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1. FIELD OF THE INVENTION
This invention relates to methods and compositions which are used to
enhance retention of administered bioactive agents at specific tissue or
organ sites in the body of man or animals. The present invention involves
covalent linkage of fibronectin to bioactive agents or their carriers to
form conjugates having high affinity for collagen-, heparin-,
fibrin/fibrinogen-, hyaluronic acid-, or ganglioside-rich body sites.
Methods and compositions described herein have a wide range of
applicability to the field of drug delivery systems. The practice of the
present invention is demonstrated herein by way of example for the
localized delivery of medicament to joints by intra-articular
administration of the medicament entrapped in liposomes with enhanced
affinity for joints conferred by fibronectin covalently cross-linked to
the lipid bilayer.
2. BACKGROUND OF THE INVENTION
2.1. TARGETED AND LOCALIZED DRUG DELIVERY SYSTEMS
In order to exert characteristic therapeutic effects an administered drug
must reach the proper site of action at an appropriate concentration.
Thus, desirable features of drug delivery systems include resistance to
rapid clearance and sustained release of drug at its site of activity.
Another desirable feature is the ability to deliver the drug to a specific
site of intended action without adversely affecting non-target tissues or
systems. This latter feature is especially important when administering
drugs such as anti-tumor agents which are particularly toxic, or drugs
such as local anesthetics or steroid hormones which may have undesirable
systemic side effects.
Although much has been written regarding the potential advantages of lipid
vesicles or liposomes as in vivo drug delivery systems, failure to deliver
drug to a specific site of activity continues to be a serious drawback
(see review by Mayhew and Papahadjopoulos, 1983, "Therapeutic Applications
of Liposomes" in Liposomes, Ostro, ed., Marcel Dekker, Inc., pp. 289-341).
Administration of targeted liposome preparations (liposomes designed to
"home" to specific tissue or cell sites upon application in vivo) has been
attempted by a number of researchers. Liposomes bearing substances such as
antigenic lipids, i.e., N-dinitrophenyl-aminocaproyl
phosphatidylethanolamine (Lesserman et al., 1979, J. Immunol. 122:
585-591), heat-aggregated IgM molecules (Weissman et al., 1975, Proc.
Natl. Acad. Sci. U.S.A. 72: 88-92) , and sialoglycoprotein which binds
lectins (Juliano and Stamp, 1976, Nature (London) 261:235-238)) have been
used in vitro to enhance specific binding to cell surfaces. Huang et al.,
1980, J. Bill. Chem. 255:8015-8018, demonstrated specific binding of
liposomes to mouse L-929 cells in vitro by incorporation of
anti-H-2-monoclonal antibody linked to palmitic acid into the lipid
bilayer. More recently, Martin and Papahadjopoulos have demonstrated
targeting of liposome preparations by the covalent linkage of Fab'
fragments via disulfide bonds to a derivative of phosphatidylethanolamine
incorporated into the lipid bilayer (1982, J. Biol. Chem. 257:286-288;
Martin, Hubbell and Papahadjopoulos, 1981, Biochem. 20:4229-4238).
The use of of liposome preparations for localized drug delivery has also
been attempted. In the liposome preparations which are designed to adhere
at the site of administration, enhanced affinity is conferred by means of
"stick" adjuvants. Liposomes with synthetic amino-saccharide compounds
conjugated to cholesterol incorporated into the lipid bilayer have been
shown to have enhanced affinity for cells or tissues when extravascularly
applied by subcutaneous administration (Wu et al., 1981, Biochim. Biophys.
Acta 674:19-29; Wu et al., 1981, Proc. Natl. Acad. Sci., U.S.A.
78:2033-2037).
Localized drug delivery has also been attempted by utilization of specific
routes of administration. For example localized administration of steroid
(cortisol palmirate) entrapped in dipalmitoylphosphatidylcholine liposomes
by means of intra-articular administration to joints has been demonstrated
to have superior anti-inflammatory activity compared to administration of
free steroid (Shaw et al., 1978, Ann. N.Y. Acad. Sci. 308: 435-436;
Philips et al., 1979, Ann. Rheum. Dis. 38: 553-557).
2.2. FIBRONECTIN
Plasma fibronectin, also called cold insoluble globulin (CIG) or large
external transformation sensitive (LETS) protein, is a dimeric
glycoprotein of approximately 440,000 daltons molecular weight. The
polypeptide chains of the dimer are linked by disulfide bonds. Fibronectin
binds to a number of naturally occurring substrates including
collagen/gelatin, heparin, hyaluronic acid, fibrin/fibrinogen,
transglutaminase substrates, gangliosides, cells, bacteria, actin and DNA.
