Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Cyclosporin preparations
The present invention relates to cyclosporin preparations in
which the cyclosporin is present colloidally in the form of
solid, X-ray-amorphous particles.
Cyclosporins, a series of nonpolar, cyclic oligopeptides, are
distinguished by their immunosuppressive action. Among them,
cyclosporin A, obtained by fermentation and consisting of 11
amino acids, has especially gained therapeutic importance.
Although cyclosporin formulations have been developed both for
oral and for intravenous administration, the oral administration
of cyclosporin is preferred, as it guarantees better patient
compliance.
However, the quite large cyclosporin A, with a molecular weight
of 1202 g/mol, has a high lipophilicity, which is simultaneously
manifested by a very low water solubility (< 0.004% m/V). Owing
to a certain solubility in oils such as olive oil as well as in
ethanol, it has been possible to develop emulsion concentrates
which lead, on peroral administration, to a bioavailability, even
if relatively variable, of about 30% (cf. ~.H. Muller et al. in
"Pharmazeutische Technologies Moderne Arzneiformen"
[Pharmaceutical Technology: Modern Pharmaceutical Forms],
Wissenschaftliche Verlagsgesellschaft, Stuttgart, 1997,
pp. 118-125).
Peroral forms which can be obtained at present on the market are
accordingly either emulsion concentrates for administration as
solutions or microemulsions filled into capsules. In both cases,
solvents such as ethanol and/or oil are employed for the
solubilization of the cyclosporin.
In these cases, the bioavailability, however, can be subject to
great variations in the range from 10 to 60%. These variations
are connected with the pharmaceutical form. and the state of the
preparation in the gastrointestinal tract. Furthermore, the
natural fat digestion has a significant influence on the
absorption of the perorally administered cyclosporin.
WO 97/07787 also describes cyclosporin formulations which in
addition to the active compound contain an alkanolic solvent such
as ethanol or propylene glycol and also a nonionic
polyoxyalkylene derivative as a surface-active substance.
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A disadvantage of such forms, however, is on the one hand that
they contain solvents, especially ethanol, and on the other hand
that the cyclosporin tends to recrystallize at low temperatures,
which is problematical with respect to the storage stability. As
a matter of fact, such precipitates are largely unabsorbed, so
that uniform bioavailability is not guaranteed under certain
circumstances.
EP-A 425 892 discloses a process for improving the
bioavailability of pharmaceutical active compounds having peptide
bonds, in which a solution of the active compound is rapidly
mixed with an aqueous colloid in a water-miscible organic solvent
such that the active compound precipitates in colloidally
disperse form.
WO 93/10767 describes peroral administration forms for peptide
drugs in which the drug is incorporated into a gelatin matrix in
such a way that the colloidal particles formed are present in
uncharged form. A disadvantage of such forms, however, is their
tendency to flocculate.
It is an object of the present invention to find administration
forms of cyclosporin suitable for oral administration, which are
free of solvents and are comparable with the microemulsions with
respect to their bioavailability.
Accordingly, we have found that this object is achieved by solid
cyclosporin preparations in which the cyclosporin is present in
the form of solid, X-ray-amorphous particles distributed in
colloidally disperse form in a matrix of a polymeric encasing
material.
All cyclosporins can be processed according to the invention, but
cyclosporin A is preferred. Cyclosporin A has a melting point of
148-151°C and is employed as a colorless crystalline substance.
In the preparations according to the invention, the cyclosporin
is embedded in particulate form in an encasing matrix consisting
of one or more polymeric stabilizers.
Polymeric stabilizers which are suitable according to the
invention are swellable protective colloids such as, for example,
bovine, porcine or fish gelatin, starch, dextrin, pectin, gum
arabic, lignosulfonates, chitosan, polystyrene sulfonates,
alginates, casein, caseinate, methylcellulose,
carboxymethylcellulose, hydroxypropylcellulose, milk powder,
dextran, whole milk or skimmed milk or mixtures of these
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protective colloids. Furthermore, homo- and copolymers based on
the following monomers are suitable: ethylene oxide, propylene
oxide, acrylic acid, malefic anhydride, lactic acid,
N-vinylpyrrolidone, vinyl acetate, a- and ~-aspartic acid.
