Cyclosporin soft capsule composition

The present invention relates to a microemulsion concentrate containing cyclosporin as an active ingredient, dimethylisosorbide as a cosurfactant, oil and a surfactant which is suitable for the formulation of a soft capsule for oral administration, said cyclosporin, dimethyl isosorbide, oil and surfactant being present in the ratio of 1:1-5:1-5:2-10, and preferably in the ratio of 1:2-4:2-5:2-4:7, by weight. Since dimethylisosorbide has substantially no membrane permeation property, the soft capsule preparation according to the present invention is outstandingly stable in comparison with the soft capsules containing ethanol, propylene glycol, transcutol, glycofurol, etc., as a cosurfactant in the prior art, and further provides an advantage in that the pharmaceutical effect, appearance and composition content of the soft capsule according to the present invention are not changed.

BACKGROUND OF THE INVENTION 
1. Field of the invention 
The present invention relates to a soft capsule composition containing a 
stable microemulsion concentrate suitable for the preparation of 
cyclosporin-containing soft capsules. More particularly, the present 
invention relates to a microemulsion concentrate containing cyclosporin as 
an active ingredient, dimethylisosorbide as a co-surfactant, an oil 
component and a surfactant, which is suitable for formulation into soft 
capsules and to a soft capsule composition containing said microemulsion 
concentrate. 
2. Background art 
Cyclosporin is a specific macromolecular (molecular weight 1202.64) cyclic 
peptide compound consisting of 11 amino acids, which has useful 
pharmacological activities, particularly immunosuppressive activity and 
anti-inflammatory activity. Therefore, cyclosporin has been used for 
suppression of immunological responses native to the living body, which 
are caused by tissue and organ transplantation, for example, 
transplantation of the heart, lung, liver, kidney, pancreas, bone marrow, 
skin and cornea, and particularly the transplantation of foreign tissues 
and organs. In addition, cyclosporin is useful for the suppression of 
autoimmune diseases and inflammatory diseases such as arthritis, etc. 
Cyclosporin is highly lipophilic and hydrophobic with a solubility in water 
at 25.degree. C. being 16 to 23 mg of cyclosporin per liter of water. The 
bioavailability of cyclosporin is also extremely low due to its low 
water-solubility. On the other hand, cyclosporin is well dissolved in an 
organic solvent such as methanol, ethanol, acetone, ether, chloroform and 
the like, due to its high lipophilic property. 
As with other conventional drugs which are sparingly soluble in water, 
cyclosporin is very difficult to formulate into a preparation for oral 
administration due to its low water solubility and bioavailability. 
Further, since the bioavailability of cyclosporin may be greately 
influenced by the condition of each individual patient, it is very 
difficult to provide an effective therapeutic effect. 
Moreover, it is very important to provide a uniform dosage amount and 
appropriate bioavailability since counterbalancing the efficacy of 
cyclosporin are its considerable toxic side effects such as 
nephrotoxicity, hepatotoxicity, among others. Accordingly, numerous 
studies have been extensively and widely conducted to find a preparation 
suitable for the effective oral administration of cyclosporin. 
The prior art preparations suitable for oral administration of sparingly 
water-soluble cyclosporin are usually formulated in the form of a 
microemulsion. In preparing the liquid microemulsion formulation, 
cyclosporin should be combined with a surfactant, an oil and a 
cosurfactant. One method using this combination is taught in U.S. Pat. No. 
4,388,307 which issued on Jun. 14, 1983. This patent discloses a liquid 
formulation of cyclosporin using ethanol as a cosurfactant. Thus, 
cyclosporin is combined with a carrier consisting of ethanol as a 
cosurfactant, a vegetable oil and a transesterification product of a 
natural vegetable oil triglyceride and a polyalkylene glycol as a 
surfactant to form the liquid formulation. However, the resulting liquid 
formulation is administered as an aqueous dilution making it very 
difficult to properly formulate the preparation to provide a uniform 
dosage for oral administration. 
In order to alleviate the requirement of diluting the cyclosporin liquid 
composition in water prior to oral administration, a liquid composition in 
the form of a microemulsion concentrate has been formulated into a soft 
capsule preparation, which is presently commercially available as 
SANDIMMUN.RTM. (trademark). In this preparation the cyclosporin soft 
capsule contains a large amount of ethanol as a cosurfactant due to the 
solubility requirements of cyclosporin. However, since ethanol, which has 
a low boiling point, permeates the gelatin membrane of the capsule to 
volatilize even at normal temperature, the constitutional ratio of the 
contents in soft capsules varies during storage. The reduced ethanol 
content results in a significant difference in the bioavailability of 
cyclosporin and making the problem worse, the cyclosporin crystallizes 
when the soft capsules are stored at low temperature. 
