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BACKGROUND OF THE INVENTION
The present invention relates to a process for producing bilayer vesicles
by forming mixed micelles in a colloidal solution from a bilayer-forming
substance and a detergent, and removing the detergent by means of
flow-through dialysis.
Substances which are capable of forming bilayers (i.e., double layers) in
the aqueous phase are known, for example phospholipids, such as lecithin.
These bilayers are frequently in the shape of small hollow spheres which
are hereinafter referred to as bilayer vesicles.
Known processes for producing bilayer vesicles, such as subjecting
bilayer-forming substances to ultrasound, injecting bilayer-forming
substances dissolved in organic solvents into an aqueous medium, removing
detergents from micelle solutions (i.e., solutions of mixed micelles of
bilayer-forming substance and detergent) by means of gel chromatography,
and conventional dialysis [compare Biochim. Biophys. Acta 457, 259-302
(1976), CRC Critical Reviews in Toxicology 6, 25-79 (1978)], produce
bilayer vesicles with undesired properties. The main disadvantages of such
processes are characterized by the inclusion of organic solvents in the
bilayer vesicles, the degradation of the bilayer-forming substance, the
formation of multi-lamellar structures and, in particular, the formation
of vesicles which are nonhomogeneous in size (20 to 200 nm in diameter).
Furthermore, undesired dilution effects can occur and these necessitate a
subsequent concentration process. If bilayer vesicles are employed as
medicament carriers and/or as pharmaceutical preparations, the resultant
plasma clearance and distribution in the organs are determined above all
by the homogeneity of the vesicles and the vesicle size. Multi-lamellar
heterogeneous structures are rapidly absorbed, in particular, by the
spleen and the liver and are no longer available to the organism as a
pharmodynamically active substance [Biochim. Biophys. Res. Comm. 63,
651-658 (1975)]. The extent and course of this process, and the
interaction of the vesicles at the cellular level, can be controlled by
selection of suitable lipid composition and morphology (size) of the
vesicles [Science, Volume 205, 1,142-1,144 (1979); Biochim. Biophys. Acta,
Volume 541, 321-333 (1979)].
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide a process which
overcomes these disadvantages and by which bilayer vesicles of a
homogeneous size can be produced.
Upon further study of the specification and appended claims, further
objects and advantages of this invention will become apparent to those
skilled in the art.
These objects have been obtained by providing a process for producing
bilayer vesicles from a colloidal solution comprising mixed micelles of a
bilayer-forming substance and a detergent, the process comprising removing
the detergent from the micelle-containing colloidal solution by means of
flow-through dialysis whereby the colloidal solution is dialyzed against a
dialysis liquid in a chamber formed at least partially by a semi-permeable
membrane, wherein the dialysis liquid is moved along one side of a
semi-permeable membrane at a velocity such that the detergent
concentration in the dialysis liquid, on at least 90% of the active
surface of the membrane, is at most 10% of the detergent concentration in
the micelle solution in contact with the other side of the membrane, and
wherein a homogeneous detergent concentration is maintained in the micelle
solution by the movement of the latter.
BRIEF DESCRIPTION OF THE DRAWINGS
Various other objects, features and attendant advantages of the present
invention will be more fully appreciated as the same becomes better
understood when considered in conjunction with the accompanying drawings,
in which like reference characters designate the same or similar parts
throughout the several views, and wherein:
FIG. 1 shows a cross section through a dialysis cell and
FIG. 2 shows a front view of a side-wall element of the cell of FIG. 1.
DETAILED DISCUSSION
The removal of detergents from micelle solutions by means of known dialysis
processes (e.g., by equilibrium dialysis, for example, by the dialysis bag
method) leads to nonhomogeneous bilayer vesicles of various sizes and to
multi-lamellar structures. It has been established that this is derived
from the uncontrolled dialysis kinetics of the detergent. These nonuniform
dialysis kinetics result from the fact that concentration gradients, which
are constantly changing and, hence, cannot be controlled, build up both in
the material to be dialyzed (the micelle solution) and also in the
dialysate. This results in a constantly changing dialysis rate of the
detergent, which can hardly be influenced, and which continuously changes
the size distribution of the mixed micelles. The complete removal of the
detergent from the micelle solution takes a very long time and exacerbates
the above-mentioned disadvantages of the process.