Particularly preferably, one of the gelatin types mentioned is
employed, in particular gelatin subjected to acidic or basic
degradation and having bloom numbers in the range from 0 to 250,
very particularly preferably gelatin A 100, A 200, B 100 and
B 200 and also low-molecular weight, enzymatically degraded
gelatin types having the bloom number 0 and molecular weights of
3000 to 30,000 D such as, for example, Collagel A and Gelitasol P
(Stoess, Eberbach) or chemically modified gelatin, such as, for
example, Gelafundin (B. Braun, Melsungen), and mixtures of these
gelatin types.
The preparations furthermore contain low-molecular weight
surface-active compounds. Those suitable are especially
amphiphilic compounds or mixtures of such compounds. Basically,
all surfactants having an HLB of 5 to 20 are suitable. Suitable
appropriate surface-active substances are, for example: esters of
long-chain fatty acids containing ascorbic acid, mono- and -
diglycerides of fatty acids and their ethoxylation products,
esters of monofatty acid glycerides with acetic acid, citric
acid, lactic acid or diacetyltartaric acid, polyglycerol fatty
acid esters such as, for example, the monostearate of
triglycerol, sorbitan fatty acid esters, propylene glycol fatty
acid esters, 2-(2'-stearoyllactyl)lactic acid salts and lecithin.
Ascorbyl palmitate is preferably employed.
The amounts of the various components are chosen according to the
invention such that the preparations contain 0.1 to 70% by
weight, preferably 1 to 40% by weight, of cyclosporin, 1 to 80%
by weight, preferably 10 to 60% by weight, of one or more
polymeric stabilizers and 0 to 50% by weight, preferably 0.5 to
20% by weight, of one or more low-molecular weight stabilizers.
The percentages by weight relate to a dry powder.
In addition, the preparations can also contain antioxidants
and/or preservatives for the protection of the cyclosporin.
Suitable antioxidants or preservatives are, for example,
a-tocopherol, t-butylhydroxytoluene, t-butylhydroxyanisole,
lecithin, ethoxyquin, methylparaben, propylparaben, sorbic acid,
sodium benzoate or ascorbyl palmitate. The antioxidants or
preservatives can be present in amounts of from 0 to 10% by
weight, based on the total amount of the preparation. It can also
be recommended to add to the preparations lipoprotein blockers to
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improve the absorption of the cyclosporin, for example
polyoxyethylene cholesterol ethers.
Furthermore, the preparations can also contain plasticizers for
increasing the stability of the final product. Suitable
plasticizers are, for example, sugars and sugar alcohols such as
sucrose, glucose, lactose, invert sugar, sorbitol, mannitol,
xylitol or glycerol. Lactose is preferably employed as a
plasticizes. The plasticizers can be present in amounts of from 0
IO to 50% by weight.
Furthermore, the preparations can also contain edible oils and/or
fats to increase the colloidal stability of the preparation
according to the invention. Suitable oils and fats are, for
example, of vegetable origin such as sunflower oil, groundnut
oil, corn oil, linseed oil, olive oil, poppyseed oil, rapeseed
oil, castor oil, coconut oil, peanut oil, soybean oil, palm oil
or cottonseed oil. Other suitable oils or fats are fish oils,
neatsfoot oil, lard, beef tallow and butterfat. The oils and fats
can be present in the preparation according to the invention in
the range from 0-50% by weight, based on cyclosporin. In the -
formulation, the oil or fat is embodied in the colloidal
particles containing solid cyclosporin. It is crucial in the
choice of the nature and amount of the oil or fat that the
colloidal particles containing cyclosporin are furthermore
present as solid particles at administration temperatures
(T < 40~C).
Further pharmaceutical excipients such as binders, disintegrants,
flavorings, vitamins, colorants, wetting agents, additives
affecting the pH (cf. H. Sucker et al., Pharmazeutische
Technologie, Thieme-Verlag, Stuttgart 1978) can also be
incorporated via the organic solvent or the aqueous phase.
