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FIELD OF THE INVENTION
The present invention relates to a high-visoisity EGF/liposome composition,
and to methods of making and using the composition.
REFERENCES
Bronaugh, R. L., et al, J. Pharm. Sci., 72:64 (1985).
Buckley, A., et al, Epidermal Growth Factor increases Granulation Tissue
Formation Dose Dependently. J. Surg. Res. 43, 322 (1987).
Buckley, A., Davidson, J. M., Kamerath, C. D., Wolt, T. B., and Woodward,
S. C., Sustained Release of EGF Accelerates Wound Repair. Proc. Natl.
Acad. Sci. USA 82, 7340, (1985).
Chowhan, Z. T., Yotsuyanagi, Y., and Higuchi, W. I., Biochem. Biophys. Acta
266:320-342, (1972).
Franklin, T. S., et al., Acceleration of Wound Healing by Recombinant Human
Urogastrone. J. Lab. Clin. Med., 108,103, (1986).
King, L. E. and Carpenter, G. F. Epidermal Growth Factor. In: Goldsmith, L.
A. (ed), Biochemistry and Physiology of the Skin. New York, Oxford
University Press, 1983, pp. 26914 281.
Knauer, D. J. et al, Relationship between Epidermal Growth Factor Receptor
Occupancy and Mitogenic Response. J. Biol. Chem. 259 (9), 5623-5631
(1984).
O'3 Keefe, E. et al, Invest. Dermatol. 78, 482 (1982).
MacDonald, R. C., and Simon, S. A., Proc. Natl. Acad. Sci. USA
84:4089-4093, (1987).
Mayhew, E., et al, Exp. Cell Res. 171:195 (1987).
Schwinke, D. L., Ganesan, M. G., and Weiner, N. D. J. Pharm. Sci.
72:244-248, (1983).
Szoka, F., et al, Proc. Nat. Acad. Sci, US, 75:4194 (1978).
Szoka, F., et al, Ann. Rev. Biophys. Bioeng., 9:467 (1980).
Tallarida, R. J., et al, in Manual of Pharmacologic Calculations with
Computer Programs, Springer-Verlag, NY, pp. 72.
BACKGROUND OF THE INVENTION
Epidermal Growth Factor (EGF) is a widely distributed endogenous
polypeptide (King). It is a powerful mitogen with high affinity receptors
in both fibroblasts and epidermal keratinocytes, and has been shown to
accelerate wound healing in vivo (O'Keef; Knauer). The first 5-10 days
after injury are the most critical period during which maximal differences
are seen between EGF treated and untreated wounds. EGF application after
this period produces no significant improvement over controls, since by
this time reepithelialization has already occurred in both groups. Due to
its relatively short half life of about one hour, (Buckley, 1987), loss of
occupied receptors through turnover and a lag time of 8-12 hours to commit
cells to DNA synthesis (Knauer), it is necessary to apply EGF frequently
to a wound to maintain effective local concentration during the critical
period of initial wound healing (Buckley, 1987; Buckley, 1985; and
Franklin, 1986). Thus, effective EGF therapy depends on frequent or
sustained application of the drug during the first several days of wound
healing.
For superficial wounds, local concentration of EGF can easily be maintained
by frequent applications. For surgical incisions and full thickness skin
wounds requiring suture repair, frequent application is not possible and a
sustained-release formulation of EGF must be used for these uses.
Implanted sponges have demonstrated the advantages of sustained EGF
release in an animal wound model (Buckley, 1985) but would not be suitable
as a dosage form.
SUMMARY OF THE INVENTION
It is therefore one object of the invention to provide a high-viscosity
EGF/liposome composition which can be applied to a wound or surgical
incision, for retention and sustained release of EGF at the site of
application.
It is another object of the invention to provide a method for treating a
wound or incision with such composition.
