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BACKGROUND OF THE INVENTION
Liquid crystalline phases of lipids are known in the prior art. These
phases are called liquid crystalline since they have degrees of order
which are intermediate between the three dimensional order of a crystal
and the random distribution of a liquid. These phases have order in one or
two dimensions characterized by onionskin or lamellar arrangements of the
lipids when dispersed in water.
When liquid crystals are subjected to energy in the form of ultrasonic
radiation, they can be broken down to single layer vessicles of small
dimensions called liposomes.
In recent years encapsulation of various medicaments in
phospholipid-chloesterol liposomes has been accomplished. The systems
described are ternary systems of a phospholipid plus cholesterol and
water. Basically, these systems are prepared by dissolving the
phospholipid and cholesterol in a solvent which is evaporated to leave a
thin film of lipid. The aqueous phase medicament is then added, which
swells the cholesterol phospholipid mixture to encapsulate the medicament.
Subsequent ultrasonic irradiation provides the liposomes.
Vanlerberghe et al. U.S. Pat. No. 4,217,344, discloses the preparation of
dispersions of liposomes containing an active substance, which after size
reduction by ultrasonic treatment, are separated from the dispersion by
gel filtration.
Preparation of phospholipid-sterol liposomes by gel filtration without
sonication has been described by J. Brunner et al. "Single Bilayer
Vesicles Prepared Without Sonication Physico-Chemical Properties"
Biochemica et Biophysica Acta, 455 (1976) 322-331.
Basically, the prior art preparation of phospholipid-sterol liposomes using
gel filtration involved two major steps:
(1) The preparation of the liposome in the traditional fashion by,
(a) Dissolving the lipid substances in organic solvents to evenly disperse
the components, and then evaporating the solvent.
(b) Adding water or other aqueous media containing drug to the lipid film
to form the liposomes.
The liposome dispersion is then sonicated or not sonicated depending on the
type of liposome desired.
(2) The liposome preparation is passed over a gel-filtration column to
remove untrapped drug and to isolate liposomes of a uniform size.
In contrast, the present liposome process using gel filtration involves the
following steps:
(1) Solubilization and homogeneous dispersion of drug and lipid components
in a detergent (rather than organic solvent).
(2) Passage of the detergent solution over a gel-filtration column which
removes the detergent, causing the formation of the liposome.
In the present invention the concentration of components may be in excess
of that necessary for liposome formation. The liposome formed will be what
is physically the most stable form. The present procedure may be made
limiting by initially adjusting the concentration of the components. Thus,
the present procedure allows for greater control over the final liposome
composition than the prior art procedure.
SUMMARY OF THE INVENTION
This invention relates to processes and apparatus for the preparation of
medicament delivery systems. It provides processes for the preparation of
liposome medicament delivery systems, wherein a medicament is encapsulated
in a liposome, comprising an aliphatic lipid-sterol-water lamellae.
Broadly, this invention provides a process for the production of medicament
containing liposomes, comprising an aliphatic lipid-sterol-water lamallae,
wherein a clear, mixed micellar medicament-detergent-aliphatic
lipid-sterol sample is applied to a gel filtration column e.g. Sephadex
G50. The column effluent is monitored to detect the liposome fraction
eluted. On passage through the column a liquid crystalline (liposome)
front comes off around the void volume. The liposomal fraction is
collected. The free medicament, detergent, lipid and/or sterol may be
recirculated back to the column.
Preferably, this invention provides a continuous process for the production
of medicament containing liposomes, comprising an aliphatic
lipid-sterol-water lamellae, wherein a clear, mixed micellar
medicament-detergent-aliphatic lipid-sterol sample is applied to a gel
filtration column e.g. Sephadex G50. The column effluent is continuously
monitored to detect the liposome fraction eluted. On passage through the
column a liquid crystalline (liposome) front comes off around the void
volume. The liposomal fraction is collected. The free medicament,
detergent, lipid and/or sterol are recirculated back to the column.
Certain medicaments in the liposome systems may be subject to alkaline
degradation such as hydrolysis. Adjustment of the hydrogen ion
concentration (pH) of these systems may be necessary to protect the
medicaments from the alkaline degradation.
This invention provides an apparatus for the preparation of liposome
medicament delivery systems which comprises a column having a loading end
and a discharge end. The discharge end has photoelectric detection means
in optical association therewith. The discharge end is in fluid
communication with a multidirectional valve, which is switchable between a
first conduit and a second conduit. The first conduit is in communication
with a fluid collection device. The second conduit is in communication
with the column loading end, an introductory circuit and a pump. A
microprocessor is in electronic communication with the photoelectric
detection means, the multidirectional valve, the fluid collection device
and the pump, whereby the microprocessor controls fluid flow from the
column by switching fluid flow to either the collection device or the
column loading end, depending upon the input electronic signal received
from the photoelectric device.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The aliphatic lipid may be any pharmacologically acceptable aliphatic
surface-active compound which forms micelles in aqueous media when present
in concentrations above the critical micelle concentration include the
sodium and potassium salts of C.sub.4 to C.sub.18 saturated and
unsaturated fatty acids, e.g., butyric acid, isovaleric acid, caproic
acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic
acid, oleic acid, linoleic acid, linolenic acid, and their amides. Other
pharmacologically acceptable aliphatic surface active agents may include
glycerol containing phospholipids both natural and synthetic (containing
choline, ethanolamine, serine or inositol), phosphotidyl glycerol and
shingosine containing lipids such as shingomyelin, cardiolipin
glucolipids, gangliosides and cerebrosides.