In an effort to prevent the volatilization of ethanol from the soft capsule 
preparations during storage and distribution, the soft capsule 
preparations are wrapped in a packing material, such as an aluminum film 
foam package. However, such specific packaging does not completely 
maintain the uniform composition of the wrapped capsule. It has been 
demonstrated through experiments that although the cyclosporin soft 
capsule is wrapped up in aluminum film foam package, the ethanol content 
is lowered to 7.9% from the initial level of 10.8% after a period of one 
week. This results in a great difference in bioavailability and may 
contribute to the increase of its price. 
To solve the above-mentioned disadvantages which accompany the use of 
ethanol as a cosurfactant, a method using a non-ethanol component as a 
cosurfactant has been proposed. Korean Laid-open Patent Publication No. 
90-4348 (Apr. 12, 1990) discloses a pharmaceutical composition in the form 
of a microemulsion concentrate containing a non-ethanol component. The 
non-ethanol components include pharmaceutically acceptable C.sub.1-5 alkyl 
or tetrahydrofurfuryl di- or partial-ether of low molecular mono- or 
poly-oxy-alkanediol, for example, diethyleneglycol monoethyl ether [e.g. 
Transcutol] or tetrahydrofurfuryl alcohol polyethylene glycol ether [e.g. 
Glycofurol], or 1,2-propyleneglycol as a cosurfactant. However, the above 
non-ethanol cosurfactants are glycols which contain the -OH group in their 
structures. It has now been identified that the OH group-containing glycol 
creates problems in the formulation of soft capsules because its strong 
absorption property is sufficient to absorb the moisture from the 
atmosphere and also because it is highly permeable to the gelatin film of 
the soft capsule. 
Thus, the present inventors have studied numerous additives, including 
various solvents, in an effort to find a cosurfactant capable of providing 
a microemulsion concentrate suitable for the formulation of cyclosporin 
into a soft capsule preparation. As a result, a certain pharmaceutically 
acceptable solvent, dimethyli- sosorbide [Trade name: ARLASOVE.RTM. DMI, 
available from ICI Speciality Chemicals] has been found as a suitable 
solvent for this purpose to complete the present invention. 
Therefore, it is an object of the present invention to provide a 
microemulsion concentrate containing dimethylisosorbide as a cosurfactant, 
which is suitable for formulation into soft capsules for oral 
administration. 
It is a further object of the present invention to provide a microemulsion 
concentrate suitable for formulation into soft capsules, which contains 
cyclosporin as an active ingredient, dimethylisosorbide as a cosurfactant, 
an oil component and a surfactant. 
Further, it is another object of the present invention to provide a soft 
gelatin capsule composition according to the present invention which is 
highly storage stable such that there is little variation of the 
composition over time. 
The foregoing has outlined some of the more pertinent objects of the 
present invention. These objects should be construed to be merely 
illustrative of some of the more pertinent features and applications of 
the invention. Many other beneficial results can be obtained by applying 
the disclosed invention in a different manner or modifying the invention 
within the scope of the disclosure. Accordingly, other objects and a more 
thorough understanding of the invention may be had by referring to the 
disclosure of invention and the drawings, in addition to the scope of the 
invention defined by the claims.

DISCLOSURE OF INVENTION 
In one aspect, the present invention relates to a microemulsion concentrate 
comprising cyclosporin as an active ingredient, dimethylisosorbide as a 
cosurfactant, an oil component and a surfactant which is suitable for the 
formulation of soft capsules for oral administration. 
Cyclosporin, the first essential component, is the pharmaceutically active 
ingredient in the microemulsion concentrate according to the present 
invention. Cyclosporin is a cyclic peptide compound having useful 
immuno-suppressive activity and antiinflammatory activity as described 
above and known in the art. Although cyclosporin A, B, C, D, G and the 
like can be used as the cyclosporin component in the present invention, 
cyclosporin A is most preferred since its clinical effectiveness and 
pharmacological properties are well established in the art. 
The dimethylisosorbide cosurfactant, which is the second essential 
component in the microemulsion concentrate according to the present 
invention, is a compound represented by the following structural formula. 
Dimethylisosorbide has been used as a skin permeation stimulator in 
topical pharmaceutical compositions. The chemical name of 
dimethylisosorbide is 1,4:3,6-dianhydro-2,5-dimethyl-D-glucitol. 