To avoid uncontrollable changes in the concentration gradient transverse to
the semi-permeable membrane, the process of this invention comprises
moving the dialysis liquid, on at least one side of a semi-permeable
membrane, at a velocity such that the detergent concentration in the
dialysis liquid, on virtually the whole of the active surface of the
membrane, in any case on at least 90% of this surface, is at most 10% of
the detergent concentration in the micelle solution in contact with the
other side of the membrane, and maintaining a homogeneous detergent
concentration in the micelle solution by the movement, e.g., stirring, of
the latter.
In this way, by keeping the detergent concentration in the dialysis liquid
which is in contact with the membrane as low as possible at all points,
preferably below about 2%, for example at about 1% or less, relative to
the detergent concentration in the micelle solution, it is possible to
ensure that, at all points on the active membrane surface, virtually
identical concentration gradients are formed normal to the membrane
surface. Homogeneous bilayer vesicles of a defined size, which can be used
as carriers for biologically and pharmacodynamically active substances
and/or can be employed as pharmaceutical preparations, are thereby
obtained after a relatively short dialysis time.
Preferably, the dialysis liquid is carried past the semi-permeable
membrane, with a constant flow velocity in laminar flow, in such a way
that a linear concentration gradient of the detergent is formed from the
entry of the dialysis liquid into the flow-through compartment up to its
discharge as dialysate. In flow-through dialysis of this type, the
intermediates resulting from the formation of the bilayer vesicles are
already in themselves homogeneous and defined. The desired homogeneous
bilayer vesicles of defined size are formed from these intermediates after
removal of the detergent. The bilayer vesicle size can be controlled and
selected conventionally by selection of the molar ratio of bilayer-forming
substances/detergent in the initially formed colloidal solution, or by
suitable choice of the detergent, or by choice of the dialysis kinetics of
the detergent. Such dialysis kinetics, (i.e., dialysis rate) in turn
depend, in a known manner, on the temperature, the ratio of membrane
surface area/solution volume, the type of membrane (thickness, pore size),
the concentration and the physical and chemical properties of the
substances to be dialyzed. See, e.g., (a) "Membrane Separation Processes",
P. Meares ed., chapter 1 and 2, p. 1-79, Elsevier Scientific Publ. Comp.,
New York (1979); (b) Biochemistry 18, 4173-4176 (1979) which disclosure is
incorporated by reference herein.
A laminar flow of the dialysis liquid over the membrane surface has the
advantage that the flow velocity can be kept approximately the same over
the whole surface, and this is important since the flow velocity should be
as high as possible, inter alia because of the desired low detergent
concentration in the dialysis liquid, but a certain maximum, beyond which
the molecular film on the membrane would be destroyed, of course, should
not be exceeded. Preferably, the flow velocity of the dialysis liquid in
the immediate vicinity of the membrane surface, at as many points as
possible, is in the region of 0.2-6 m/minute, advantageously in the region
of 1-3 m/minute.
The preferred laminar flow can be ensured, for example, by arranging, in
the flow-through compartment, guide elements which are in contact with the
membrane and which carry the dialysis liquid along the surface of the
membrane in laminar flow. For example, the dialysis liquid can be carried
over the surface of the membrane in a meandering or spiral channel in an
element which is in contact with the membrane. Alternatively, it is also
possible simply to construct the flow-through compartment with very low
thickness (measured normal to the membrane), preferably a thickness of
less than 1 mm. If this thickness is not more than about 2 mm and the
dialysis liquid is introduced into the flow-through compartment,
distributed over the width of the latter, an approximately laminar flow is
likewise achieved at virtually all points.
The movement of the micelle solution in contact with the other side of the
semi-permeable membrane, which movement is employed to maintain a
homogeneous detergent concentration in the micelle solution, can be
achieved, for example, by stirring with a mechanical stirring member (for
example a magnetic stirring rod), or by forcing an inert gas into the
chamber containing the micelle solution, or by moving the whole dialysis
device to and fro (tilting movements) with this chamber.
The drawing shows a dialysis device with which an embodiment of the process
of this invention can be carried out.