(See Yamada, 1981, "Fibronectin and Other Structural Proteins" in Cell
Biology of the Extracellular Matrix, Hay, ed., Plenum Press, pp. 95-114).
Fibronectin has been cross-linked to collagen and various amines including
dansylcadaverine, spermine, spermidine, and putresane, and bacterial cell
membranes by Factor XIII or transglutaminase enzyme (Mosher and Proctor,
1980, Science 209:927-929; Mosher et al., 1980, J. Biol. Chem.
255:1181-1188; Mosher, 1975, J. Biol. Chem. 250:6614-6621).
Transglutaminase, a calcium dependent enzyme, catalyzes an acyl transfer
reaction in which gamma-carboxamide groups of glutamyl residues are acyl
donors and primary amines are acyl acceptors. Thus, transglutaminase forms
epsilon-gamma glutamyl-lysine linkages between proteins.
Although several studies have suggested the utilization of fibronectin
non-covalently bound to phospholipid vesicles as a possible means of
targeting liposomes to particular body sites rich in collagen (see Ross
and Wallace, 1983, J. Biol. Chem. 258:3327-3331; Chazon et al., 1981,
Proc. Natl. Acad. Sci., U.S.A. 78:5603-5607) no study to date has
demonstrated such specific targeting in vivo. Hsu and Juliano, 1982,
Biochim. Biophys. Acta 720:411-419, have demonstrated enhanced adherence
of liposomes coated with fibronectin (by a non-covalent association) to
macrophages in vitro. While such preparations may be useful to treat
intracellular infections of the reticuloendothelial system, the liposomes
would presumably be cleared rapidly from the circulation. Moreover, the
non-covalently attached fibronectin-lipid associations suffer from several
disadvantages. Such associations are unstable, and the amount of
fibronectin associated cannot easily be controlled.
2.3. N-HYDROXYSUCCINIMIDE METHOD OF CONJUGATION
Activated esters of N-hydroxysuccinimide (NHS) have long been used to form
peptide linkages between free carboxyl groups and primary amines.
(Anderson et al., 1964, J. Am. Chem. Soc. 86:1839-1842).
Huang et al. utilized an activated ester of palmitic acid and NHS to form a
conjugate of palmitic acid and monoclonal anti-H-2 antibody which was then
incorporated into liposome bilayers. (1980, J. Biol. Chem. 255:
8015-8018). Peptide linkage between the antibody and palmitic acid was
performed in the presence of detergent (deoxycholate) in order to
solubilize the lipid which would precipitate in an aqueous buffer.
Formation of liposomes with antibody conjugate incorporated was
accomplished by dialysis to remove detergent.
Recently this group of investigators has incorporated this derivatized
palmitoyl-antibody into liposomes by mixing the derivatized antibody in
detergent with preformed liposomes made by a modified reverse evaporation
method (Shen et al., 1982, Biochim. Biophys. Acta 689:31-37). A high level
of attachment of antibody, capable of binding to antigen, was achieved
without inducing leakage of vesicles; however, a lengthy, 40-hour dialysis
was necessary to remove the detergent. (See also, Huang et al., 1982,
Biochim. Biophys. Acta 716:140-150). Although these liposomes could be
shown to adhere to cells in vitro, targeting in vivo may be obscured by
removal of vesicles by the reticuloendothelial system.
The covalent attachment of long chain fatty acids to the hydrophilic
soybean protein glycinin was reported by Haque and Kito (1982, Agric.
Biol. Chem. 46(2):597-599). Active NHS esters of lipids were reacted with
glycinin in a reaction buffer consisting of tetrahydrofuran (THF) and 8M
urea. The conjugated product in solution was dialyzed exhaustively for
three days against deionized water to remove THF before freeze-drying.
3. SUMMARY OF THE INVENTION
This invention presents new and substantially improved compositions and
methods for enhancing localized retention of administered bioactive agents
at specific sites in vivo which are rich in collagen, heparin, hyaluronic
acid, fibrin/fibrinogen, gangliosides, or transglutaminase substrates. The
invention is based upon the fact that fibronectin has a strong affinity
for such sites in vivo. When fibronectin is covalently linked to a
bioactive agent or to its carrier, the affinity of the resulting conjugate
for appropriate sites in vivo is greatly increased. For example, enhanced
affinity for collagenous sites is conferred by the covalent binding of
fibronectin to a bioactive agent or to its carrier (e.g., the liposome in
which the bioactive agent is entrapped).
According to the present invention, fibronectin conjugates are prepared by
either of two methods. According to one method, thrombin-activated
calcium-dependent Factor XIII, or transglutaminase enzyme, is used to
crosslink fibronectin to a number of substrates containing a plurality of
amines; such substrates include but are not limited to lipids which are
incorporated within a liposome, peptides, proteins, aminoglycoside
antibiotics, etc. A second method entails a substantially improved
modification of the N-hydroxysuccinimide (NHS) protocol for formation of
peptide bonds between fibronectin and lipid compounds.
