Lyophilized emulsion containing an active substance

The invention relates to a lyophilized emulsion composition which contains an active substance and which can be redispersed with water to give the original emulsion, and to a process for the preparation thereof. The redispersed emulsion is suitable for parenteral use.

The present invention relates to a lyophilized emulsion which can be 
redispersed with water to give the original emulsion. 
In particular, the invention relates to a fat emulsion which contains an 
active substance and whose external aqueous phase has been removed by 
freeze-drying and which can be redispersed by addition of water 
spontaneously to give the original emulsion with a particle size 
distribution corresponding to the initial formulation. 
Emulsions are disperse systems composed of two mutually immiscible liquids, 
one of which, the internal, disperse phase, is finely dispersed in the 
other, the external, continuous phase. 
Fat emulsions are emulsion systems in which the internal, disperse phase 
consists of very fine fat particles which are homogeneously dispersed in 
the external phase which is composed of water. Emulsion formulations of 
this type are preferably used parenterally and are particularly used for 
intravenous nutrition of patients unable to take food by mouth. 
Fat emulsions which can be administered intravenously make high demands on 
the tolerability of their ingredients and the particle size of the fat 
particles. Preferably used as fat component are oils with a high content 
of unsaturated fatty acids such as soya bean, safflower and cottonseed 
oils, as emulsifiers are lecithins such as egg, soya and cerebral 
lecithins, as well as antioxidants such as tocopherol acetate and other 
auxiliary substances. 
The fat particles should, in order to avoid changes in blood pressure and 
the risk of embolism, not exceed an average particle size of 1 .mu.m. 
The emulsion is normally prepared by preemulsifying the heated oil and 
aqueous phases with a mixer, followed by microfine emulsification using a 
high-pressure homogenizer and subsequent sterilization with superheated 
steam. 
The "Handbook on Injectable Drugs" (American Society of Hospital 
Pharmacists, pages 237-244 (1986), Lawrence A. Trissel) describes some 
commercially available formulations. They contain soya bean oil or 
safflower oil, egg lecithin, glycerol and water and have average particle 
sizes of .ltoreq.0.5 .mu.m. 
Fat emulsions are also repeatedly used as vehicle systems for lipophilic 
medicinal substances to be administered parenterally. The aim in this case 
is to increase the therapeutic efficacy and safety of medicinal substances 
by controlled release from emulsion systems. 
In accordance with their solubility properties, lipophilic active 
substances in emulsions are partly or completely incorporated in the fat 
particles. This means that their pharmacokinetic behaviour is crucially 
determined by the pharmacokinetic behaviour of the vehicle formulations 
from which the active substance is first released. Delayed release avoids 
high local concentrations of active substance, reduces degradation and 
thus increases the duration of action. 
Emulsion systems of this type are particularly advantageous for 
prostaglandins, especially prostaglandin E.sub.1 (PGE.sub.1). PGE.sub.1 is 
a highly active tissue hormone which is successfully used, for example, 
for the treatment of arterial occlusive disease. Used for this purpose is 
a PGE.sub.1 -.alpha.-cyclodextrin complex which, dissolved in 
physiological saline solution, is infused parenterally, preferably 
intraarterially, as close as possible to the body region to be treated. 
However, high pressure conditions and small dilution effects during the 
intraarterial infusion make high demands on the equipment and the training 
of the treating physician. Although intravenous infusion is simpler to 
perform by comparison, even in this case infusion is possible only slowly 
and in relatively high dilution because of the local irritant effect of 
PGE.sub.1. Overall, the extended residence time of the active substance in 
the vascular system before reaching the target site and, in particular, 
the additional passage through the pulmonary circulation leads to 
increased degradation of active substance. Both intra-arterial and 
intravenous infusions make high demands on the equipment and careful 
adjustment of the infusion rate and are therefore usually performed in 
hospital and not by the established physician, which impedes wide use of 
the valuable active substance in the therapy of arterial occlusive 
disease. 
These problems can be avoided by incorporating PGE.sub.1 in a fat emulsion. 
Delayed release of active substance avoids high local concentrations, 
reduces degradation of active substance and increases the duration of 
action so that formulations of this type are also suitable for intravenous 
bolus injection. 
The process for preparing such fat emulsions containing active substances 
substantially corresponds to the above-mentioned preparation of a fat 
emulsion, with the difference that the active substance to be incorporated 
is dissolved in the oil phase before carrying out the preemulsification. 
Although PGE.sub.1 -containing fat emulsions of this type are suitable for 
solving the disadvantages described for the conventional use of PGE.sub.1, 
they have low storage stability owing to hydrolytic degradation of the 
active substance, which impedes their general utilizability. 
One possibility for stabilizing the active substance within the emulsion 
comprises removal of the substances destabilizing the active substance. 