For carrying out the process according to the invention, a
solution of the cyclosporin in a suitable solvent is first
prepared, solution in this connection meaning a true molecularly
disperse solution or a melt emulsion. Suitable solvents are
organic, water-miscible solvents which are volatile and thermally
stable and only contain carbon, hydrogen and oxygen. Expediently,
they are miscible to at least 10% by weight with water and have a
boiling point of below 200°C and/or have less than 10 carbon
atoms. Appropriate alcohols, esters, ketones and acetals are
preferred. In particular, ethanol, n-propanol, isopropan-1-ol,
1,2-butanediol 1-methyl ether, 1,2-propanediol 1-n-propyl ether
or acetone is used.
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According to one embodiment of the process, a molecularly
disperse solution of the cyclosporin is dissolved in the chosen
solvent at temperatures in the range from preferably 20 to 150°C,
within a period of time of less than 120 seconds, it optionally
5 being possible to work at an overpressure of up to 100 bar,
preferably 30 bar.
According to a further preferred embodiment, the cyclosporin
solution is prepared such that the mixture of cyclosporin and
solvent is heated above the melting point of the cyclosporin to
150 to 240°C within a period of time of less than 10 seconds, it
optionally being possible to work at an overpressure of up to
100 bar, preferably 30 bar.
The concentration of the cyclosporin solution prepared in this
way is in general 10 to 500 g of cyclosporin per 1 kg of solvent.
In a preferred embodiment of the process, the low-molecular
weight stabilizer is added directly to the cyclosporin solution.
In a process step connected thereto, the cyclosporin solution is
mixed with an aqueous solution of the polymeric encasing -
material. The concentration of the solution of the polymeric
encasing material is 0.1 to 200 g/1, preferably 1 to 100 g/1.
In order to obtain particle sizes which are as small as possible
in the mixing process, a high mechanical energy input is
recommended when mixing the cyclosporin solution with the
solution of the encasing material. Such an energy input can be
effected, for example, by vigorous stirring or shaking in a
suitable apparatus, or by injecting the two components into a
mixing chamber using a powerful jet, so that vigorous mixing
occurs.
The mixing process can be carried out batchwise or, preferably,
continuously. As a result of the mixing process, precipitation of
the cyclosporin occurs in the form of solid, X-ray-amorphous
particles. The suspension or the colloid thus obtained can then
be converted into a dry powder in a manner known per se, for
example by spray-drying, freeze-drying or drying in a fluidized
bed.
A procedure is used in the production of the preparations
according to the invention in which the pH of the solution of the
encasing material, in particular of gelatin, and a solution of
the cyclosporin is adjusted such that no neutral charge occurs in
the cyclosporin particles formed, i.e. the pH of the gelatin
solution does not have to be adjusted to such a value that a
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charge-neutral state is established on formation of the
particles.
The mean particle diameter of the solid cyclosporin particles in
the matrix of the polymeric encasing material is 20 to 1000 nm,
preferably 100 to 600 nm. Surprisingly, the spherical cyclosporin
particles are completely X-ray-amorphous. X-ray-amorphous in this
connection means the absence of crystal interferences in X-ray
powder diagrams (cf. H.P. Klug, L.E. Alexander, "X-Ray
Diffraction procedures for Polycristalline and Amorphous
Materials, John Wiley, New York, 1959).
The dry powders obtained according to the invention can be
employed in all customary oral pharmaceutical forms. It is thus
possible, for example, to fill the powders into hard or soft
gelatin capsules or to compress them to give tablets using the
excipients customary therefor.
Furthermore, the powders, on account of their good
redispersibility in water, are suitable for use as beverage
forms, for example as beverage granules, in effervescent tablets, -
juices, syrup forms or in sachets and for parenteral
administrations. Even after redispersion, uniform finely divided
suspensions (hydrosols) or colloids are obtained.
The preparations according to the invention not only offer the
advantage that they are completely free of solvents such as
ethanol, but also have a good bioavailability, which is perfectly
comparable to that of the microemulsions. kith respect to the
prior art, such a good bioavailability was not to be expected for
a preparation which contains cyclosporin in solid form.
The results of a canine study confirm the good bioavailability of
the preparation in comparison to a commercially available
product.