In order to effect localized delivery in vivo, conjugates of the present
invention may be administered directly to the site in vivo or may first be
incorporated into liposome preparations which provide sustained release of
entrapped medicament. Covalent linkage of the fibronectin to lipid is
essential for stability of liposomes containing fibronectin. The
fibronectin conjugates described herein offer the following advantages:
(1) they adhere to body sites rich in collagen, fibrin/fibrinogen,
hyaluronic acid, heparin, transglutaminase substrates, actin, etc.;
(2) they reduce systemic side effects by localizing medicament at the site
of administration; and
(3) they concentrate medicament at the site of action.
As a result, these conjugates overcome problems of rapid clearance and
non-specificity associated with conventional drug delivery systems.
4. DETAILED DESCRIPTION OF THE INVENTION
The present invention involves methods and formulations of conjugates of
fibronectin (i.e., fibronectin covalently bound to amine-containing
compounds such as lipids, peptide hormones, proteins, or aminoglycosides
or to carboxyl-containing lipids such as fatty acids, acidic
phospholipids, derivatized phospholipids or liposomes) which have numerous
advantages for use as drug delivery or carrier systems. Fibronectin
covalently bound to bioactive agents may be administered directly or
incorporated into a liposome preparation which is then administered in
vivo. Alternatively, if fibronectin is bound to a lipid, this conjugate
may be incorporated into the lipid bilayer of liposomes containing an
entrapped bioactive agent.
The following sections describe how the conjugates are prepared and used.
4.1 ENZYMATIC PREPARATION OF FIBRONECTIN CONJUGATES
Fibronectin can be obtained from the fibrinogen fraction of vertebrate
blood, and is commercially available as lyophilized preparations. Rather
than using commercially available fibronectin, it is possible to isolate
naturally occurring substance from the plasma of an intended recipient
and, after appropriate purification, administer the same, thereby
minimizing the possibility of immune reactions.
According to one embodiment of the present invention, conjugation of
fibronectin to compounds containing a plurality of amines may be achieved
by the enzymatically catalyzed cross-linkage of the glutamine of the
N-terminus of the fibronectin glycoprotein to the compound.
Blood coagulation Factor XIII or transglutaminase, a calcium dependent
enzyme, has been demonstrated to crosslink fibronectin (via glutamyl
residues) to various amine containing substrates such as bacterial
membranes (Mohser and Proctor, 1980, Science 209:927-929); polyamines such
as putrescine, spermidine, spermine, dansylcadaverine,
N-(5-aminopentyl)-5-dimethylamino-naphthalene-1 sulfonamide (Mosher et al.
1980, J. Biol. Chem. 255:1181-1188; 1977, Hoppe-Seyler's Z. Physiol. Chem.
Bd. 358:1165-1168); and to fibronectin itself (Mosher, 1975, J. Biol.
Chem. 250:6614-6621).
Factor XIII is utilized in the present invention to catalyze the
crosslinking of fibronectin to a number of substrates. Presumably the
crosslinking occurs via an acyl transfer reaction in which the
gamma-carboxamide group(s) of peptide-bound glutamyl residue(s) of
fibronectin function as acyl donors. Substrates containing a plurality of
amines function as acyl acceptors. These substrates include but are not
limited to: lipids, such as phosphatidylethanolamine, phosphatidylserine,
etc. which are incorporated into liposome membranes; proteins such as
fibrinogen, etc.; peptide hormones such as somatotropin or growth hormone,
luteinizing hormone, etc.; aminoglycosides such as gentamicin, neomycin,
tobramycin, and kanamycin, etc. Thus, fibronectin may be conjugated
directly to a lipid molecule which is incorporated into a liposome or to a
bioactive agent. It should be noted that the transglutaminase catalyzed
reaction is carried out in an aqueous buffer; therefore, lipid substrates
(which are not soluble in aqueous solutions) must be incorporated into
liposomes in order to function as the enzyme substrate in the aqueous
reaction mixture.
Fibronectin-liposome conjugates of the present invention may be prepared
using the following protocol: liposomes, prepared as described herein
(Section 4.3.) using egg phosphatidylcholine (EPC) and between 0.5 to 40
mole % phosphatidylethanolamine (PE) (e.g., 3.71 mg PE) are incubated at
room temperature for 2 hours with greater than 0.3 mg (e.g., 0.65 mg)
fibronectin, 20 to 200 ug Factor XIII (transglutaminase), and at least 1
unit Thrombin (range 1 to 10 units) in 5 to 50 mM CaCl.sub.2.6H.sub.2 O
(one unit thrombin will clot a 250 mg % fibrinogen solution in 15 seconds
at 37.degree. C.). As a result the fibronectin is covalently linked to the
PE which is incorporated into the liposome bilayer.