One example of this is given in U.S. Pat. No. 4,684,633 which describes 
the stabilization of active substances brought about by using 
phosphatidylethanolamine-free egg lecithin in an emulsion composition 
containing prostaglandin, soya oil, egg lecithin, glycerol and water. 
However, stabilization of active substances is shown only for the 
condition of brief sterilization at 125.degree. C. for 2.2 min. There are 
no data on the stability on long-term storage. The formulation also 
contains water so that degradation of active substance as a result of 
hydrolysis cannot in principle be ruled out. A fundamental disadvantage is 
that the stabilization occurs only to the stated active substances and is 
not generally applicable. 
Besides stabilization of the active substance in a fat emulsion ready for 
administration, fat emulsions with intact active substance can also be 
used by being prepared only immediately before use. One example of this is 
given by EP 0 331 755. It describes a kit consisting of a conventional fat 
emulsion and either an active substance solution in water, liquid 
polyalkylene glycols, liquid alkylethanolamines or liquid alcohols 
containing a plurality of hydroxyl groups, or an active substance 
composition consisting of active substance, saccharides and/or amino 
acids, which are combined and vigorously mixed immediately before use. 
Vigorous mixing is absolutely necessary to make it possible to disperse 
the active substance in the fat emulsion. Thus, for example, the active 
substance emulsion described in Example 5 is prepared by mixing for 2-3 
minutes. However, long mixing times are disadvantageous on use. 
An example relating to prostaglandins which is mentioned in EP 0 331 755, 
Example 3, describes an active substance composition consisting of a 
prostaglandin and triethanolamine. However, triethanolamine is not without 
objections physiologically so that its use in pharmaceutical formulations, 
especially in injectable products, should be avoided where possible. 
There has thus been a continuing need for an emulsion formulation which 
avoids PGE.sub.1 degradation as a consequence of hydrolysis, is 
physiologically unobjectionable and is simple to handle. A formulation of 
this type ought to preclude in principle hydrolytic degradation of active 
substance on storage due to absence of water, be easily redispersible by 
addition of water, and ensure dispersion of the active substance in the 
fat phase of the emulsion from the outset. 
A formulation of this type is to be prepared by lyophilization of a fat 
emulsion containing active substance, which as anhydrous formulation leads 
to the expectation of storage stability and can be reconstituted with 
water to give the original formulation before administration. However, 
investigations show that lyophilization of emulsions lead to coalescence 
of the fat particles and thus to enlargement of the particles thereof or 
even to their complete destruction. Various research groups have 
repeatedly attempted to prevent this coalescence of emulsions as a 
consequence of lyophilization by adding cryoprotection agents. However, 
their results show that even this measure is unable to prevent an increase 
in the average particle diameter [Lladser, M. et al.: The use of supports 
in the lyophilization of oil-in-water emulsions, J. Pharm. Pharmacol 20, 
450-455 (1968); Rambhan, D. et al.: Stability Studies on Lyophilized O/W 
Emulsions, Indian J. Pharm. 39, 52-55 (1977)]. Bensouda et al. further 
show that the increase in the average particle diameter as a consequence 
of lyophilization increases with decreasing size of the emulsion particles 
[Bensouda, Y. et al.: Freeze-drying of Emulsions--Influence of Congealing 
on Granulometry Research of a Cryoprotection Agent, Pharm. Acta Helv. 64, 
40-44 (1989)]. However, emulsions for intravenous administration should, 
in order to avoid embolism, have an average particle size less than or 
equal to 1 .mu.m. Prevention of an increase in particle size as a 
consequence of lyophilization therefore appears to be particularly 
difficult, but is indispensible for safety reasons. 
Lyophilized reconstitutable emulsions which are said also to be suitable 
for intravenous use are described in the documents JP 60239417 and ZA 86 
04 032. JP 60239417 describes the preparation of lyophilized emulsion 
systems with the addition of cryoprotection agents such as saccharides and 
sugar alcohols and water-soluble polymers such as polyvinylpyrrolidone, 
gelatin and hydroxypropylcellulose. However, it is evident from the 
examples that the particle diameters of the redispersed emulsions are all 
higher than in the initial emulsion. ZA 86 04 032 discloses lyophilized 
emulsion compositions and a method for their preparation. After 
preparation of a conventional fat emulsion by known processes and addition 
of a bulking sugar, the emulsion is sprayed into a boiling liquid with a 
boiling point below -20.degree. C. and subsequently lyophilized. The 
examples show, however, that even in this case the average particle 
diameter of the fat particles after reconstitution with water is higher 
than in the initial emulsion. 
Lyophilized emulsions which have, after reconstitution with water, a 
particle dispersion corresponding to the initial formulation are not 
present in the prior art. 
