncharged glycosidic cholesterol derivatives, and with charged, but non-glycosidic cholesterol derivatives, including
aminohexylcholesterol, and amino ethyl cholesteryl carbamate, suggest that the pharmocokinetic effects observed are related primarily to glycoside interactions with tissue specific receptors, rather than to non-specific charge interactions, as in the
present invention. Where charge-related effects were observed, e.g., in liposome stability in vivo (Mauk), the mole percentage of charged lipid was relatively low (less then ten percent).
II. Liposome Preparation
A. Lipid Components
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 lipsome retention are presented in
Examples VIII, below, and are shown in FIGS. 1 and 2. FIG. 1 is a plot of ocular retention against time with liposomes containing increasing amounts of lysinyl PE, from 0 to 40 percent. As seen in FIG. 1, liposomes containing no lysinyl PE (solid
squares) were retained at less than about 5% after 1 hour. Increasing the amount of lysinyl PE from 10 (open circles), to 20 (open triangles), to 30 (open squares) to 40 (solid circles) mole percent gave progressively enhanced retention, with 40 mole
percent liposomes showing nearly 50% retention after 1 hour. FIG. 2 shows the same increased-retention effect, but with increasing mole percentages of lysine lysinyl PE. Here both 20 (solid triangles) and 30 (open circles) mole percent charged lipid
gave strong enhancement of ocular retention. Generally, 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, of 10 mole percent of a doubly charged component, such as lysine lysinyl PE, and so on.
The charge concentration noted above apply only to the outermost lipid layer in the liposomes, and therefore the liposomes themselves need not have a uniform charge density through their one or more bilayer regions. In fact, studies conducted in
support of the present invention indicated that in small unilamellar vesicles (SUVs), positive charge seems to localize preferentially on the outer of the two lipid layers forming the vesicle bilayer. The study is described in Example VII. As seen
there, SUVs formed with 20 mole percent lysinyl PE have contain about 75% of the total positive lipid charge in their outer lipid layer, and SUVs formed with 20 lysine lysinyl PE contain about 92% of the positive lipid charge in the outer layer. This
unequal transbilayer distribution of charge is probably related to the greater polar charge repulsion which is present on the inner side of the bilayer. The studies indicate in any case that the requisite 20 mole percent charge distribution on the outer
liposome surface can be achieved with less than 20 mole percent actual lipid used in formulating the liposomes.
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.
In the following discussion, the considerations governing the choice of 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. Experiments conducted in support of the invention show that the enhanced liposome retention on mucosal surfaces which is achieved with
amine-derivatized lipid components is enhanced still further using lipid components which tend to increase the packing density of lipids in the vesicle bilayers. In particular, a enhanced retention is seen either with addition of cholesterol or a
cholesterol derivative, or with a high percentage of saturated phospholipid acyl chain components, both of which are known to increase lipid packing (Papahadjopoulos, 1973). Studies on the effect of cholesterol on lipsome binding to an ocular surface
are detailed in Example VIII. With reference to FIG. 5, which summarizes the data, it is seen that the addition of 40 mole percent cholesterol (open symbols) significantly increased the binding affinity of liposomes containing either lysinyl PE
(circles) or lysine lysinyl PE (triangles). Cholesterol alone, in the absence of added amine-derivatized PE, produced no appreciable binding enhancement. Similar results were seen in PC/lysine PE liposomes, where PC with saturated acyl chains gave
signficantly greater retention of the amine-derivatized liposomes than did egg PC.
The degree of saturation of phospholipid components can also have a major effect on the rate of release of entrapped drug from the liposomes. In general, 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. For many drugs, 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, by contrast, 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.
Still anothe consideration in the choice of the lipid components is extent of lipid oxidative/peroxidative damage which can be tolerated. It is known, and experiments conducted in support of the invention have confirmed, that both unsaturated
phopholipids and cholesterol are susceptible to lipid oxidative damage, particularly where the lipids are stored over an extended period at above refrigeration temperatures. The problem of lipid oxidation damage would therefore be quite sever for a
lipsome product, such as an ophthalmic eye-drop composition, which is normally sold and stored at room temperature. Reduced oxidation can be achieved by using predominantly saturated lipid components, such as saturated phospholipids and diglycerides and
cholestane sterols.
Lipid peroxidative damage can also be reduced by a combination of a lipophilic free radical scavenger, such as .alpha.-tocopherol (.alpha.-T), and a water-soluble iron-specific chelator, such as desferrioxamine. This combination of protective
agent is discussed in U.S. patent application "Liposome/Anthraquinone Drug Composition and Method", Ser. No. 806,084, filed Dec. 6, 1985 and now U.S. Pat. No. 4,797,285 , and is based on the ability of the chelator/free-radical scavenger combination
to inhibit both the initiation and propagation of free-radical reactions in a liposome suspension.
The lipophilic free radical scavenger used in the composition is preferably .alpha.-T, or a pharmacologically acceptable analog or ester thereof, such as .alpha.-T succinate. Other 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.sup.30) and a relatively low binding
constant for 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. Alternatively, 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.
B. Liposome Preparation
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. 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 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 (REVs) have typical average
sizes beween 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 REV | | |