According to one embodiment of the present invention, enzyme catalyzed
cross-linkage of fibronectin to stable plurilamellar lipid vesicles (SPLVs
prepared as described infra) was effected using a number of formulations.
Formulation 1: SPLV-entrapped streptomycin (control SPLVs) was prepared
using 100 mg EPC and 100 mg streptomycin sulfate; Formulation 2: control
SPLVs were prepared except 0.65 mg fibronectin was added externally to the
lipid ingredients in order to non-covalently associate with the liposomes;
Formulation 3: control SPLVs were incubated with 0.65 mg FN, 90 ug Factor
XIII, 1 unit Thrombin in 20 mM CaCl.sub.2.6H.sub.2 O; Formulation 4:
SPLV-entrapped streptomycin, prepared using 100 mg EPC, 3.71 mg
phosphatidylethanolamine (PE), and 100 mg streptomycin sulfate was
incubated with 0.65 mg FN, between 90 to 100 ug Factor XIII, 1 unit
Thrombin in 20 mM CaCl.sub.2.6H.sub.2 O in order to covalently attach the
fibronectin to the lipid bilayer. In all cases a trace amount of .sup.125
I-p-hyroxyphenylpropionic acid derivatized gentamicin sulfate (.sup.125
I-GS) was incorporated as radiolabel. All preparations were incubated at
room temperature for 2 hours, centrifuged at 15,000.times.g for 10
minutes, and washed 2 times in PBS. Aliquots of the FN-modified SPLVs were
applied to collagen Sepharose columns (prepared as described below) to
determine binding of fibronectin-modified vesicles.
The following collagen or gelatin Sepharose assay system was developed to
measure the affinity of FN-modified SPLVs for collagen: 5 gm of cyanogen
bromide activated Sepharose 4B was washed on a glass filter with 1 liter
of 1 mM HCl to remove dextran/lactose stabilization additives. Fifty mg of
gelatin was dissolved with gentle heating in 50-100 ml of coupling buffer
containing: 0.1M NaHCO.sub.3 and 0.5M NaCl at pH 8.3. The Sepharose was
washed in coupling buffer and mixed with the cooled gelatin mixture. After
2 hours incubation at room temperature (with mechanical shaking), the
unbound gelatin was washed away with coupling buffer. The remaining active
groups were reacted with 100 ml of blocking groups (either 0.2M glycine at
pH 8 or 1M ethanolamine at pH 8) for 1-2 hours. Three washing cycles were
used each consisting of wash with acetate buffer (0.1M sodium acetate plus
1M NaCl at pH 4), followed by bicarbonate buffer wash (0.1M NaHCO.sub.3
plus 1M NaCl pH 8.3).
FN-modified SPLVs migrated into the collagen column whereas liposomes
without FN did not, thus indicating successful attachment of the
fibronectin to the SPLVs. Radiolabel entrapped in the FN-modified SPLVs
could be eluted in significant amounts only with 8M urea.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of
fibronectin-modified liposomes demonstrated that covalent cross linkage of
fibronectin to the lipid bilayer was essential for prolonged stability of
vesicle preparations. One month after SPLVs were prepared lipids were
extracted and protein was precipitated using ethanol-chloroform (9:1).
When applied to 7.5% SDS polyacrylamide gels only preparations of SPLVs
with fibronectin covalently attached (i.e., SPLVs incubated with
fibronectin and Factor XIII; or SPLVs incubated with fibronectin, Factor
XIII, and thrombin) demonstrated the characteristic fibronectin band of
about 200,000 daltons molecular weight. SPLVs prepared with fibronectin
non-covalently associated (i.e., SPLVs incubated with fibronectin but no
enzyme) did not exhibit this banding, indicating dissociation of the
fibronectin-lipid complex and possible breakdown of the fibronectin
molecule itself. Thus, for stable liposome preparations with enhanced
affinity for collagen-rich areas it is desirable to covalently cross-link
the fibronectin to the lipid.
4.2. PREPARATION OF FIBRONECTIN CONJUGATES BY A MODIFIED NHS METHOD
According to another embodiment of the present invention, fatty acids,
acidic phospholipids such as phosphatidic acid or phosphatidylserine,
derivatized phospholipids or liposomes may be conjugated with fibronectin
using a modified NHS procedure. These fibronectin-lipid conjugates may
then be incorporated into liposomes for therapeutic use.
Procedures for this modified NHS method of conjugate preparation are
disclosed in U.S. Patent Application by Robert P. Lenk, Sharon S.