Production Example 1
Production of a cyclosporin dry powder having an active compound
content in the range of 10~ by weight
a) Production of the micronizate
3 g of cyclosporin A were stirred into a solution of 0.6 g of
ascorbyl palmitate in 36 g of isopropanol at 25°C, a clear
solution resulting.
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For the precipitation of the cyclosporin A in colloidally
disperse form, this molecularly disperse solution was fed
into a mixing chamber at 25°C. Mixing with 537 g of an
aqueous solution of 14.4 g of gelatin B 100 bloom and 12.6 g
of lactose in completely deionized water adjusted to pH 9.2
by means of 1 N NaOH was carried out therein. The entire
process was carried out with pressure restriction to 30 bar.
After mixing, a colloidally disperse cyclosporin A dispersion
having a cloudy white color shade was obtained.
By means of quasi-elastic light scattering, the mean particle
size was determined to be 256 nm with a variance of 31%. By
means of Fraunhofer diffraction, the mean value of the volume
distribution was determined to be D(4,3) = 0.62 Eun with a
I5 fine component of the distribution of 99.2 < 1.22 Eun.
b) Drying of the dispersion a) to give a nanoparticulate dry
powder
Spray-drying of the product la) afforded a nanoparticulate
dry powder. The active compound content in the powder was
determined to be 9.95% by weight by chromatography. The dry
powder dissolves in drinking water with formation of a cloudy
white dispersion (hydrosol).
c) X-ray wide-angle scattering
Figure 1 illustrates the scattering curves of active compound
(above) and dry powder according to lb) (below). The
cyclosporin starting material is crystalline, as the X-ray
diagram, which is distinguished by a number of sharp
interferences, confirms. In contrast to this, the scattering
curve of the dry powder exhibits only diffuse, broad
interference maxima, such as are typical of an amorphous
material. The active compound is accordingly present in X-ray
amorphous form in the dry powder produced according to lb).
This also applies to the otherwise crystalline excipients
lactose and ascorbyl palmitate.
d) Cryo-replica transmission electron microscopy (Cryo-TEM)
Figure 5 shows a Cryo-TEM photograph of the dry powder
according to lb) redispersed in tap water. The spherical
nanoparticulate cyclosporin particles having a mean diameter
of D = 500 nm can be readily detected. This illustration
confirms that after the redispersion of the dry powder a
colloidally disperse cyclosporin solution again forms in
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which the individual colloid particles are present in
nonaggregated form.
Production Example 2
Production of a cyclosporin dry powder having an active compound
content in the range of 15% by weight
a) Production of the micronizate
3 g of cyclosporin A were stirred into a solution of 0.6 g of
ascorbyl palmitate in 18 g of isopropanol and 18 g of
completely deionized water at 25°C. This solution was
converted into the molecularly dissolved state by heating in
a heat exchanger. The residence time of the cyclosporin
solution in the heat exchanger was 90 min, a temperature of
at most 135°C not being exceeded.
For the precipitation of the cyclosporin A in colloidally
disperse form, this molecularly disperse solution was fed
into a mixing chamber at 135°C. Mixing with 393.9 g of an
aqueous solution of 9.2 g of gelatin A 100 bloom and 6.1 g of
lactose in completely deionized water adjusted to pH 9.2 by
means of 1 N NaOH was carried out therein. The process was
carried out with pressure restriction to 30 bar in order to
prevent evaporation of the water. After mixing, a colloidally
disperse cyclosporin A dispersion having a cloudy white color
shade was obtained.
By means of quasi-elastic light scattering, the mean particle
size was determined to be 285 nm with a variance of 48%. By
means of Fraunhofer diffraction, the mean value of the volume
distribution was determined to be D(4,3) = 0.62 ~.m with a
fine component of the distribution of 99.8% < 1.22 Vim.
b) Drying of the dispersion 2a) to give a dry powder
Spray-drying of the dispersion led to a nanoparticulate dry
powder. The active compound content in the dry powder was
determined to be 15.9% by weight by chromatography. The dry
powder dissolved in drinking water with formation of a cloudy
white dispersion.
By means of quasi-elastic light scattering, the mean particle
size immediately after redispersion was determined to be
376 nm with a variance of 38%. By means of Fraunhofer
diffraction, the mean value of the volume distribution was
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determined to be D(4,3) = 0.77 Eun with a fine component of
the distribution of 84.7% < 1.22 Eun.