Surprisingly, an anhydrous emulsion composition which contains active 
substance and which contains at least one cryoprotection agent/bulking 
agent and can be redispersed by addition of water to give the original, 
water-containing, active-substance containing emulsion with corresponding 
particle size distribution has now been found. An emulsion composition 
having no coherent external aqueous phase is termed anhydrous. A 
composition of this type avoids hydrolytic degradation of the active 
substance(s) and shows high storage stability. 
The emulsion composition advantageously contains hydrophilic emulsifiers 
and acetylated monoglycerides. Hydrophilic emulsifiers are surfactants 
whose emulsifying behaviour is crucially determined by their hydrophilic 
groups and preferably form fat-in-water emulsions. 
The emulsion composition preferably contains ethoxylated triglycerides or 
polyoxyethylene hydroxy fatty acid esters and acetylated monoglycerides 
with unsaturated double bonds. 
The emulsion composition particularly contains glycerol polyethylene glycol 
ricinoleate (macrogol glycerol ricinoleate) or polyoxyethylene 660 
12-hydroxystearate (macrogol 660 12-hydroxystearate) and diacetylated 
monoglycerides or a mixture of diacetylated and partially acetylated 
monoglycerides. 
Particularly advantageous in this case are acetylated monoglycerides which 
contain 2% by weight to 40% by weight, preferably 20% by weight, of 
partially acetylated monoglycerides. 
According to an expedient embodiment of the present invention, the 
hydrophilic emulsifiers and the acetylated monoglycerides are present in a 
ratio of 1:10 to 2:1 by weight, preferably in a ratio of 2:3 by weight. 
According to a particularly preferred embodiment of the anhydrous emulsion 
composition according to the invention, the latter contains at least one 
active substance from the active substance group of prostaglandins, 
especially PGE.sub.1. 
Another advantageous embodiment of the emulsion composition according to 
the invention contains as cryoprotection agent/bulking agent 
physiologically tolerated mono-, di- or oligosaccharides, especially 
lactose or sugar alcohols such as sorbitol and/or mannitol. 
According to another preferred embodiment of the emulsion composition 
according to the invention, the latter contains at least one customary 
antioxidant, advantageously from the group of tocopherols such as 
.alpha.-, .beta.-, .gamma.- or .delta.-tocopherol, preferably 
.alpha.-tocopherol, and the physiologically tolerated salts thereof, such 
as phosphates, succinates and acetates and/or physiologically tolerated 
buffer salts. 
According to another preferred embodiment of the emulsion composition 
according to the invention, its internal disperse phase has, after 
reconstitution with water, average particle diameters of 0.1 .mu.m to 5 
.mu.m, preferably 0.2 .mu.m to 1.0 .mu.m. 
Another particularly preferred embodiment of the anhydrous emulsion 
composition according to the invention contains cryoprotection 
agents/bulking agents and can be redispersed by addition of water to give 
the original water-containing emulsion with identical particle size 
distribution. 
The emulsion composition according to the invention can be prepared by 
removing the aqueous phase by lyophilization from an emulsion which has 
been prepared by the processes and technologies customary in the 
production of pharmaceuticals. 
The invention therefore also relates to a process for the preparation of 
the active-substance containing anhydrous emulsion composition, according 
to the invention, which is characterized in that an active-substance 
containing emulsion is prepared in a conventional way and its external 
aqueous phase is subsequently removed by freeze-drying. 
It is possible to incorporate in the emulsion composition according to the 
invention hydrophilic active substances by dissolving in the aqueous 
phase, and lipophilic active substances by dissolving in the phase 
containing emulsifier and fat. Alternatively, the active substance can 
also be added immediately before the lyophilization is carried out, which 
is particularly advantageous for active substances which are sensitive to 
hydrolysis and/or unstable to heat. This means that the process for 
preparing the emulsion composition containing an active substance 
according to the invention can be adapted in an advantageous manner to 
suit the physicochemical properties of the active substances. Therefore, 
in an expedient embodiment of the process according to the invention, at 
least one active substance is dissolved either in the aqueous phase or in 
the phase containing emulsifier and fat before emulsification, or at least 
one active substance is added to the emulsion before it is freeze-dried. 
Care should be taken in this case that the active substance dispersion 
within the disperse system is ensured before lyophilization, which can 
easily be checked by customary methods such as equilibrium dialysis, 
differential dialysis and ultrafiltration. 
Preparation

EXAMPLE 1 
emulsion preparation 
3.42 g of citric acid monohydrate, 1.57 g of trisodium citrate dihydrate 
and 60.0 g of lactose were dissolved by heating in 475 g of water for 
injections. Subsequently, 12.0 g of polyoxyethylene 660 
12-hydroxy-stearate and 18.0 g of diacetylated monoglycerides with a 
hydroxyl number of 25 were dissolved in 30.0 g of absolute ethanol by 
heating to about 30.degree. C. under an inert atmosphere (nitrogen). 