In the dialysis cell of FIGS. 1 and 2, two semipermeable membranes 1 are
arranged between the ring 2 and, in each case, a side-wall element 3. A
colloidal solution of mixed micelles is brought (e.g., through closable
orifices in ring 2, which are not shown) into the interior space 4 between
the two membranes 1.
The semi-permeable membranes must be impermeable to the bilayer-forming
substances and to the aggregates or associates (e.g., the micelles,
unilamellar or multilamellar vesicles) formed therefrom, but permeable to
solvents and to auxiliaries and active substances dissolved therein. The
following are particularly suitable as components of the membranes:
cellulose, hydrated cellulose, regenerated cellulose (e.g., cellophane) as
well as cellulose derivatives such as acetyl cellulose, furthermore
polyamides, polyalkylenes such as polyethylene or polypropylene,
polyesters, polyvinyl chloride, polytetrafluoroethylene, polycarbonates,
etc.
The preferred membrane thickness is about 5 to about 20 .mu.m.
The mixed micelles are produced from a detergent (solubilizer) and
bilayer-forming substances.
Suitable micelle-forming solubilizers are nonionic, anionic, cationic or
amphoteric detergents.
The following are particularly suitable as detergents: cholic acid, their
salts and derivatives such as desoxycholic acid, taurocholic acid,
chenodesoxycholic acid, lithocholic acid, glycocholic acid and their
salts, preferably their sodium salts; glycosides, above all monomeric or
oligomeric sugar derivatives with lipophilic side chain, e.g,
1-O-n-hexyl-.beta.-D-glucopyranoside,
1-O-n-heptyl-.beta.-D-glucopyranoside or
1-O-n-octyl-.beta.-D-glucopyranoside.
Among the anionic solubilizers, there are suited in particular the Na and K
salts of fatty acids of, preferably, 8 to 24 C atoms, amine soaps (e.g.,
triethanolamine stearate), salts of sulfuric and sulfonic acid esters of
higher fatty alcohols such as sodium lauryl sulfate, docusate sodium salt
or sodium lauryl sulfonate; among the cationic, quaternary ammonium
compounds. Suitable nonionic solubilizers include, e.g., partial fatty
acid esters of polyvalent alcohols such as glycerol monostearate,
pentaerythritol monostearate; partial fatty acid esters of sorbitan (e.g.,
Span.RTM., Crill.RTM.) and of polyoxyethylene sorbitan (e.g., Tween.RTM.),
reaction products of castor oil or hydrogenated castor oil with ethylene
oxide (e.g., Cremophor.RTM.EL), ethoxylated saturated fatty alcohols
(e.g., cremophor.RTM.A and O, Brij.RTM.), polyethyleneglycol esters of
fatty acids (e.g., Cremophor.RTM.AP, Myrj.RTM.), polyetheralcohols (e.g.,
Pluronic.RTM.), etc.
Only amphiphilic substances which are capable of forming bilayers (i.e.,
double layers) in the aqueous phase can be used as bilayer-forming
substances; that is, substances of polar (hydrophilic) as well as apolar
(lipophilic) properties.
Suitable bilayer forming substances include, particularly, phospholipids,
for instance phosphoglycerides (diesters, monoesters, diethers, monoethers
wherein the ester and ether groups preferably are of 8 to 24 carbon atoms
each) such as lecithins (phosphatidylcholines), kephalins
(phosphatidyl-ethanolamines, phosphatidylserines), inositolphosphatides,
phosphatidylic acids, phosphatidylglycerols, cardiolipin; sphingolipids,
e.g., sphingomyelin; glycolipids, e.g., cerebrosides, gangliosides;
furthermore, e.g., fatty acids of, preferably, 8 to 24 carbon atoms as
well as their esters, salts and amides; alkyl ethers of, preferably 8 to
24 carbon atoms; alkyl ether derivatives of, preferably, 8 to 24 carbon
atoms, such as 1,3-propanediol-phospholipids; higher alkylamines of,
preferably, 8 to 24 carbon atoms, e.g., stearyl amine; fatty alcohols of
preferably 8 to 24 carbon atoms, e.g., stearyl alcohol, higher alkylthiols
of, preferably, 8 to 24 carbon atoms; etc. Furthermore, mixtures of these
substances are also suitable. In general, the alkyl chains of the cited
substances can be straight or branched.