Freeze-drying of the product led to a nanoparticulate dry
powder. The active compound content in the powder was
determined to be 16.1% by weight of cyclosporin by
chromatography. The dry powder dissolved in drinking water to
give a cloudy white hydrosol.
By means of quasi-elastic light scattering, the mean particle
size immediately after redispersion was determined to be
388 nm with a variance of 32%. By means of Fraunhofer
diffraction, the mean value of the volume distribution was
determined to be D(4,3) = 0.79 N.m with a fine component of
the distribution of 82.4% < 1.22 N,m.
c) Microelectrophoresis
Figure 3 illustrates the pH-dependent mobility curves of an
aqueous dispersion of active compound, gelatin and dry powder
according to 2b) (spray-drying). The mobility curve of the
crystalline cyclosporin A employed as starting material
differs markedly in the height of the mobilities and the
position of the isoelectric point from that of the micronized
dry powder according to 2b). The isoelectric point of the
redispersed dry powder coincides with the gelatin used. This
shows that the nanoparticulate cyclosporin particles are
embedded by a gelatin casing.
Production Example 3
Analogously to Example 2a), a colloidally disperse cyclosporin A
dispersion was produced from 4.5 g of cyclosporin A, 0.9 g of
ascorbyl palmitate, 9.6 g of gelatin A 100 bloom and 7.2 g of
lactose.
By means of quasi-elastic light scattering, the mean particle
size was determined to be 280 nm with a variance of 21%. By means
of Fraunhofer diffraction, the mean value of the volume
distribution was determined to be D(3,4) -- 0.62 N.m with a fine
component of the distribution of 99.2% < 1.2 Vim.
b) Drying of the dispersion 3a) to give a nanoparticulate dry
powder
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By means of spray-drying, a nanoparticulate dry powder having
a cyclosporin A content (determined by chromatography) of
19.9% by weight was obtained. The dry powder dissolved in
drinking water with formation of a cloudy white dispersion
5 (hydrosol).
By means of quasi-elastic light scattering, the mean particle
size immediately after redispersion was determined to be 377
nm with a variance of 45%. By means of Fraunhofer
10 diffraction, the mean value of the volume distribution was
determined to be D(4,3) = 0.62 N.m with a fine component of
the distribution of 83.3% < 1.2 N.m.
Figure 3: X-ray wide-angle scattering of dry powders 2b) and 3b)
Figure 3 presents the scattering curves of the dry powders
according to 2b) (upper curve) and 3b) (lower curve) obtained in
each case by spray-drying. In contrast to the scattering curve of
the crystalline cyclosporin starting material, which contains
sharp interferences, contained in Figure 1, the scattering curves
of the dry powders exhibit only diffuse, broad interference
maxima, such as are typical of amorphous materials.
Production Example 4
Analogously to Production Example 3, a preparation was produced
in which the encasing matrix material was fish gelatin having
molecular weight components of 103 to 10~ D.
Pharmacokinetic properties of the dry powders
Blood level kinetics in the dog: general method
Cyclosporin was administered either orally as a solid form or by
means of stomach tube in liquid form in the appropriate
preparation to beagle dogs having a weight in the range from 8 to
12 kg. Liquid forms were given in 50 ml of water and washed down
with a further 50 ml of water. Solid forms were administered
without water. The animals were not fed for 16 h before substance
administration; feeding continued 4 h after substance
administration. Blood was taken in heparinized vessels from the
jugular vein or the antebrachial cephalic vein of the dogs before
substance administration and at intervals up to 32 h after
substance administration. The blood was deep-frozen and stored at
-20°C until the analytical work-up. The blood levels were
determined by a validated, internally standardized GC-MS method.
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Form 1 (for comparison): Sandimmun~ Optoral, capsule, 100 mg of
active compound
Form 2: dry powder according to Production Example 1, active
compound dose 100 mg; administration as a hydrosol
Form 3: dry powder according to Production Example 3, active
compound dose 100 mg; administration as a hydrosol
Figure 4 indicates the medians of the corresponding blood levels.