The EGF/liposome composition of the invention includes a high-viscosity
suspension of negatively charged EGF/liposomes, i.e., liposomes containing
EGF in liposome-entrapped form. The EGF/liposomes (i) contain neutral
phospholipid, and at least about 10 weight percent negatively charged
phospholipid, and preferably, between 20-50 weight percent each of neutral
phospholipid, negatively charged phospholipid, and cholesterol. The total
lipid concentration of the EGF/liposomes in the composition is at least 50
mg/g composition and preferably between 50-200 mg/g composition. The EGF
may be entrapped in the EGF/liposomes by encapsulation or surface
adsorption or a combination of both.
The high viscosity EGF/liposome composition may be prepared in a gel or
paste-like state. The gel state is produced, according to another aspect
of the invention, by mixing the above-noted vesicle-forming lipids with a
low conductivity aqueous medium, preferably a low-conductivity (low
ionic-strength) buffer which contains a zwitterionic compound whose
isoelectric point is between about 5.5 and 8.5.
At a pH substantially above or below the isoelectric point of the
zwitterionic compound, the medium contains charged electrolyte (the
charged zwitterionic compound), and the composition exists in a non-gelled
or only partially gelled state. In this state, the composition is easily
processed, for example to absorb EGF, remove free EGF, and/or size and
filter sterilize the composition. By adjusting the pH of the suspension to
the isoelectric point of the zwitterionic compound, the aqueous medium
becomes essentially non-ionic, and the composition assumes a gel state.
The composition is formed in a paste-like state by mixing a fluidic
EGF/liposome composition with empty liposomes, to a final lipid
concentration of at least about 300 mg/g composition and preferably
between about 400-500 mg/g composition. The empty liposomes are preferably
uncharged, and contain between about 20-50 mole percent cholesterol.
The EGF/liposome composition is used, according to another aspect of the
invention, in a method for treating a surface wound or surgical incision,
by sustained release of the composition from the wound or incision site.
These and other objects 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
FIGS. 1 and 2 are Scatchard plots of EGF binding to EPG/EPC and
EPG/EPC/cholesterol liposomes, respectively;
FIGS. 3 and 4 are plots of surface pressure, at an air/water interface, of
aqueous EGF (FIG. 3) and EPG/EPC/cholesterol (FIG. 4), respectively, as a
function of EGF and liposome concentration;
FIG. 5 is a plot of change in surface pressure, as a function of initial
surface pressure, in the presence and absence of EGF in
EPC/EPG/cholesterol liposomes (open triangles) and PC/PG liposomes (solid
circles);
FIGS. 6-9 show the change in free EGF available in the donor compartment of
a two compartment flux chamber, plotted as a function of time for free EGF
(FIG. 6) and for three EGF/liposome compositions (FIGS. 7-9);
FIGS. l0-13 show the retention of radiolabled EGF, plotted as a function of
time, for free EGF (FIG. 10), and for three EGF/liposome compositions
(FIGS. 11-13);
FIG. 14A-14C illustrate surgical steps in a corneal implant operation; and
FIGS. 15A-15C are diagrammatic cross sections of the surgical region of an
eye seen in FIGS. 14A-14C, showing in FIG. 15B the introduction of an
EGF/liposome formulation prepared according to the invention, and in FIG.
15C, the residual composition in the eye after an extended release period.
DETAILED DESCRIPTION OF THE INVENTION
I. Viscous EGF/Liposome Compositions
This section describes components and methods used in forming the
high-viscosity EGF/liposome composition of the invention.
A. Definitions
As used herein, the terms below have the following meaning:
1. "Neutral phospholipid" refers to any vesicle-forming lipid having (i)
two hydrocarbon-chain moieties which are effective to produce a stable
bilayer formation, and (ii) a polar head group with no net charge at a pH
between about 5.5-8.5.
2. "Negatively charged phospholipid" refers to any vesicle-forming lipid
having (i) two hydrocarbon-chain moieties which are effective to produce a
stable bilayer formation, and (ii) a polar head group with a net negative
charge at a pH between about 5.5-8.5.
3. "Cholesterol" refers to cholesterol or any related sterol capable of
combining with phospholipids to form stable lipid-bilayer vesicles.
4. "Epidermal Growth Factor" or "EGF" refers to human-EGF (h-EGF),
typically recombinantly produced human EGF (rh-EGF), and to related
peptides having the requisite ability to promote the growth of a variety
of cells of epithelial origin in vitro.