The detergent and bilayer-forming substances form a ternary system with
water, which is referred to here as a mixed micelle. The colloidal
solution of the mixed micelle, which is subsequently called the micelle
solution, can additionally contain electrolytes (predominantly
physiologically compatible inorganic salts such as sodium chloride, sodium
mono- and di-hydrogenphosphate, potassium mono- and di-hydrogenphosphate,
etc.), sorption promoters (such as organic solvents, fatty alcohols and
fatty acid esters, etc.), auxiliaries (such as stabilizers and
preservatives), peptides, proteins, nucleic acids, lipids, antigens and
antibodies, and also active substances with biological and pharmacodynamic
properties, etc. Suitable active substances include, for instance,
medicinally active compounds such as sterols, e.g., cholesterol,
sitosterol, etc.; estrogens, e.g., estrone, estradiol and its esters,
ethinylestradiol, etc.; gestagens, e.g., norethisterone acetate,
chlormadinone acetate, etc.; corticoids, e.g., hydrocortisone,
prednisolone, prednisone, dexamethasone, betamethasone, etc. and their
esters, e.g., hydrocortisone acetate, betamethasone-17-valerate, etc.;
antibiotics, e.g., penicillins, cephalosporins, aminoglysides such as
gentamicin, etc.; antimycotics and dermatics, such as clotrimazol,
miconazol, dithranol, benzoyl peroxide, etc.; antiphlogistics such as
indometacin, methyl, benzyl or 2-butoxyethyl nicotinate, etc.; etc.
Furthermore, cosmetically active agents are suitable, e.g., light
protecting agents or agents for the care of the skin.
The micelle solution can contain about 5 to 150, preferably 10 to 100 mg/ml
of bilayer-forming substance and about 1 to 200, preferably 5 to 100 mg/ml
of detergent. Suitable concentrations of active substances and other
mentioned micelle solution components may vary within broad limits; e.g.,
the active substances concentrations are usually 0.3 to 40, preferably 1
to 20 mg/ml. The concentration ranges for the other mentioned micelle
solution components can also vary in these same ranges.
Suitably, the micelle solution is stirred, for example at about 75 rpm, in
the interior space 4 of the dialysis cell by means of a magnetic stirring
rod (which is not shown), in order to keep the detergent concentration
virtually homogeneous.
Suitably, a dialysis liquid (the composition of which, generally,
corresponds to that of the micelle solution except that the bilayer
forming substance and the detergent are absent) is moved along the
external sides of the membranes 1 in two flow-through compartments and
with a sufficiently high velocity such that the detergent concentration in
the dialysis liquid, which is built up by the detergent passing through
the membranes, will remain below about 1% of the detergent concentration
in the interior space 4 at virtually all points in this liquid and at all
times (or for all dialysis detergent concentrations of this invention), in
particular, including those where the liquid is in contact with the
surfaces of the membranes 1 (=active membrane surfaces). In order to
achieve this, it is advantageous to have a laminar flow of the dialysis
liquid along the surfaces of the membranes 1. This laminar flow can be
ensured by forming, in each of the side-wall elements 3, a meandering
channel 5 through which the dialysis liquid must flow. The partitions 5a,
between the mutually parallel sections of the channel, form flow-guiding
elements for the dialysis liquid, which are in contact with the respective
membrane 1. The dialysis liquid is introduced into the channel 5, at the
bottom, through an inlet 6 in the side-wall element 3, and withdrawn from
the channel 5, at the top, through an outlet 7. The average flow velocity
in the channel 5 is advantageously between 20 and 600 cm/minute and
preferably about 300 cm/minute, for a channel cross-section of, for
example, about 1 mm.sup.2 (width 2 mm, depth 0.5 mm).
Of course, it is also possible to use a spiral channel in place of the
meandering channel 5. If desired, it is also possible to arrange several
mutually parallel channels, separated from one another by partitions,
between the inlet 6 and the outlet 7.