The sodium and potassium salts of C.sub.4 to C.sub.18 unsaturated fatty
acid are preferred in the practice of this invention.
Most preferred are the sodium and potassium salts of C.sub.14 to C.sub.18
unsaturated fatty acids, e.g., sodium oleate and potassium oleate.
The sterols of this invention are those sterols capable of forming
liposomes (as described above) with the aliphatic lipids and medicaments
of this invention. Among the sterols which may be used are cholesterol,
.beta.-sitosterol, desmosterol, 7-keto-cholesterol, .beta.-cholestanol,
estradiol and the like. Cholesterol and .beta.-sitosterol are the
preferred sterols.
The nature of the medicament to be encapsulated is not critical. Suitable
medicaments include vaccines and antigens, as well as drugs. Drugs useful
in connection with this invention are those drugs capable of being
encapsulated in an aliphatic lipid-sterol-water liposome. The drug
delivery system of this invention is especially useful for intestinal
absorption of labile drugs.
Among the drugs which may be used in the practice of this invention are
insulin, ergot alkaloids, e.g., dihydroergocornine, dihydroergocristine,
dihydrooergokryptine, and mixtures thereof, thioridazine, enzymes,
hormones, and the like.
Liposomes of this invention may be prepared having the following aliphatic
lipid (e.g., sodium or potassium salts of C.sub.4 to C.sub.18 saturated or
unsaturated fatty acid)-sterol (e.g., cholesterol)-water weight percent
(%) compositions;
Aliphatic lipid from about 0.03% to about 20% sterol from about 1.0% to
about 55%, and water from about 45% to about 97%.
Preferably the liposomes may contain the aliphatic lipid from about 1.0% to
about 15%, sterol from about 1.0% to about 40%, and water from about 50%
to about 97%.
More preferably, the liposomes may contain the aliphatic lipid from about
5.0% to about 10%, sterol from about 1.0% to about 10%, and water from
about 75% to about 97%.
The processes of this invention are preferably carried out in an inert
atmosphere, e.g., nitrogen, to prevent auto-oxidation of the lipid and/or
sterol.
The detergent of this invention does not enter into the final liposome
product. Therefore, its selection is not critical. The exception to this
is that, it must have sufficient detergent properties under the process
conditions of salt concentration, pH and medicament properties not to be a
limiting factor in dispersing the proportions of coating lipid and
medicament for the liposome formation.
Among the detergents which may be used in the practice of this invention
are: anionics such as sodium taurocholate, sodium dodecylsulfate, cholic
acid and the like;
Nonionics such as polyethyleneglycol sterol ester (Solulan),
polyoxyethylene sorbitan monolaurate (Tween) and the like.
The gel filtration of this invention maybe carried out with gels such as
Sephadex, G-10 though G-200, preferably Sephadex G-50 and G-70 (Pharmacia
Fine Chemicals, Piscataway, NJ), Sepharase, Agarose, Acrylamide, or
Cellulose.
The Processes of this invention may be carried out from a temperature
(C..degree.) at which the critical micelle concentration of the lipid is
reached, to about 60.degree. C. Preferably the processes may be carried
out at from about 20.degree. C. to about 50.degree. C. More preferably the
processes may be carried out at from 25.degree. C. to about 45.degree. C.
When it is necessary to protect the medicament from alkaline degradation,
the pH of the liposome system may be adjusted from an alkaline pH to a
neutral or acid pH, e.g. from about pH 8 to about pH 5. The pH adjustment
may be affected by contacting the liposome system with a pharmaceutically
acceptable mineral acid, e.g. hydrochloric acid, organic acid, e.g. citric
acid, or buffer solution.
The liposome medicament delivery system of this invention are useful for
both oral and parenteral administration of medicaments. Oral
administrations is preferred, however, as the liposome encapsulation may
serve to protect drugs such as insulin which are labile in the digestive
system. For oral administration the liposome suspension may be admixed
with pharmacologically acceptable dilutents or carriers and with
conventional adjuvants such as flavorings and colorings, and administered
in such forms as syrups, elixirs, capsules, tablets, etc. Suppositorial
administration may also be utilized.
The drawing illustrates a continuous process for producing liposomes using
the process teachings of this invention.
A column 10 e.g. chromatography column packed with Sephadex particles 11,
has at its discharge end 12 a photoelectric detection means, e.g. a light
source 13 and a photoelectric tube 14, and a multidirectional valve 16.
The valve 16 is in fluid communication via conduit 17 with a conventional
sample fraction collector 18, and via conduit 19 with the loading end 21
of the column 10. A pump 22 on conduit 19 and an introductory circuit 23
complete the external fluid circuitry.