5. "High-viscosity" refers to a gel-like or paste-like consistency which
can be applied by squeezing from a tube or syringe, but which is
sufficiently non-flowable, once applied, to be retained in bolus form at a
wound or incision site for at least several hours.
6. A "low-conductivity aqueous medium" refers to an aqueous medium having
an ionic strength which is less than that of a fully
B. Lipid Components
EGF/liposomes formed in accordance with the 30 invention are prepared to
contain between 10-90 weight percent neutral phospholipid, and 10-50
weight percent negatively charged phospholipid, and preferably between
about 20-50 weight percent each of neutral phospholipid, negatively
charged phospholipid, and cholesterol.
Neutral phospholipids having a variety of acyl chain groups of varying
chain length and degree of saturation are available, or may be isolated or
synthesized by well-known techniques. In general, partially unsaturated
phosphatidylcholine (PC), such as egg PC (EPC) is readily obtained and
provides suitable liposome characteristics, such as ease of extrusion and
rate of release of liposome-entrapped EGF.
Likewise, negatively charged phospholipids having a variety of acyl chain
groups of varying chain length and degree of saturation are available, or
may be isolated or synthesized by well-known techniques. A variety of
negatively charged phospholipids, such as phosphatidylglycerol (PG),
phosphatidylserine (PS), and phosphatidylinositol (PI), can be used. One
preferred phospholipid is partially unsaturated PG, such as egg PG (EPG).
The negatively charged phospholipid in the composition serves two
important roles. First, it imparts a negative charge to the lipid bilayer
membranes, providing an electrostatic interaction between the membrane and
the positively charged EGF The adsorption of EFG to the liposomal membrane
will be discussed below. Secondly, the relatively high surface charge is
important in the formation of a gel like liposome state which is
characterized by a low lipid concentration and high viscosity.
Cholesterol is known to increase the stability of liposomes and, in the
case where the phospholipid components are relatively unsaturated, to
increase the packing density of the lipids in the liposomal bilayers. The
effect of cholesterol on the rate of EFG release from EFG composition has
been examined both in vitro and in vivo, as detailed below in Section II.
Briefly, cholesterol significantly increased the half-life of EGF release
in vivo.
One advantage of cholesterol in the EGF/liposome composition is potentially
reduced toxicity due to lipid exchange between the liposomes and cells at
the wound or surgical site. It has been demonstrated, for example, with
several cultured tumor cell lines that EPG/EPC liposomes inhibit cellular
growth in vitro, and that for at least some cell lines, this inhibition
can be greatly reduced by the addition of cholesterol to EPG/EPC liposomes
(Mayhew).
It is understood that the liposomes in the EGF/liposome composition may
contain a variety of other lipid components which may enhance liposome
stability, viscosity, or EGF release characteristics, and/or materials
cost. For example, the liposomes may include .alpha.-tocopherol, or
pharmaceutically acceptable analogue thereof, at a total concentration of
between about 0.1 to 2 weight percent, to improve lipid stability on
storage.
C. Preparing an EGF/Liposome Gel Composition
The EGF/liposome gel composition of the invention can be prepared
conveniently by a modified thin-film hydration method. ln this method,
vesicle-forming lipids are dissolved in a suitable organic solvent or
solvent system and dried under vacuum or an inert gas to form a thin lipid
film. If desired, the film may be redissolved in a suitable solvent, such
as tertiary butanol, and then lyophilized to form a more homogeneous lipid
mixture which is in a more easily hydrated powder like form.
This film is covered with hydration medium and allowed to hydrate,
typically over a 15-60 minute period with agitation. The size distribution
of the resulting multi lamellar vesicles (MLVs) can be shifted toward
smaller sizes by hydrating the lipids under more vigorous agitation
conditions. The final concentration of EGF/liposomes is at least 50 mg/g
and preferably between about 50-200 mg/g composition, and this can be
achieved readily by hydrating a given quantity of lipid with approximately
5-20 volumes/weight hydration medium.
