Liposomes preparation method and plant

Details Liposomes preparation method and plant Liposomes preparation method and plant

1998-10-29

2001-04-17

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C264S004100, C264S004300, C428S402200

Reexamination Certificate

active

06217899

ABSTRACT:

FIELD OF THE INVENTION
The present invention is related to a new liposomes preparation method and plant
The present invention concerns also the liposomes populations obtained and their use.
BACKGROUND OF THE INVENTION
Liposomes are currently extensively studied as potential drugs or cosmetics carrier. A wide variety of liposomes preparations has been described and reviewed [1, 2, 6].
However, at the present time, most of the methods have not been scaled up from the laboratory level to the industrial production [3]. To ensure an optimal reproducibility of drug laden liposomes in vivo, the assessment of physico-chemical parameters (size, number of lipid bilayers, encapsulation efficiency, . . . ) characterizing the dispersion is essential.
In general, the mentioned parameters only refer to average data. But not only average values have to be considered; attention should also be paid to the homogeneity of the dispersions [3].
The preparation method in combination with the composition of the lipid mixtures and the nature of the aqueous dispersing solution decide upon morphology and homogeneity of the obtained liposome population and on their behaviour in vivo.
A well-defined preparation technique together with a fixed lipid composition and validated operating procedures are the key conditions to produce a liposome population with an acceptable reproducibility, suitable for pharmaceutical use.
STATE OF THE ART
In general, liposomes preparation includes two major steps: lipid hydration of a mixture of lipid with possibly liposoluble molecule and sizing to the desired particle size distribution. The difference between the various methods is the way in which these two steps are performed individually or how they are combined.
For convenience, these methods have been classified in three categories
mechanical dispersion methods such has hand-shaking and vortexing [2], sonication and use of a French press;
detergent-solubilizing dispersion methods [22];
solvent dispersion methods such as ethanol injection [12], ether infusion [15] and reverse-phase evaporation [14].
However, all these methods suffer from one or more drawback(s) in term of suitability for bulk manufacture for pharmaceutical applications (see tables 1 and 2 below).
In addition, for the pharmaceutical production at the industrial scale, a standard procedure to obtain lipidic vesicles in a specific size range is still missing.
Especially, liposome populations of large size liposomes (diameter >0.2 &mgr;m) with narrow size distributions are particularly difficult to produce.
TABLE 1
Characteristics of the liposome preparation methods
Volume
Encap-
%
sulated
encap-
Methods
Struc-
Diameter
(&mgr;l/&mgr;mol
sulation
of preparation
ture
(&mgr;m)
lipids)
(%)
Ref.
Hydration film
MLV
1.8-8.5
 [4]
0.5
6
 [5]
0.05-30 
3
 [6]
0.4-5
4.1
 5-15
 [7]
1
1.4-1.8
 9-27
 [8]
Ultrasonication of
SUV
0.02-0.05
0.2-1.5
0.1-1.0
 [9]
MLV
French press extrusion
SUV
0.02-0.08
0.2-1.5
 5-25
[10]
of MLV
Polycarbonates
membranes
extrusion of MLV
0.1 &mgr;m
SUV
0.06-1  
1-3
5-30
[11]
1-0.2 &mgr;m
MLV
0.05-2  
1.8-3.7
15-60
 [7]
Microfluidisation
SUV
<0.1 
0.7-1.0
 5-78
 [9]
Ethanol injection
SUV
 0.03
0.5
[12]
 0.12
0.4-1.5
[13]
Reverse phase
LUV
0.1-1.0
 7-11
30-68
[14]
evaporation
Ether infusion
LUV
0.05-0.25
13-25
2
[15]
Lyophilisation/
MLV
0.02-0.2 
26-72
 [4]
hydration
Freezing/thawing
LUV
 0.09
25-30
[16]
(SUV)
Congelation/
MLV
2-5
31-89
[17]
decongelation (MLV)
Elimination of
LUV
0.6
12
[18]
detergent
0.1
2.4
12
[19]
0.04-0.18
1.75-2.4 
[20]
0.2-0.3
  6-7.4
22
[21]
SUV
 0.037
 0.47
[22]
TABLE 2
Advantages and inconveniences of the liposome
preparation methods
Preparation methods
Advantages
Inconveniences