Carpenter-Green, Alan L. Weiner, and Michael W. Fountain entitled
"Improved Methods For Conjugate Formation Using N-hydroxysuccinimide Lipid
Esters" filed concurrently herewith now abandoned.
In an N-hydroxysuccinimide reaction, dicyclohexylcarbodiimide is utilized
to activate a free carboxyl group on the lipid molecule and effect an
activated ester linkage with the amine groups of NHS in order to form a
reactive intermediate (NHS-lipid intermediate). For example, the
intermediate composed of either NHS-fatty acid or NHS-phospholipid ester
is prepared by incubation of dicyclohexylcarbodiimide, NHS, and either a
fatty acid or phospholipid at room temperature for several hours. When
fibronectin is added to the reaction mixture containing the NHS-lipid
intermediate, the fibronectin-lipid conjugate is formed.
The conventional reaction mixture used to make lipid conjugates is an
emulsion of the NHS-lipid intermediate and the protein prepared using
detergent in an aqueous environment. In contrast, the reaction solutions
of the present invention comprise approximately equal volumes of an
organic solvent containing the NHS-lipid ester intermediate and aqueous
buffer containing fibronectin (organic-aqueous reaction buffer). The
organic solvent must (1) be miscible with aqueous solutions, (2)
solubilize the NHS-lipid intermediate, and (3) not denature the
fibronectin. Solvents which may be used in the process include but are not
limited to dimethylformanide (DMF), tetrahydrofuran (THF), dioxane, and
lower number carbon aliphatic alcohols (i.e., not greater than 5 carbon
atoms including but not limited to methanol, ethanol, propanol, butanol,
pentanol, etc.)
When the reaction in organic-aqueous reaction buffer is complete, a large
amount of aqueous buffer is added to the reaction mixture. The unreacted
fibronectin will be solubilized by the aqueous buffer and the lipid
conjugate and unreacted lipids will precipitate out of solution. The
suspension is separated by centrifugation or filtration, and the
supernatant or filtrate containing unreacted compound is removed.
Organic solvent is then added to the mixture of conjugate and unreacted
lipid in order to solubilize the unreacted lipid, but not the conjugate.
Of course, the addition of organic solvent to remove unreacted lipids may
precede the addition of aqueous buffer to remove the unreacted
fibronectin.
Conjugates of the present invention may be incorporated into the lipid
bilayer of liposomes. Incorporation of preformed lipid conjugates may be
accomplished either by mixing the lipid conjugate with preformed
liposomes, or by adding the conjugate to a mixture of lipids used to
prepare the liposomes. Alternatively, NHS-lipid intermediates may be
incorporated into the liposome bilayer and fibronectin added to such
preparations to form the fibronectin-lipid conjugate covalently attached
to the liposome.
In one experiment a liquid phase synthesis was employed to prepare a
fibronectin-palmitate conjugate. An NHS-palmitate intermediate was
prepared as follows. The reaction mixture composed of: 500 mg NHS
dissolved in 15 ml ethyl acetate, 750 mg palmitic acid, and 1.25 ml ethyl
acetate containing 773 mg dicyclohexylcarbodiimide was incubated overnight
at room temperatures. Following incubation, the solution was filtered
(Whatman #1), rotoevaporated to remove ethyl acetate, and redissolved in
40 ml ethanol at 60.degree. C. The solution was then cooled to -20.degree.
C., filtered, and the NHS-palmitate crystals thus formed were washed with
ice cold ethanol.
The reaction mixture to form the conjugate, composed of 1 mg fibronectin
[dialyzed against phosphate buffered saline (PBS)] with trace amount of
.sup.125 I-fibronectin as radiolabel in 1 ml PBS and 5 mg NHS-palmitate in
1 ml DMF, was incubated for 30 minutes at room temperature. Following
incubation, 5-7 ml Tris-HCl pH 7.4 was added and the suspension was
centrifuged at 16,000.times.g for 30 minutes. The aqueous phase was
removed, the insoluble pellet containing the reaction product was
redissolved in 2 ml DMF to remove unreacted NHS palmitate, and the mixture
was centrifuged again. After washing with Tris-HCl buffer, radioactivity
of the remaining pellet was determined. Recovery of 56% of initial
.sup.125 I-fibronectin radiolabel as derivatized product indicated that
this method affords a rapid, easy and efficient method of covalently
linking lipid compounds to fibronectin.
Stable plurilamellar vesicles (SPLVs) were prepared (as described infra
using 40 mg egg phosphatidylcholine (EPC). The liposome preparation was
then incubated with a trace amount of .sup.125 I-FN-palmitate conjugate
for 4-5 hours at 4.degree. C. with shaking in order to form the
fibronectin modified SPLVs. Partitioning of the radioactive marker into
the SPLVs occurred (60% of the radiolabel was detected in the pelleted
liposomes). Aliquots of the fibronectin-modifed SPLVs (FN-SPLVs) were
applied to collagen Sepharose columns prepared as previously described.