According to an important feature of the invention, it has been discovered
that hydration of the vesicle-forming lipids with a low-conductivity
aqueous medium containing a zwitterionic compound at the isoelectric point
of the compound between pH 5.5 and 8.5, produces a liposome composition
which is both gel-like in consistency and viscosity, and has a relatively
low lipid concentration.
More specifically, the combination of net negative surface charge on the
liposomes (due to the presence of at least about 10 weight percent
negatively charged phospholipid) and the low-ionic strength of the aqueous
medium and the unchanged zwitterionic compound produces a liposome
composition characterized by (a) a viscous, gel-like consistency and (b) a
relatively low lipid concentration, e.g., 50-200 mg/g composition.
Neutral amino acids, such as glycine, isoleucine alanine, proline, and
valine are preferred zwitterionic compounds. The concentration of
zwitterionic compound in the buffer is at least about 0.5 percent by
weight and preferably between about 1-5 percent by weight, and the buffer
is adjusted in pH to the isoelectric point of the compound to achieve the
gel state.
The aqueous medium buffered with zwitterionic compound may initially be
adjusted to a pH at which the zwitterionic compound is substantially in a
charged form, so that the medium has a relatively high electrolyte
concentration, i.e., a relatively high conductivity By adjusting the pH to
the isoelectric point of the zwitterionic compound, typically after lipid
hydration and liposome formation, the compound becomes non-electrolytic,
i.e., has the desired low conductivity.
In forming the liposome gel composition directly, the low conductivity
buffer is added to the lipid film, and the mixture is agitated until the
desired liposome gel forms. The hydration step is generally effective to
produce a homogeneous liposome suspension, where relatively small lipid
quantities are involved For larger lipid amounts, the hydrated suspension
may contain particles of non-hydrated or partially hydrated lipids This
suspension can be converted to a homogeneous suspension by further
processing, preferably by extrusion through a defined-pore size membrane,
such as a 2 micron pore size polycarbonate membrane The extrusion step, of
course, also reduces the size heterogeneity in the suspension. This
general procedure for preparing a liposome gel suspension is illustrated
in Example 1.
Alternatively, the liposome gel composition can be formed in two stages,
involving initial liposome formation of a fluidic liposome suspension, by
addition of the aqueous buffer in an electrolytic condition (e.g., where
the zwitterionic compound is not at its isoelectric point, and subsequent
pH adjustment of the aqueous medium to a non-electrolytic state
(isoelectric point of the medium), to produce the desired gel formation.
This approach allows the liposome suspension to be processed, after
liposome formation for example, to size and sterilize the suspension, as
discussed below, in a convenient, low viscosity form, then brought to a
final gel state by pH adjustment.
The aqueous medium used in forming the composition may contain dissolved
EGF, at a suitable concentration The suspension formed in this manner
includes encapsulated, liposome-adsorbed, and free EGF. Free EGF can be
removed, if desired, by conventional methods, such as molecular sieve
filtration or the like.
Alternatively, free EGF may be added to preformed liposomes at a suitable
concentration, producing a suspension with liposome-adsorbed and free EGF.
According to one aspect of the invention, it has been found that the in
vivo release kinetics of EGF from EGF/liposomes containing absorbed EGF
only is comparable for EGF/liposomes prepared to include both adsorbed and
encapsulated EGF (Example 5).
It will be appreciated that a variety of liposome preparation methods,
including reverse-phase evaporation and solvent-injection methods (Szoka,
1978, 1980), can be adapted for preparation of the above EGF/liposome gel
composition, using a non-electrolyte aqueous buffer in the liposome
formation step.
D. Preparing an EGF/Liposome Paste Composition
The EGF/liposome paste composition of the invention is prepared from a
fluidic, i.e., non viscous, EGF/liposome composition having a lipid
composition detailed above and lipid concentration of at least 50 mg/g and
preferably between about 50-200 mg/g. This composition is then mixed with
empty, preferably neutral, liposomes to a final lipid concentration at
which the composition has a desired paste like consistency, typically
between 300 and 500 mg lipid/g suspension.