In operation, a drug containing micellar sample, is applied to the Sephadex
column 11 and passes down through the column. The light source 13 and
photoelectric tube 14 monitor the fractions eluted from the column and
send a continuous input signal to a conventional microprocessor 24. Clear
solution eluted from the column is continuously recirculated back into the
column by valve 16 and pump 22, via conduit 19.
When the milky liposome fraction is detected by the photoelectric tube 14,
the microprocessor 24 switches valve 16 to circuit 17 to collect the
eluting liposome fractions in the fraction collector 18.
When the liposome fractions have been collected valve 16 is again switched
back into fluid communication with circuit 19 for continuous recirculation
of the eluting material.
From time to time, additional micellar sample is added by the
microprocessor to the column, via introductory circuit 23 and pump 26 from
a storage source not shown, to maintain the desired concentration of
material on the column. Thus, a continuous process for the production of
liposomes is maintained.
In this specification and claims, the following statements are descriptive
of the terms indicated:
Micelle--collodial particles, consisting of oriented molecules, e.g., lipid
molecules surrounding a medicament.
Liquid crystals--states of matter having characteristics of both liquids
and crystalline solids. Liquid crystals are formed when the micelles of a
lipid-medicament have penetrated a sterol.
Liposomes--the product of the particle size reduction of liquid crystals.
Lamellae--the layers of a liquid crystal or liposome.
In each of the following examples a clear, mixed micellar sample was
applied to a Sephadex G50 column.
The column conditions were:
Type of Column: Sephadex G-50 medium
Column Size: 0.9 cm.times.48.0 cm
Flow Rate: 1 ml/min.
Volume of Eluted Fraction Collected: 3.0 ml
Elution Buffer: Potassium Phosphate pH 6.0-6.2
The Micellar Sample was 2.0 ml comprising:
Sodium Taurocholate: 10.76 mg/ml
Sodium Oleate: 2.36 mg/ml
Cholesterol: 0.671 mg/ml
In each of the examples one of the micellar components was radioactive in
order to monitor the fractions eluted from the Sephadex Column. In
addition each fraction was visually inspected for turbidity. The micelle
fractions are clear solutions while the liposome fractions are milky.
The total radioactivity eluted from the Sephadex column was determined by
pipetting 0.1 ml aliquots from each eluted fraction into vials. ACS Liquid
Scintillation Fluid was added and the radioactivity determined using a
Packard Tri Card Liquid Scintillation Counter. Counts per Minute (CPM) and
Disintegrations per Minute (DPM) were determined, with the total DPM
indicating total radioactivity.
EXAMPLE I
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Fraction # CPM DPM/Fractions
Visual
______________________________________
1 27 <2 .times. 's Bkg.
clear
2 25 " "
3 28 " "
4 31 0 milky
5 65 1,438 milky
6 46 738 turbid
7 154 4,712 "
8 2,047 74,922 "
9 7,575 280,961 "
10 8,126 300,077 clear
11 5,961 222,446 "
12 2,219 81,664 "
13 453 15,927 "
14 81 1,940 "
15 39 <2 .times. 's Bkg.
"
16 "
17 "
18 "
19 "
31 "
Bkg. 26 TOTAL 984,825
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Bkg. = Background radiatiation
C.sup.14 Na taurocholate 21.52 mg/2 ml = 939,766 DPM
Fractions #4 and #5 contain 0.15% of the total C.sup.14 Na tauracholate
eluted from the column. Because of the low CPM value this is considered to
be equivalent to 0% C.sup.14 Na taurocholate.
EXAMPLE II
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Fraction CPM DPM/Tube Visual
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1 28 <2 .times. Bkg.
clear
2 25 " "
3 28 " "
4 2,585 94,710 milky
5 3,107 115,917 milky
6 170 5,340 turbid
7 86 2,208 "
8 71 1,612 "
9 56 1,042 clear
10 52 896
11 39 <2 .times. Bkg.
12 26
13 28
14 26
15 30
16 27
27
20 28
Bkg. 28 TOTAL 221,725
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C.sup.14 Na Oleate 4.72 mg/2 ml = 349,996 DPM
Fractions #4 and #5 contained 95% of the C.sup.14 Na Oleate eluted from the
column.
EXAMPLE III
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Fraction CPM DPM/Fraction
Visual
______________________________________
1 28 <2 .times. 's Bkg.
clear
2 28 " "
3 27 " "
4 1,083 39,987 milky
5 521 18,661 milky
6 90 2,245 turbid
7 81 2,032 "
8 67 1,471 "
9 49 <2 .times. 's Bkg.
clear
10 33
11 26
12 29
13 30
14 26
15 24
16 24
17 26
18 27
19 27
20 31
Bkg. 27
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C.sup.14 cholesterol 1.342 mg/2 ml = 48,030 DPM
Fractions #4 and #5 contain 91% of the C.sup.14 cholesterol eluted from the
column.
The results of Examples I, II, and III show the Na taurocholate can be
removed by the Sephadex column from mixed micelles. Thus, Na
Oleate-cholesterol liposomes were generated.
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Description |
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