In certain cases, it is also possible to dispense with the carrying
channel, i.e., to let the dialysis liquid flow on the external sides of
the membranes through cylindrical flow-through compartments which are not
interrupted by flow-guiding elements. In fact, in this case, in particular
if the flow-through compartments are relatively thick, measured normal to
the membranes, the results (uniformity of the vesicle size) are somewhat
less good, but are still satisfactory, provided that it is ensured that
the flow velocity of the dialysis liquid in the immediate vicinity of the
membrane surface is in the region of 0.2-6 m/minute, preferably 1-3
m/minute, at virtually all points, and that there are virtually no regions
with stagnating dialysis liquid, in which the detergent concentations
could become too high.
Thus, homogeneous bilayer vesicles of defined size, which, if appropriate,
can contain auxiliaries, peptides, proteins, nucleic acids, lipids,
antigens or antibodies, or also active substances with biological and
pharmacodynamic properties, are obtained in the interior space 4 after a
relatively short dialysis time, (e.g., 1-3 hours), in the form of an
aqueous dispersion. Depending on their solubility properties, these
additives are encapsulated inside the bilayer vesicles and/or incorporated
in the double layer and/or taken up on the outside of the double layer,
whereupon the bilayer vesicles can be used, for example, as carriers for
biologically and/or pharmacodynamically active substances and/or
themselves constitute pharmaceutical preparations.
The dispersion obtained contains about 5 to 150, preferably 10 to 100 mg/ml
of the bilayer forming substance and, if desired, up to 40, preferably up
to 20 mg/ml of the active substance. If desired, an obtained dilute
dispersion can also be concentrated, e.g., by partial evaporation or by
partical lyophilization, suitably up to a concentration of about 150,
preferably about 100 mg/ml of the bilayer forming substance only,
however.
Without further elaboration, it is believed that one skilled in the art
can, using the preceding description, utilize the present invention to its
fullest extent. The following preferred specific embodiments are,
therefore, to be construed as merely illustrative, and not limitative of
the remainder of the disclosure in any way whatsoever. In the following
examples, all temperatures are set forth uncorrected in degrees Celsius;
unless otherwise indicated, all parts and percentages are by weight.
EXAMPLES OF A PREPARATION
Example 1
65 mg of egg lecithin in ethanolic solution are evaporated to dryness and
the residue is resuspended in 5 ml of 1 mM phosphate buffer (composed of
Na.sub.2 HPO.sub.4.2H.sub.2 O, KH.sub.2 PO.sub.4 and 0.9% NaCl) of pH 7.3
and ionic strength 0.16. 58.3 mg of solid sodium cholate are added to this
suspension, while stirring constantly, and the mixture is left to stand
for two minutes at room temperature under a nitrogen atmosphere, until the
formation of the mixed micelles is complete (the micelle solution becomes
clear). To remove the sodium cholate, the micelle solution is subjected at
room temperature, to the flow-through dialysis system described in FIGS. 1
and 2, the micelle solution being stirred constantly (75 rpm). Cellulose
membranes with a molecular exclusion limit of about 10,000 are used as the
dialysis membranes. The flow rate of the dialysate is about 3 ml/minute in
each side-wall element 3. Bilayer vesicles are obtained after a dialysis
time of 20-24 hours, the residual cholate contents of which are less than
1%, relative to the initial cholate content. Bilayer vesicles produced
under these conditions are homogeneous and have a diameter of 60.+-.3 nm.
The comprehensive physicochemical characterization of these vesicles is
described in Biochim. Biophys. Acta. 512, 147-155 (1978).
Examples 2 to 17
The size of the bilayer vesicles can be influenced, for example, by using
dialysis membranes with different permeation properties, and/or by varying
the bilayer-forming substances, and/or by varying the molar ratio of
bilayer-forming substances/detergent, and/or by selection of detergent.
Results are summarized in the following table.