The initial fluid EGF/liposome suspension can be prepared by one of a
variety of known liposomepreparation methods, as above. These methods
include, but are not limited to, thin-film hydration, reverse-phase
evaporation (Szoka), and solvent injection. As above, EGF may be
incorporated into the liposomes on formation or by addition to EGF to
already formed liposomes to produce an EGF liposome composition with
absorbed EGF only.
The empty liposomes used in preparing the final high viscosity preparation
are preferably uncharged or substantially uncharged liposomes. One
preferred composition includes between about 50-70 mole percent neutral
phospholipid, preferably EPC or partially hydrogenated EPC and 30-50 mole
percent cholesterol.
The empty liposomes may be prepared prior to mixing with the EGF/liposomes
(or negatively charged liposomes, if EGF is added subsequently to the
combined liposome suspension), employing conventional liposome methods
such as those discussed above. Alternatively, the relatively dilute
EGF/liposome suspension may be employed as a hydration medium, for the
formation of uncharged liposomes, also yielding a liposome suspension
containing both EGF/liposomes and empty liposomes.
As an illustration of the latter method, a dilute EGF/liposome suspension
which has been treated to remove free EGF, and sized and sterilized as
below, is added to a thin film of sterile dehydrated neutral lipids to a
final lipid concentration of preferably between 300-500 mg/g. Lipid
hydration may be carried out with vigorous agitation and/or at a
temperature of at least about 37.degree. C. to promote a uniform liposome
suspension, preferably under sterile conditions. The resulting composition
will include relatively small negatively charged EGF/liposomes, and
relatively large neutral liposomes.
E. Liposome Processing
The above EGF/liposome compositions may be further processed to (a) achieve
smaller and or more uniform liposome sizes, (b) remove free EGF and/or (c)
sterile the EGF/liposome preparation. As indicated above, these processing
steps are preferably carried out with the compositions in a non viscous
state. This is accomplished, in the gel formulation, by employing an
aqueous medium whose pH is adjusted to produce a substantial electrolyte
concentration, as described above. In the EGF/liposome paste formulation,
the processing steps are preferably carried out before addition of the
empty liposomes.
Where it is desired to produce a sterile EGF/liposome composition, the
liposomes are preferably sized down to 0.2 to 0.3.mu., to allow
sterilization by filtration through a convention depth filter. Several
techniques are available for reducing liposomes to this size range,
including sonication, homogenization and extrusion through a defined pore
size membrane. Extrusion of liposome through a small pore polycarbonate
membrane has been used successfully, as has extrusion through asymmetric
ceramic membranes (co-owned U.S. Pat. No. 4,737,323). The liposomes may be
extruded through successively smaller-pore membranes, to achieve a gradual
reduction in liposome size.
Free EGF can be removed, if desired, by conventional centrifugation,
ultrafiltration, or gel filtration (molecular sieve) methods. When the EGF
liposomes are sized by extrusion, free EGF is preferably removed following
the extrusion step.
The EGF liposomes are preferably sterilized, where required, by filtration
through a conventional depth filter, typically having a 0.22 micron
particle exclusion size. This method can be carried out on a practical,
high through-put basis only if the liposomes have first been sized down to
about 0.2-0.3 microns or less.
From the foregoing, several advantages of the method of preparation of the
gel and paste like EGF/liposome compositions can be appreciated. The gel
composition can be prepared at a low lipid composition and thus is
relatively inexpensive to manufacture. The final viscosity of the
composition can be controlled by small changes in final ionic strength,
produced either by addition or removal of ionic components, or by
relatively small pH changes in a medium containing a zwitterionic buffer.
The gel liposomes can easily be prepared and processed in a dilute form,
for example to remove EGF, size and sterilize the liposomes, then brought
to a final viscous state by pH adjustment.
The paste composition can likewise be prepared in an initial dilute form
convenient for EGF removal, and if desired, for sizing and sterilization,
then brought to a paste-like consistency by addition of empty liposomes
Since the only step in the liposome processing which is difficult to carry
out under sterile conditions is EGF removal, the empty liposomes can be
added after sterilization of the EGF/liposomes, without additional sizing
and sterilization requirements
Finally, as discussed in Section II below, EGF is adsorbed readily to the
negatively charged liposomes in the EGF/liposome composition, allowing the
composition to be prepared simply by mixing free EGF with preformed
liposomes.