##STR1## 
Since dimethylisosorbide has a high boiling point, 234.degree. C., it does 
not volatilize even at high temperature such as the temperature necessary 
for manufacturing soft capsules. In addition, dimethylisosorbide does not 
contain any hydroxy groups, --OH, and therefore, its hygroscopic property 
is very low and it does not permeate the gelatin membrane of the soft 
capsule. Most importantly, cyclosporin dissolves well in 
dimethylisosorbide which contributes to the formulation of a suitable 
microemulsion as defined above. 
Accordingly, the use of dimethylisosorbide as a cosurfactant in the 
microemulsion concentrate of the present invention provides certain 
advantages. That is, when the microemulsion concentrate is formulated into 
a soft capsule, a change in the composition of the concentrate does not 
appear during storage and the contents of the components contained therein 
are substantially uniformly maintained so that the uniformity of the 
composition content can be assured over a greater time period than ethanol 
based compositions. 
In the microemulsion concentrate of the present invention, 
dimethylisosorbide is used in the ratio of 1 to 5 parts by weight and 
preferably 2 to 4 parts by weight, per 1 part by weight of cyclosporin. 
The third component used in the microemulsion concentrate according to the 
present invention is an oil(s). The oil component suitable for use in the 
present invention includes any animal or vegetable oils which are 
pharmaceutically acceptable. Thus, neutral animal oils and vegetable oils, 
for example, castor oil or corn oil, particularly animal oil rich in 
unsaturated fatty acids having an iodine value of 180 to 200, for example, 
refined fish oil and vegetable oil having the hydroxyl value of 150, for 
example, castor oil, are preferably used. Among such oils, the most 
preferred oil is refined fish oil. Refined fish oil is ideally fit for the 
absorption of cyclosporin since it contains EPA (eicosapentaenoic acid), a 
highly unsaturated fatty acid, and DHA (docosahexaenoic acid) in the 
amount of 18%, or more, and 12%, or more, respectively, having a total 
unsaturated fatty acid content of 30%, or more, which allows for the 
absorption of cyclosporin. 
The oil component according to the present invention may include only a 
single oil selected from the above-mentioned oils or a mixture selected 
from the above-mentioned oils. When a mixture of two or more oil 
components is used, it is preferred that at least one animal oil and at 
least one vegetable oil be present in said mixture. 
In addition, if required, in the oil component the appropriate amount of 
saturated fatty acids, for example, caprilic acid/capric triglyceride 
[Trademark: MIGLYOL.RTM. 812; Dynamit Nobel Chemikalien] can be included 
in order to control the absorption of cyclosporin. When such saturated 
fatty acid is added to the oil component, it can modulate the absorption 
pattern of cyclosporin to reduce the occurrence of typical side effects 
involved in cyclosporin preparations. 
The fourth essential component used in the microemulsion concentrate 
according to the present invention is a surfactant. The suitable 
surfactants for use in the present invention include any of 
pharmaceutically acceptable surfactant capable of stably emulsifying the 
lipophilic portion of the composition comprising the 
cyclosporin-containing oil component and the hydrophilic part comprising 
the cosurfactant in water to form a stable microemulsion. The most 
preferred surfactants according to the present invention include 
polyoxyethylene glycolated natural or hydrogenated vegetable oils, 
transesterification reaction products of natural vegetable oil 
triglyceride and polyalkylene polyol, sorbitan fatty acid esters, 
polyoxyethylene-sorbitan fatty acid esters, polyoxyethylene alcohols, 
etc., such as the reaction products of castor oil and ethylene oxide, 
which are commercially available under the trade mark CREMOPHOR.RTM. 
(BASF), the esterification reaction products of natural vegetable oil and 
polyethylene glycol, which are commercially available under the trade mark 
LABRAFIL.RTM. (Etablissement Gattefosse), the sorbitan fatty acid esters 
which are commercially available under the trade mark SPAN.RTM. (Lippo 
Chemicals) and the polyoxyethylene alcohols which are commercially 
available under the trade mark BRIJ.RTM. (BASF). Among these surfactants, 
LABRAFIL.RTM. M 1944 CS (apricot kernel oil PEG-6 ester) having the HLB 
Value of 14.0 to 15.0 is the most preferred. The surfactant may include 
any one of the abovementioned surfactants alone or, preferably, in a 
combination of mentioned surfactants alone or, preferably, in a 
combination of one or more surfactants selected from the above 
surfactants. 
In the microemulsion concentrate according to the present invention, four 
essential components are present preferably in the ratio of 
cyclosporin:cosurfactant:oil component:surfactant=1:1-5:1-5:2-10, and more 
preferably in the ratio of cyclosporin: cosurfactant:oil 
component:surfactant=1:2-4:2-5:2-7 by weight. The most preferable 
microemulsion concentrate according to the present invention consists of 
cyclosporin A, dimethylisosorbide, LABRAFIL.RTM. and refined fish oil in 
the ratio of 1:4:3:2.7 by weight. In addition to this composition, the 
composition illustrated in the following examples can be mentioned as the 
further preferable compositions according to the present invention. 
