Note: Descriptions are shown in the official language in which they were submitted.
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1 This invention relates to a new method ~or preparing
injectable liposomes.
Liposomes are a pharmaceu-tical composition in which the
drug is contained in corpuscles or globules consisting of aqueous
and lipidic concentric layers. The drug may be present in either
or both of the aqueous and/or lipidic layers. The lipidic layer
comprises or consists generally of a phospholipid, such as
lecithin and sphyngomyelin; a steroid, for example cholesterol;
and an ionic tensioactive or surface active substance, such as
dicetylphosphate, stearylamine and phosphatidic acid. The dia-
meter ofliposomes ranges from 0.01 to 5 ~. The procedure usually
employed for preparing liposomes comprises two main stepso
1. Preparation of liposomes. The lipidic components are
dissolved in chloroform, which is then evaporated under vacuum.
In the flask containing the residue as a thin layer, the drug
solution is added and the whole is subjected to ultrasonic shaking
for a time ranging from 30 seconds to 12 hours. The liposomic
suspension so obtained contains an important fraction of non-
entrapped drug which must be separated ~rom the liposomes.
2. Separation of the liposomec, from the non-entrapped
drug. This procedure is carried out by column chromatograph~ with `
e.g. Sepharose 6B as a molecular sieve material. Sepharose is a
registered trademark for spherical agarose gel beads manufactured
by Pharmacia Fine Chemicals AB. The "6B" refexs to an agarose
concentration of about 6% and a particle size in the wet state of
about 40 to 210,u. The liposomes eluate first, whereas the free
drug is retained by the Sepharose.
Another suitable procedure is ultracentrifuga-tion at
lOO,OOOg and subsequent washing, always by unltracentrifugation,
with buffered solution.
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1 The liposomes have demons-trated, in the animal, an abil-
ity to direct antitumoral drugs selectively toward neoplastic
cells.
The presen-t invention relates to a new procedure for
separatiny liposomes from non-entrapped drug. It utilizes ion-
exchange and adsorbent resins without the necessity of employing
chromatography. The ion-exchange resin is added to the liposomic
suspension of the drug which is to be purified, and the whole
shaken for 1-120 minutes.
The free liposomic suspension is ob-tained after filtra-
tion through porous sheet that retains the resin on which the free
drug has been adsorbed. A suitable porous sheet is one made e.g.
of a sin-tered glass filter material, such as manufactured by the
German firm Jena er Glaswerk Schott 8 Gen. Mainz that produces a
wide range o~ suitable glasses. These glasses are named with
different alphabetical letters and numbers (e.g. Gl : in this case
number 1 reEers to the broader pores generally used in filtration).
The pore size abviously changes. The alphabetical letter refers
to the kind of sintered glass that also changes depending on the
eventual desired transfer o~ residue due to filtration.
This new purification procedure, utilizing ion-exchange
resins, shows the great advantage of giving very concentrated
liposomic suspensions (up to 4 mg/ml of Adriamycin hydrochloride)
which cannot be achieved by chromatography with molecular sieves
~max 200 r/ml). The liposomic suspension so obtained is very
stable and is not inclined to sedimentation in contrast to the
suspensions obtained by ultracentrifugation at high r.p.m.'s.
The desired chemical stabilization is achieved by
lyophilization of the liposomic suspension.
The following examples are provided to still further
illustrate the invention but without limiting it:
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1 FXAM E 1
In a saponiEication flask, l.5 y of egg lecithin, 0.4 g
of cholesterol, and 0.2 g of dicetylphosphate were dissolved in
chloroform and the solvent evaporated to dryness. An adriamycin
hydrochloride solution (concentration 20 mg/ml) in 0.007 N buffer
phosphate was then added and the resulting solution was subjected
to ultrasonic shaking for one minute.
The suspension was then allowed to stand at room temper-
ature under a nitrogen atmosphere for 30 minutes, then 2 g of
BDH IR-50 resin (a granular-polymeric resin manufactured by R~hm
Haas), previously activated in the sodium form, were added
(weight refers to dry weight and is equivalent to 5 ml of inflated
resin. By "inflated resin" is meant that the resin has been
activated by two successive treatments, first in a 4% HCl solution
and then in a 4~ NaOH solution).
The flask was subjected for 30 minutes to shaking; the
suspension was then filtered over a porous sintered glass sheet
Gl.
Liposomes of size varying from 0.5 to 2~1 and containing
~O 20% of the starting amount of adriamycin have been obtained. They
were made stable by lyophilization.
EXAMPLE 2
Operating as described in Example l, and employing the
same amounts of adriamycin and lipids, the ultrasonic shaking time
was extended to lQ minutes in order to obtain liposomes of size
less than l,u. A non-granular resin, having also the function of
a sieve, was selected in view of the fact that the whole body of
the liposomes is not perfectly homogeneous in size.
Accordingly, l0 ml of DOWEX 50W-X 4 100-200 mesh resin
(a macroreticular resin available from Dow), and previously
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1 a~tivated in sodium form, were introduced into the flask. After
filtration, a suspension was ob-tained containing liposomes ranging
from 0.2 and ~.8~ in size and containing 12% of the starting
adriamycin. The liposomes were then made sta~le by lyophilization.
EXAMPLE 3
A solution of 5-fluorouracil at a concentration of
10 mg/ml in 0.007N buffer phosphate and pH 8, was poured into the
saponification flask containing the lipidic phase prepared as
previously described. The suspension was treated as in Example 1,
using as filtering resin 10 ml of Amberlite IRA-400 (Cl), a
granular polymeric resin produced by Rohm & Haas, previously
activated as the hydrochloride. The liposomes so obtained were
made sta~le by lyophilization.
EXAMPLE 4
Liposomes of 5-fluorouracil were prepared as prevlously
described utilizing 1.5 g of egg lecithin, 0.4 g of cholesterol,
and 0.2 g of stearylamine. As the purification agent 10 ml of a
previously activated DOWEX 1 resin (50-100 mesh), a granular poly-
meric resin available from Dow, were used. The liposomes so ob-
tained were stabilized by lyophilization.
EXAMPLE 5
Operating under the same conditions and with the same
amounts as in Example 2, an adriamycin liposomic suspension was
prepared, purified by usiny 15 g of adsorbing Rohm h Haas XAD 7
resin (a granular polymeric resin), directly placed into the pre-
paration flask.
A~ter shaking for 40 minutes and filtration through
sintered filter glass Gl, a suspension of liposomes containing
about 50% of the startiny quanti-ty of adriamycin was obtained,
which was then stabilized as before by lyophilization.
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