Liposomes containing FN-palmitate in the bilayer migrated into and adhered
to collagen columns.
4.3. LIPOSOME PREPARATIONS
According to the present invention, liposome preparations in which
fibronectin (FN) constitutes an active component incorporated into the
lipid bilayer by the methods described supra may be utilized to provide
for enhanced retention of entrapped bioactive agent at the site of
administration. While it is possible to use as little as 0.1% fibronectin
(by weight of phospholipid in the liposomes), generally the amount will be
from 1 to 5%. Upon administration in vivo such liposome compositions are
localized to body sites rich in collagen (or elastin), heparin, hyaluronic
acid, fibrin/fibrinogen, gangliosides, or transglutaminase substrates due
to the affinity of FN for these substances. Various liposome preparations
which may be used in the present invention are described in the
subsections below.
Liposomes used in the present invention can be prepared by a number of
methods, including but not limited to: the original methods of Bangham et
al. (1965, J. Mol. Biol. 13: 238-252) which yield multilamellar vesicles
(hereinafter MLVs); methods described in U.S. Pat. No. 4,522,803 granted
on Jun. 11, 1985 based on U.S. patent application Ser. No. 476,496 filed
Mar. 24, 1983 which yield stable plurilamellar vesicles (hereinafter
referred to as SPLVs); and methods described in U.S. patent application
Ser. No. 521,176 filed August 8, 1983, now U.S. Pat. No. 4,588,578 which
yield monophasic vesicles (hereinafter referred to as MPVs). The
procedures for the preparation of SPLVs and MPVs are described below.
SPLVs are prepared as follows: an amphipathic lipid or mixture of lipids is
dissolved in an organic solvent. Many organic solvents are suitable, but
diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated
hydrocarbons and ether are preferred. To this solution are added an
aqueous phase and the active ingredient to be entrapped. This biphasic
mixture is converted to SPLVs by emulsifying the aqueous material within
the solvent and evaporating the solvent. Evaporation can be accomplished
during or after sonication by any evaporative technique, e.g., evaporation
by passing a stream of inert gas over the mixture, by heating, or by
vacuum. The volume of solvent used must exceed the aqueous volume by a
sufficient amount so that the aqueous material can be completely
emulsified in the mixture.
In practice, a minimum of about 3 volumes of solvent to about 1 volume of
aqueous phase may be used. In fact, the ratio of solvent to aqueous phase
can vary up to 100 or more volumes of solvent to 1 volume aqueous phase.
The amount of lipid must be sufficient so as to exceed that amount needed
to coat the emulsion droplets (about 40 mg of lipid per ml of aqueous
phase). The upper boundary is limited only by the practicality of
cost-effectiveness, but SPLVs can be made with 15 gm of lipid per ml of
aqueous phase.
Most amphipathic lipids may be constituents of SPLVs. Suitable hydrophilic
groups include but are not limited to: phosphato, carboxylic, sulphato and
amino groups. Suitable hydrophobic groups include but are not limited to:
saturated and unsaturated aliphatic hydrocarbon groups and aliphatic
hydrocarbon groups substituted by at least one aromatic and/or
cycloaliphatic group. The preferred amphipathic compounds are
phospholipids and closely related chemical structures. Examples of these
include but are not limited to: lecithin, phosphatidylethanolamine,
lysolecithin, lysopnatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and
the cerebrosides. Specific examples of suitable lipids useful in the
production of SPLVs are phospholipids which include the natural lecithins
(e.g., egg lecithin or soybean lecithin) and synthetic lecithins, such as
saturated synthetic lecithins (e.g., dimyristoylphosphatidylcholine, or
dipalmitoyl-phosphatidylcholine or distearoylphosphatidylcholine) and
unsaturated synthetic lecithins (e.g., dioloyl-phosphatidylcholine or
dilinoloylphosphatidylcholine. The SPLV bilayers can contain a steroid
component such as cholesterol, coprostanol, cholestanol, cholestane and
the like. When using compounds with acidic hydrophilic groups (phosphato,
sulphato, etc.) the obtained SPLVs will be anionic; with basic groups such
as amino, cationic liposomes will be obtained; and with polyethylenoxy or
glycol groups neutral liposomes will be obtained. The size of the SPLVs
varies widely. The range extends from about 100 nm to about 10,000 nm (10
microns) and usually about 100 nm to about 1,500 nm.