II. Properties of the Composition
A. Viscosity of the Gel Composition
The EGF/liposome gel composition of the invention is characterized by a
high-viscosity gel like consistency which is maintained at a low ionic
strength, but which collapses as ionic strength is increased. This feature
is illustrated in the study described in Example 2. Here liposomes
containing equal-weight amounts of EPG, EPC, and cholesterol were prepared
in a 2.3% w/v glycine buffer at isotonic pH (pH 6.0) buffer, as detailed
in Example 1, except that EGF was not added. The mean viscosity for the
samples was 13.3.times.10.sup.3 Cps (centipoise) at 1.0 per second shear
rate, characterized by a thick, relatively non-flowing gel consistency.
With addition of NaCl to a concentration of only 0.05% w/v (at about 8.5
mM), the material lost its gel-like properties, being quite fluid, with a
mean viscosity of only about 2.7.times.10.sup.3 Cps at 1 per second.
Further relatively small decreases in viscosity were seen with further
addition of NaCl to a final concentration of 0.2% w/v.
B. EGF Binding to Negatively Charged Liposomes
According to one aspect of the invention, it has been found that EGF may be
entrapped in negatively charged liposomes by surface adsorption, and that
the binding affinity of EGF for the liposomes is effective to produce slow
release of adsorbed peptide both in vitro and in vivo. In the binding
study reported in Example 3, liposome gel compositions formed from either
PC/PG (equal weight ratios) or PC/PG/cholesterol (equal weight ratios)
were prepared as in Example 1. Increasing amounts of EGF (iodine
radiolabeled) were added to aliquots of each of the two compositions, and
the mixtures were allowed to equilibrate for one week at 4.degree. C. The
ratio of bound to free EGF was determined from total radiolabel measured
before and after centrifugation, and these values were plotted as a
function of amount bound, yielding the plots in FIGS. 1 and 2 for the
EPC/EPG and EPC/EPG/cholesterol compositions, respectively. Affinity
constants K.sub.d were determined from these plots as described in Example
3. As seen from the two figures, the K.sub.d values are in the range
1-2.times.10.sup.-5 molar for both compositions.
The number of EGF binding sites on the liposomes was determined from the
x-axis intercept in the FIG. 1 and 2 plots, along with the calculated
K.sub.d values, also as detailed in Example 3. From this, it was
determined that at a peptide concentration of about 200 .mu.g/ml, about
30% of the EGF is adsorbed at the lipid/water interface.
The adsorption of EGF to EPC/EPG and EPC/EPG/cholesterol monolayers was
also examined in a lipid monolayer system, also as detailed in Example 3.
Briefly, the method measures the ability of EGF to interpenetrate the
lipid monolayer, as evidenced by changes in the interfacial surface
pressure as EGF is added to the monolayer.
FIG. 4 is a plot of the interfacial surface pressure, .pi., as a function
of lipid concentration for a EPC/EPG/cholesterol (equal wight ratios)
lipid monolayers, as a function of lipid concentration. Similar plots were
was made for EPC/EPG monolayers, and EPC/EPG/cholesterol and EPC/EPG
monolayers containing 40 .mu.g/ml EPG, at each of several lipid
concentrations. These plots were used to construct the graph of change in
surface pressure due to the presence of 40 .mu.g/ml EGF in the monolayer,
as a function of surface pressure, for each of the two lipid compositions.
This graph is shown in FIG. 5.
Using linear regression analysis to extrapolate to the y-axis intercept it
can be seen that the change in interface surface pressure produced by EGF
in the EPC/EPG/cholesterol composition is about 15 dynes/cm, and in the
EPC/EPG composition, about 13.5 dynes/cm. The interface surface pressure
attributable to EGF alone (no lipid interaction) is plotted in FIG. 3, and
is .mu..6 dynes/cm at 40 .mu.g/ml. Thus for both lipid compositions, the
measured change in surface pressure due to EGF in the presence of lipid is
greater than that produced by EGF alone, indicating that the peptide is
interacting with the monolayer.