__________________________________________________________________________
Molar ratio:
bilayer-
forming
Molecular substance
exclusion (or lipid
Tem-
Diameter
Limit of
Bilayer-forming substance
mixture,
pera-
of bilayer
Example
dialysis
or lipid mixture, respectively
respectively)/
ture
vesicles
No. membrane
(Molar ratio) Detergent
detergent
.degree.C.
in nm
__________________________________________________________________________
2 2000 EL NaC 0.625 20 75
3 10000 EL/10% PA NaC 0.625 20 50
4 10000 EL/20% PA NaC 0.625 20 40
5 10000 EL NaC 0.60 20 54
6 10000 EL NaC 0.95 20 69
7 10000 EL NaC 1.15 20 80
8 10000 EL OG 0.18 20 170
9 10000 EL/cholesterol (8:2)
NaC 0.60 20 80
10 10000 EL/cholesterol (7:3)
NaC 0.52 20 61
11 10000 EL/phosphatidyl-ethanolamine
NaC 0.22 20 36
(3:7)
12 10000 EL/phosphatidyl-inositol
NaC 0.60 20 60
(8:2)
13 10000 EL/phosphatidic acid (10:2)
NaC 0.62 20 42
14 10000 EL/stearylamine (10:2)
NaC 0.62 20 49
15 10000 bovine brain cerebroside/
NaC 0.60 20 81
EL (100 g/mol)
16 10000 dimyristoylphosphatidylcholi-
NaC 1.25 30 143
ne/phosphatidylinositol (10:2)
17 10000 EL OG 0.20 20 177
__________________________________________________________________________
EL = egg lecithin
PA = phosphatidic acid from egg lecithin
NaC = sodium cholate
OG = noctyl-.beta.-D-glucopyranoside
The bilayer vesicles of defined size, produced in accordance with the
process described, can be used as carriers for biologically and
pharmacodynamically active substances and/or can be employed as a
pharmaceutical preparation. Pharmaceutical preparations can thus be
produced in such a way that the bilayer vesicles are mixed as the active
constituent, with a carrier suitable for therapeutic administration, and,
if appropriate, the mixture is converted to a particular galenic form.
The following galenic forms of administration are possible:
ampoules, in particular sterile injection and infusion solutions, the
colloidal solution of the bilayer vesicles containing pharmacodynamically
active substances being subjected to an antimicrobial treatment;
solutions, in particular syrups, eye drops and nose drops, which can
contain diverse auxiliaries in addition to the bilayer vesicle solution
described above;
non-metering aerosols and metering aerosols, which can contain propellent
gas and stabilizers in addition to the bilayer vesicle solution described
above;
emulsions, such as water-in-oil or oil-in-water emulsions, for parenteral,
oral and topical, (e.g., creams) administration, and also emulsions of
these types which have been processed to give corresponding nonmetering
aerosols or metering aerosols. Water-in-oil emulsions form, for example,
the contents of soft gelatin capsules which can be administered perorally
or rectally.
Furthermore, gels and the most recently developed therapeutic systems based
on diffusion, osmotic and soluble units, such as, for example,
Ocusert.RTM., Biograviplan.RTM., the displacement pump Alzet.RTM., and
Oros (oral therapeutic system), can also be used as possible forms of
administration, which again comprise the colloidal solutions of the
bilayer vesicles containing pharmacodynamically active substances.
Bilayer vesicles in the lyophilized state can be processed, together with
corresponding pharmaceutical auxiliaries to give tablets or dragees.
Example of an Application: Hydrogel
(a) In analogy to Example 1, 320 mg of egg lecithin, 80 mg of cholesterol
and 40 mg of betamethasone 17-valerate are dissolved in ethanol. The
solution is evaporated to dryness, the residue is resuspended in 20 ml of
phosphate buffer, and 400 mg of sodium cholate is added. Thereafter, the
procedure of Example 1 is followed.
(b) In 75 ml of water, there are dissolved 0.2 g of potassium sorbate,
0.224 g of Na.sub.2 HPO.sub.4.12H.sub.2 O and 0.64 g of KH.sub.2 PO.sub.4.
With light warming and vigorous stirring, 2 g of hydroxyethyl cellulose is
dissolved in the solution obtained. After 0.5 hour of standing, 2 g of
glycerol is added with stirring, followed by the liposome dispersion
obtained according to (a). The volume of the mixture is adjusted to 100 ml
by adding water.
The obtained hydrogel contains 0.04% of active substance and shows a pH
value of 5.8 to 6.3.
The preceding examples can be repeated with similar success by substituting
the generically or specifically described reactants and/or operating
conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easily ascertain
the essential charcteristics of this invention, and without departing from
the spirit and scope thereof, can make various changes and modifications
of the invention to adapt it to various usages and conditions.
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