The following is an example of the proportions that may be used in SPLV
synthesis: SPLVs may be formed by adding 50 micromoles of phospholipid to
5 ml of diethyl ether containing 5 micrograms of butylatedhydroxytoluene
(BHT) and then adding 0.3 ml of aqueous phase containing the active
substance to be encapsulated. The resultant solution which comprises the
material to be entrapped and the entrapping lipid is sonicated while
streaming an inert gas over the mixture thus removing most of the solvent.
See also Lenk et al., 1982, Eur. J. Biochem. 121:475-482 which describes a
process for making liposome-encapsulated antibodies by sonicating and
evaporating a solution of cholesterol and phosphatidylcholine in a mixture
of chloroform and ether with aqueous phase added, but does not set forth
the relative proportions of lipid to aqueous phase.
Another suitable liposome preparation which may be used is lipid vesicles
prepared in a monophasic solvent system, hereinafter referred to as
monophasic vesicles or MPVs. MPVs are particularly stable and have a high
entrapment efficiency. MPVs are prepared by a unique process as follows: a
lipid or a mixture of lipids and an aqueous component are added to an
organic solvent or a combination of organic solvents in amounts sufficient
to form a monophase. The solvent or solvents are evaporated until a film
forms. Then an appropriate amount of aqueous component is added, and the
film is resuspended and agitated in order to form the MPVs.
The organic solvent or combination of solvents used in the process must be
(1) miscible with water and (2) once mixed with water should solubilize
the lipids used to make the MPVs.
For example, an organic solvent or mixture of solvents which satisfies the
following criteria may be used in the process: (1) 5 ml of the organic
solvent forms a monophase with 0.2 ml of aqueous component and (2) the
lipid or mixture of lipids is soluble in the monophase.
Solvents which may be used include but are not limited to ethanol, acetone,
2-propanol, methanol, tetrahydrofuran, glyme, dioxane, pyridine, diglyme,
1-methyl-2-pyrrolidone, butanol-2, butanol-1, isoamyl alcohol,
isopropanol, 2-methoxyethanol, or a combination of chloroform methanol
(e.g., in a 1:1 ratio).
The evaporation should be accomplished at suitable temperatures and
pressures which maintain the monophase and facilitate the evaporation of
the solvents. In fact, the temperatures and pressures chosen are not
dependent upon the phase-transition temperature of the lipid used to form
the MPVs. The advantage of this latter point is that heat labile products
which have desirable properties can be incorporated in MPVs prepared from
phospholipids such as distearoylphosphatidylcholine, which can be formed
into conventional liposomes only at temperatures above the
phase-transition temperature of the phospholipids. The process usually
allows more than 30-40% of the available water-soluble material to be
entrapped during evaporation and 2-15% of the available water-soluble
material to be entrapped during the resuspension; and up to 70-80% of the
available lipid-soluble material can be entrapped if the lipid:drug ratio
is increased significantly. With MLVs the entrapment of aqueous phase,
which only occurs during the rehydration step since no aqueous phase is
present during the drying step, usually does not exceed 10%.
Most lipids may be constituents of MPVs. Suitable hydrophilic groups
include but are not limited to: phosphato, carboxylic, sulphato and amino
groups. Suitable hydrophobic groups include but are not limited to:
saturated and unsaturated aliphatic hydrocarbon groups and aliphatic
hydrocarbon groups substituted by at least one aromatic and/or
cycloaliphatic group. The preferred amphipathic compounds are
phospholipids and closely related chemical structures.
Specific examples of suitable lipids useful in the production of MPVs are
phospholipids which include but are not limited to the natural lecithins
or phosphatidylcholines (e.g., egg lecithin or soybean lecithin) and
synthetic lecithins, such as saturated synthetic lecithins (e.g.,
dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or
distearoylphosphatidylcholine) and unsaturated synthetic lecithins (e.g.,
dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine). Other
phospholipids include but are not limited to phosphatidylethonolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid,
ceramides and the cerebrosides. The MPV bilayers can contain a steroid
component such as cholesterol, coprostanol, cholestanol, cholestane and
the like. When using compounds with acidic hydrophilic groups (phosphato,
sulphato, etc.) the obtained MPVs will be anionic; with basic groups such
as amino, cationic liposomes will be obtained.
MPVs may advantageously be used in delivery systems wherein a bioactive
agent is entrapped within the MPV ("entrapped" is defined as entrapment
within the aqueous compartment or within the membrane bilayer). In order
to entrap one or more agents in MPVs, the agent or agents may be added to
the monophase prior to evaporation and formation of the film.
Alternatively, the agent or agents may be added with the aqueous component
used to resuspend the film and form the MPVs. In fact, to obtain a high
entrapment efficiency, the agent or agents may be added to both the
monophase and to the aqueous component used to resuspend the film. Two or
more agents can also be entrapped in one MPV preparation by adding one
agent to the monophase and the other to the aqueous component used to
resuspend the film.