It can be clearly discerned that with the forms F2 and F3
according to the invention at the start a more rapid increase in
the blood level values is achieved than with the comparison form
F1.
Areas under the blood level curves and relative bioavailability
Table
Form 1 (Sandimmun Optoral)
Para- Animal)Animal AnimalAnimal Animal AnimalMean Median
meter 1 2 3 4 5 6
tmax 2 2 2 1 2 2 2
Cmax 1030.01062.0 865.0 387.0 1799.0 869.0 1002.0 949.5
AUC 5781.86487.8 6497.02403.6 7892.2 7047.86018.4 6492.4
AUC/ 734.7 855.9 759.9 288.5 947.4 846.1 738.8 803.0
dose
BA$ 99 116 103 39 128 114 100 100
AUC: Area under the curve
BA: Bioavailability
tmax: [h]
Cmax: [ng/ml]
45
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Form 2
Para- Animal AnimalAnimal Animal AnimalAnimal Mean Median
meter 1 2 3 4 5 6
tmax 0.5 1 2 1 0.5 1 1
Cmax 3529.0 718.0 676.0 459.0 853.0 580.0 1135.8 697.0
AUC 123x9.74410.13889.5 2727.2 5049.74251.3 5452.9 4330.7
AUC/ 1574.3 581.8 454.9 327.4 606.2 510.4 675.8 546.1
dose
BA% 213 79 62 44 82 69 91 74
Form 3
para- Animal Animal AnimalAnimal AnimalAnimal Mean Median
meter 1 2 3 4 5 6
tmax 0.5 2 0.5 2 2 0.5 1.25
Cmax 4208.0 567.0 829.0 439.0 956.0 4086.0 1847.5 892.5
AUC 17049.34565.2 3889.82849.6 5222.51s7o3.o8379.9 4893.9
AUC/ 2166.4 602.3 454.9 342.1 627.0 2005.2 1003.0 614.6
dose
BA$ 293 81 62 46 85 271 140 83
production Example 4
Production of a cyclosporin dry powder having an active compound
content in the region of 15% by weight
a~ production of the micronizate
3 g of cyclosporin A were stirred into a solution of 0.6 g of
ascorbyl palmitate and 0.3 g of soybean oil in 18 g of
isopropanol and 18 g of completely deionized water at 25°C
such that a cloudy, coarsely disperse solution resulted. This
solution was converted into the molecularly dissolved state
by heating in a heat exchanger. The residence time of the
cyclosporin solution in the heat exchanger was about 90 min,
a temperature of at most 135°C not being exceeded.
For the precipitation of the cyclosporin A in colloidally
disperse form, this molecularly disperse solution was fed
into a mixing chamber at 135°C. Mixing with 412.3 g of an
aqueous solution of 8.9 g of gelatin A 100 bloom and 6.5 g of
lactose in completely deionized water, adjusted to pH 9.2 by
means of 1 N NaOH, was carried out therein. The entire
process was carried out with pressure restriction to 30 bar
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in order to prevent evaporation of the solvent. After mixing,
a colloidally disperse cyclosporin A dispersion having a
cloudy white color shade was obtained.
By means of quasi-elastic light scattering, the mean particle
size was determined to be 273 nm with a distribution breadth
of t 37%. By means of Fraunhofer diffraction, the mean value
of the volume distribution was determined to be D(4,3) _
0.62 N.m with a fine component of the distribution of 99.8% <
1.22 Nm.
b) Drying of the dispersion from a) to give a nanoparticulate
dry powder
Spray-drying of the product from Production Example [lacuna]
led to a nanoparticulate dry powder. The active compound
content in the powder was determined to be 15.21% by weight
of cyclosporin A (theoretical value: 15.54% by weight) by
chromatography. The dry powder dissolves in drinking water
with formation of a cloudy white dispersion (hydrosol).
By means of quasi-elastic light scattering, the mean particle
size immediately after redispersion was determined to be
352 nm with a distribution breadth of ~ 42%. By means of
Fraunhofer diffraction, the mean value of the volume
distribution was determined to be D(4,3) = 0.73 ~.m with a
fine component of the distribution of 86.3% < 1.22 Vim.
35
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