The greater EGF-induced change in pressure seen in the EPC/EPG/cholesterol
composition indicates a greater degree of EGF interaction with the
addition of cholesterol to EPC/EPG.
C. In vitro EGF Release Characteristics
The kinetics of release of EGF from EGF/liposome compositions prepared
according to the invention were examined in a standard two-chamber
percutaneous absorption cell, as detailed in Example 4. The samples placed
in the donor cell were suspended in 25% human serum in isotonic saline,
for passage across a membrane filter into a donor collector compartment
which was continually perfused with 25% human plasma in saline.
FIG. 6 shows release kinetics of EGF in the system for three independent
kinetic Studies. The mean halflife of EGF release, calculated from the
slope of the availability of free EGF in the donor compartment, as a
function of time, is about 1.8 hours.
The EGF available in the donor compartment from various EGF/liposome
compositions were similarly measured. FIG. 7-9 shows plots of EGF
available in the donor compartment, as a function of time, from
(Composition I) EPC/EPG liposomes with encapsulated EGF (FIG. 7),
(Compositions II) EPC/EPG/cholesterol liposomes with encapsulated EGF
(FIG. 8), and (Composition III) EPC/EPG/cholesterol with adsorbed EGF. All
three compositions contain free EGF, and thus also are expected to contain
liposome-adsorbed EGF.
The model used to determine the half lives of EGF release from the
liposomal formulations is discussed in Example 4. Briefly, the free EGF
available in the donor compartment is determined from the measured rate of
appearance of EGF in the receiver compartment, the rate constant K.sub.b
of the membrane, and the volume V.sub.b of the external phase in the donor
compartment. The calculated free available EGF in the donor compartment is
then plotted as a function of time, as seen in FIGS. 7-14 9. The half
lives of EGF release during the slow phases is determined from the
resulting plots.
The half lives determined from above are 14.1 hours for the EPC/EPG
composition (encapsulated EGF); 10.1 hours for the EPC/EPG/cholesterol
composition (encapsulated EGF); and 6.2 hours for the EPC/EPG/cholesterol
composition (adsorbed EGF). It is clear that all of the liposome
formulation enhanced the half life of EGF release in vitro severalfold
over free EGF.
D. In vivo EGF Release Characteristics
According to an important feature of the invention, the high-viscosity
EGF/liposome compositions of the invention are effective to (a) remain
physically localized at a site of injection or administration and (b)
provide a source of therapeutic levels of EGF over a several-day period.
The enhanced retention of EGF in an EGF/liposome composition has been
demonstrated with conjunctival placement of the various EGF compositions
by subconjunctival-injection, and monitoring of levels of EGF retained at
the conjunctival site over a several day period. The retention of
radiolabeled EGF at a conjunctival site of administration, as a function
of time after injection is shown in FIGS. 11-13 for free EGF (FIG. 10),
and EGF/liposomes composed of: (Composition I) EPC/EPG and containing free
and encapsulated EGF (FIG. 11), (Composition II) EPC/EGF/cholesterol and
containing free and encapsulated EGF (FIG. 12), and (Composition III)
EPC/EPG/cholesterol and containing free and adsorbed EGF only (FIG. 13).
As seen, all of the EGF/liposome compositions give biphasic EGF release
characteristics, indicating a burst of EGF released into the site of
administration, followed by a slow phase EGF release over a several-day
period.
Table 3 in Example 5 gives the half-lives of EGF release, and the percent
EGF released in the burst for free EGF and the three EGF/liposome
compositions, calculated from the mean values of the data plotted in FIGS.
10-13. The half life of EGF retention was extended from 1 hour for free
EGF to 14-35 hours for the liposomal compositions. Interestingly, and in
contrast to the in vitro release kinetics observed, the largest half lives
(32 and 35.6 hours) were obtained with Composition II and III
(cholesterol-containing EPC/EPG liposomes), whereas the shortest half life
(14 hours) was obtained with the Composition I. This discrepancy with the
in vitro kinetics data may be due to the greater stability of
cholesterol-containing liposomes in vivo, perhaps related to the reduced
extent of lipid exchange which would be expected between liposomes and
cells at the site of administration in the presence of cholesterol.