Thin lipid film
Simple, rapid
Poor encapsulation
hydration by
efficiency
mechanical shaking
Heterogeneous dispersion
(MLV)
of MLV
No upscaling feasibility
Ultrasonic irradia-
Small liposomes
Poor encapsulation
tion (SUV)
(20 nm diameter)
efficiency
Homogeneity
Contamination by
titane particles
Production of aerosols
Warming and lipid
degradation can occur
No upscaling feasibility
French press
Simple, reproducible,
Not economical
extrusion
non aggressive
Need the preparation of
High concentration
MLV
of lipids can be used
Poor encapsulation
High encapsulation
efficiency
efficiency
Scaling-up
Extrusion through
Rapid and reproducible
Need the preparation of
polycarbonate
Homogeneity
MLV
membranes
High concentration
Only small volumes can
of lipids
be used
High encapsulation
efficiency
Microfluidisation
High encapsulation
Need the preparation of
efficiency
MLV
Scaling-up
Not economical
Organic solvent
Homogeneity
Poor encapsulation
replacement
Simple, rapid
efficiency
Ethanol injection
Scaling-up
Reverse phase
High encapsulation
Lipids exposed to ultra-
evaporation
efficiency
sonic and solvents
Complicated technic
Low solubility in the
organic solvents
No upscaling feasibility
Ether infusion
High encapsulation
Poor encapsulation
efficiency
efficiency
Scaling-up
Lipids exposed to high
temperature and solvents
Heterogeneity
Lyophylisation/
High encapsulation
Heterogeneity
rehydration
efficiency
Need the preparation of
Simple
SUV
Scaling-up
Stability of the prepara-
tion
Freezing/thawing
High encapsulation
Need the preparation of
(MLV)
efficiency
SUV
Rapid
Freezing/thawing
Process simple and rapid
Need the preparation of
(SUV)
SUV
Difficult to prepare in
the presence of neutral
phospholipids, sugar and
high concentration of
divalent ions
Removal of
Homogeneity
Low encapsulation
detergents
Smooth condition
efficiency
Lengthy process
Difficulty to remove the
detergent
At the present time, the most direct methods for producing liposomes population on a large scale are based on high-shear homogenization. The major drawback of this method is that it allows only the production of small liposomes (<0.2 &mgr;m) with a relatively narrow size distribution.
For the production of large multilamellar liposomes (>1 &mgr;m), the lipid film hydration method can be used. However, said method is difficult to scale up due to the small surfaces available to dry the thin lipid film before the hydration step, which leads to the formation of vesicles characterized by large size distributions [3].
Nowadays, no method is available for large scale production of unilammelar or oligolamellar liposomes (i.e., consisting of a few bilayers) with a narrow size distribution in the range of 0.2-1 &mgr;m.
Usually, when the thin lipid film hydration method is applied, the particles size distribution of the liposomes produced is much broader and in many cases, the liposome population is bi- or even polymodal.
However, it is known that the size of the liposome particles is essential for their therapeutical use. For instance, according to their size, particles could reach different sites in the respiratory system. The table 3 represents the different sites reached by the particles according to their size.
TABLE 3
Particle mean
diameter size (&mgr;m)
Deposit
>100
No deposit possible
>10
Nasal cavity
>5
Trachea, bronchus
>1
Alveolus
>0.1
Stable particles - low deposit
<0.1
Important deposit
Therefore, for some specific therapeutical uses, it is very important to provide a liposomes population having a very narrow size distribution. In addition, if the distribution of the liposomes population is homogeneous, it is possible to obtain a reproducible and reliable pharmaceutical composition with liposomes having a specific encapsulation efficiency of a specific active compound.
The U.S. Pat. No. 4,737,323 describes a method of producing a suspension of liposomes which have approximately an uniform size and a selecte

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