Virtually any bioactive compound can be entrapped within an SPLV or an MPV.
Such compounds include but are not limited to nucleic acids,
polynucleotides, antibacterial compounds, antiviral compounds, antifungal
compounds, anti-parasitic compounds, tumoricidal compounds, proteins,
toxins, enzymes, hormones, neurotransmitters, glycoproteins,
immunoglobulins, immunomodulators, dyes, radiolabels, radio-opaque
compounds, fluorescent compounds, polysaccharides, cell receptor binding
molecules, anti-inflammatories, antiglaucomic agents, mydriatic compounds,
anesthetics, etc.
4.4. THERAPEUTIC USES OF FIBRONECTIN CONJUGATES
The covalently linked fibronectin conjugates of the present invention are
particularly well suited for a wide range of applications such as drug
delivery or drug carrier systems. The strong affinity of fibronectin for
collagen, heparin, hyaluronic acid, fibrin/fibrinogen, and gangliosides
permits localization and retention of the FN-conjugates or the FN-modified
liposomes. Depending upon the route of administration, retention at a
variety of collagen rich body sites including skin, bone, tendon,
cartilage, blood vessels, teeth and ocular tissues (especially conjunctiva
and cornea) can be accomplished.
Some uses of the fibronectin conjugates are described below but do not
limit the scope of the present invention.
Localized delivery of anti-inflammatory and/or analgesic agents including
but not limited to aspirin, indomethacin, ibuprofen, flurbiprofen
pinoxicam, naproxen, prednisolone, dexamethasone, cortisone, cortisol, as
well as therapeutic gold compounds can be achieved by practice of the
present invention employing fibronectin conjugates incorporated into
liposome preparations which are then injected directly into joints. Such
localization is particularly advantageous in that the therapeutic agent is
slowly released into collagen-rich joints where needed and undesirable
systemic effects particularly those seen with steroidal hormones are
avoided. Inflammation of joints due to rheumatoid arthritis or traumatic
injury may thus be treated. Sustained release from adherent liposomes is
especially advantageous in obviating repeated injections into joints in
conditions where prolonged therapy is anticipated.
Fibronectin conjugates of the present invention may also be employed to
treat ocular afflictions. Adherence of fibronectin conjugates (applied
directly as FN-conjugates or as FN-liposomes) to collagenous surfaces of
the cornea and conjunctiva will retard flushing of drug from the eye and
permit sustained delivery of medicament to localized site. Ocular
conditions which may thus be treated include but are not limited to:
glaucoma, dry eye, and infections ("pink-eye"). A number of organisms
cause eye infections in man and animals. Such organisms include but are
not limited to: Moraxella spp., Clostridia spp., Corynebacteria spp.,
Diplococcus spp., Leptospira spp., Mycobacteria spp., Neisseria spp.,
Propionibacteria spp., Proteus spp., Pseudomonas spp., Serratia spp.,
Escherichia spp., Staphylococcus spp., Streptococcus spp., and
bacteria-like organisms including Mycoplasma spp., Chlamydia spp., and
Rickettsia spp. Most recent evidence has shown that ocular application of
fibronectin has been effective in treating recurrent corneal erosion
(Nishida et al., The Lancet, Aug. 27, 1983 pp. 521-522).
Another area in which fibronectin conjugates may be used with advantage is
the treatment of both accidentally inflicted and surgical wounds in which
collagenous fibers of tissues are particularly exposed. Topical
application of fibronectin conjugated with antimicrobial agents especially
antibiotics and antifungals (or fibronectin conjugated to liposomes
containing the antimicrobial agent) may permit adherence of the active
agent at vulnerable sites for both prevention and treatment of wound
infections.
Because fibronectin conjugates injected intraperitoneally adhere especially
to the body cavity, such administration of fibronectin-antimicrobial
conjugates may be used to prevent and/or treat conditions such as
peritonitis.
Intramammary infusion of fibronectin conjugates may have particular
application for treatment of conditions such as mastitis in cows, goats or
other milk producing animals.
4.4.1. METHODS OF ADMINISTRATION
The present invention encompasses fibronectin conjugated with either free
drug or materials incorporated into a liposome bilayer immobilized at
specific body sites by a variety of routes of administration. In man and
animals drugs or drug-carrier complexes may be administered by a number of
routes including but not limited to: injection (e.g., intravenous,
intrathecal, intraperitoneal, intramuscular, subcutaneous, intraarticular,
intramammary, intraurethral, etc.); topical application (e.g., on
afflicted areas); and by absorption through epithelial or mucocutaneous
linings (e.g., ocular epithelia, | | |