The aqueous phase was transferred into a suitable presterilized reaction 
vessel (IKALR-A 1000 laboratory reactor, IKA-Werke, Jahnke & Kunkel GmbH, 
Staufen, Germany) with temperature-control device, stirrer tool and 
toothed rim disperser (Ultraturrax, IKA-Werke, Jahnke & Kunkel GmbH, 
Staufen, Germany) and heated to 80.degree. C. while stirring under a 
vacuum of &lt;1 mbar. While maintaining the vacuum and the stirring, the 
ethanolic emulsifier lipid phase was slowly injected through a cannula 
directly into the aqueous phase with simultaneous vigorous homogenization 
using the Ultraturrax. The mixture was subsequently cooled, with 
continuous stirring and maintenance of the vacuum, to room temperature 
while vigorous homogenization was carried out using the toothed rim 
dispersing rod for about 1 min in several periods. The cooled emulsion was 
transferred into a presterilized bottle and stored in a refrigerator until 
processed further. 
To incorporate the active substance, 163.9 mg of PGE.sub.1 
-.alpha.-cyclodextrin dissolved in 5 ml of water for injections were 
introduced into a presterilized 500 ml graduated flask and made up to 500 
ml with the prepared emulsion, and a sample was taken for particle 
measurements. The solution containing active substance was transferred 
under aseptic conditions and using a Dispensette with a capacity of 2 
ml/single dose to a height of about 1 cm into presterilized vials. The 
charged vials were then provided with stoppers, placed in the lyophilizer 
and frozen at -45.degree. C. for 5 hours. The subsequent lyophilization 
process was carried out as shown in the following table. 
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Initial Final 
Time temperature temperature 
Pressure 
(hours) (.degree.C.) (.degree.C.) 
(.mu.bar) 
______________________________________ 
30 -40 -40 100 
10 -40 25 100 
15 25 25 1 
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Subsequently the vacuum was removed with simultaneous introduction of 
nitrogen, the vials were closed by hydraulic lowering of the stoppers and 
were removed from the lyophilizer under aseptic conditions after it had 
been opened. 
They contained cakes which appeared dry and homogeneous and which 
spontaneously disintegrated on addition of water and formed an emulsion. 
Example 2 
1.5 g of citric acid monohydrate, 2.34 g of trisodium citrate dihydrate and 
60 g of lactose were dissolved by heating in 476 g of water for 
injections. In addition, 12.0 g of glycerol polyethylene glycol 
ricinoleate and 18.0 g of diacetylated monoglycerides with a hydroxyl 
number of 4 were dissolved in 30 g of absolute ethanol with gentle heating 
and under a nitrogen atmosphere. Subsequently, in accordance with the 
preparation process described in Example 1, the emulsion was prepared, the 
active substance was incorporated, samples were taken for particle 
measurement, and lyophilization was carried out. 
Likewise as in Example 1, the product cakes which have been formed appeared 
dry and homogeneous and spontaneously disintegrated to an emulsion on 
addition of water. 
Particle size distribution 
To examine the effect of lyophilization and redispersion on the particle 
size, the volume distributions of the emulsion particles before 
lyophilization and after freeze-drying and redispersion with water had 
taken placed were determined by laser light scattering (Malvern Master 
Sizer, Series 3.01, Malvern Instruments Limited, Spring Lane South, 
Malvern, Worcestershire, WR14 1AQ, UK). Comparison of the volume 
distributions reveals even the slightest changes in the particularly 
critical larger particles because the latter by their nature contribute a 
larger portion of the total volume. The following table shows the results 
of measurement of the particle size determination of emulsions prepared 
according to the invention as in the examples. They contain the maximum 
particle sizes which characterize the volume distributions and which, 
together with the smaller particles lying below them in each case, include 
10%, 50%, 90% or 99% of the total volume. 
______________________________________ 
Maximum size of the particles 
which, together with the smaller 
particles in each case, comprise 
the following volume proportions 
10% 50% 90% 99% 
______________________________________ 
Example 1 
before lyophilization 
0.20 .mu.m 
0.39 .mu.m 
0.98 .mu.m 
5.07 .mu.m 
after lyophilization 
0.19 .mu.m 
0.35 .mu.m 
1.02 .mu.m 
3.32 .mu.m 
and redispersion with 
water 
Example 2 
before lyophilization 
0.19 .mu.m 
0.38 .mu.m 
1.09 .mu.m 
4.56 .mu.m 
after lyophilization 
0.20 .mu.m 
0.40 .mu.m 
1.17 .mu.m 
4.56 .mu.m 
and redispersion with 
water 
______________________________________ 
It is clear that the particle size distributions of the emulsions 
reconstituted by addition of water agree with the particle size 
distributions of the initial emulsions.