The long-term availability of EGF in the region of the EGF/liposomes is
seen from the data in Table 4 of Example 5. For free EGF, substantially no
EGF was available at the conjunctival site one day after administration.
With the EGF/liposome formulations, more than 1% of the total EGF was
available at the site 4 days after administration for Composition I, six
days after administration, for Composition III, and seven days after
administration, for Composition II.
From the foregoing, it can be appreciated that the degree of retention of
EGF/liposomes at a site of administration can be selectively varied
according to the amount of cholesterol included in the liposomes, at least
over the range from about 0-33 weight percent.
The above results also indicate that liposome-adsorbed EGF in the
EGF/liposomes is released from the liposomes in vivo at substantially the
same rate as encapsulated EGF. This result confirms that EGF is tightly
bound to negatively charged liposomes (containing at least 20 mole percent
negatively charged phospholipid), and that an effective EGF/liposome
formulation can be made by surface adsorption to liposomes.
III. Utility
A. Topical Administration
The EGF/liposome composition is designed for application to burns and other
skin wounds, to promote healing. The viscous material may be applied
directly to the skin or in a skin dressing.
The material is preferably supplied in gel form from a tube or the like
which can be easily applied to the skin or to a skin dressing. One unique
property of the gel material, when applied directly to the skin as a film,
is that salts in the skin will break down the gel structure, producing a
fluid lipid dispersion as the material is rubbed in the skin.
B. Surgical Wound Administration
The viscous EGF/liposome composition is also useful for treating surgical
incision, by application to the incision area before suturing. In this
application, the high viscosity of the material reduces loss of material
from the incision site, and the slow release of EGF from the liposomes
provides a therapeutic level of EGF at the site over a several day healing
period.
The gel or paste material is preferably applied directly to the incision
area from a tube or syringe. In one embodiment, designed especially for
long term storage, the liposome composition is reconstituted immediately
before use, with addition of sterile water One of the problems which may
be encountered when EGF/lipid material is reconstituted is incomplete
mixing and lipid hydration, due to the viscous consistency of the material
as it rehydrates. The problem of incomplete or nonuniform hydration can be
overcome, in forming the EGF/liposome gel composition of the invention, by
suitable adjustments in pH during the gel-forming process, to allow
initial lipid hydration in a relatively fluidic form, with gradual
transformation to a gel form. The required change in pH may be produced by
addition of an acid or base following lipid hydration, or may result from
the release of an acid or base species from a reservoir present in the
dehydrated lipid mixture.
As an example, the dehydrated lipid mixture may be prepared from EGF
liposomes having an encapsulated zwitterionic compound, where the
zwitterionic compound is in a predominantly charged form and the initial
suspension is largely freed of extraliposomal (nonencapsulated) compound
prior to dehydration. The rehydration medium, in turn, may be an
unbuffered medium whose initial pH is different from the isoelectric pH of
the encapsulated compound, but which after complete equilibration with the
encapsulated compound occurs, yields the desired isoelectric pH.
Following rehydration and reformation of liposomes, slow release of the
charged zwitterionic compound from the liposomes would produce a gradual
pH shift toward the desired pH at which the compound is largely non-ionic,
producing increasing suspension viscosity. After final set up, the
material is forced from the tube or syringe in gel form into the incision
site.
C. Ophthalmic Uses
FIGS. 14A-14C illustrate surgical incision and incision repair step in a
corneal replacement or transplant operation. An initial arcuate incision
in the conjunctiva, illustrated in FIG. 14A, allows the conjunctiva to be
pulled away, exposing the underlying episclera and cornea. A second
arcuate cut in the cornea, shown in FIG. 14B, allows the cornea to be
pulled back to provide access to the lens (not shown) After surgical
removal or replacement of the lens, the cornea is first closed by
stitching, seen at 20 in FIG. 15C, followed by closure of the conjunctiva
by stitching, indicated at 20. Post-operative healing involves healing of
the two incisions, and regrowth of the episclera layer between the
conjunctiva and cornea.
FIGS. 15A-15C illustrate the use of the EGF/liposome composition of the
invention to | | |