For oral administration, the microemulsion concentrate having the 
above-mentioned composition, according to the present invention, can be 
formulated into the form of a soft capsule. In formulating the soft 
capsule, the capsule preparation can further contain, if necessary, 
pharmaceutically acceptable adjuvants, excipients and additives which are 
conventionally utilized in the preparation of soft capsules, in addition 
to the above microemulsion concentrate. Such additives include, for 
example, lecithin, viscosity regulator, perfume (e.g. peppermint oil, 
etc.), antioxidant (e.g. tocopherol, etc.), preservative (e.g. parabens, 
etc.), coloring agent, glyerin, sorbitol, gelatine, etc. 
The soft capsule preparation according to the present invention can be 
prepared according to conventional methods for the preparation of soft 
capsules. For example, cyclosporin is first dissolved in 
dimethylisosorbide while gently warming at the temperature of 
approximately 60.degree. C. The oil component and the surfactant are then 
added to the resulting mixture and the whole mixture is uniformly mixed. 
The resulting microemulsion concentrate is then introduced into a machine 
for preparing soft capsules, with or without the above-mentioned 
pharmaceutically acceptable additives conventionally utilized in 
preparation of soft capsules, to prepare the desired suitable cyclosporin 
soft capsule. The soft capsule composition thus prepared according to the 
present invention exhibits a blood level of cyclosporin comparable to that 
of the prior art ethanol-containing soft capsule preparation when they are 
administered by mouth, i.e. orally. Further the soft capsule composition 
according to the present invention is stably maintained without any change 
over a prolonged storage perior. Accordingly, it is readily apparent that 
the present invention provides a significant improvement in the field of 
preparation of the cyclosporin soft capsules. 
The present invention will be more specifically illustrated by the 
following examples. However, it should be understood that the present 
invention is not limited by these examples in any manner. 
EXAMPLE 1 
2.5 g of cyclosporin A was added to 4.5 g of dimethylisosorbide 
(ARLASOLVE.RTM. DMI). The mixture was warmed to about 60.degree. C. with 
stirring to dissore cyclosporin. To the resulting solution were added 7.5 
g of LABRAFILRM.RTM. M 1944CS (apricot kernel oil PEG-6 ester) and 11.5 g 
of refined fish oil and the mixture was sufficiently stirred until the 
homogeneous microemulsion concentrate was formed. The microemulsion 
concentrate thus obtained was introduced into a machine for preparation of 
soft capsules, which is adjusted so that 0.26g of the microemulsion 
concentrate is injected into one soft capsule, to prepare the soft gelatin 
capsules containing the cyclosporin microemulsion concentrate within 
gelatin sheets. 
EXAMPLE 2 
The soft gelatin capsules having the following compositions were prepared 
according to the same procedure as Example 1 except that the amount of 
dimethylisosorbide as a cosurfactant is varied. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
2-A. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
25 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Total 240 mg 
2-B. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
65 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Total 280 mg 
______________________________________ 
EXAMPLE 3 
The soft gelatin capsules having the following compositions were prepared 
according to the same procedure as Example 1 except that as the surfactant 
the mixture of LABRAFIL.RTM. and another surfactant is used instead of 
LABRAFIL.RTM. alone. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
3-A. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
CREMOPHOR.sup.R EL 5 
Refined fish oil 115 
Total 265 mg 
3-B. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
SPAN.sup.R 20 5 
Refined fish oil 115 
Total 265 mg 
3-C. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
BRIJ.sup.R 35 5 
Refined fish oil 115 
Total 265 mg 
______________________________________ 
EXAMPLE 4 
The soft gelatin capsules having the following compositions were prepared 
according to the same procedure as Example 1 except that the oil component 
and its content are changed. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
4-A. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 55 
Total 200 mg 
4-B. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Total 320 mg 
4-C. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Caprilic acid/Capric triglyceride 
40 
Total 300 mg 
4-D. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
Corn oil 115 
Total 260 mg 
4-E. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 100 
Castor oil 115 
Total 285 mg 
4-F. Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 80 
Refined fish oil 69 
Castor oil 46 
Total 265 mg 
______________________________________ 
EXAMPLE 5 
The soft gelatin capsule having the following composition was prepared 
according to the same procedure as Example 1, except that peppermint oil 
(perfume) and tocopherol (antioxidant) are additionally added to the 
microemulsion concentrate before it is formulated into the soft capsule. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
45 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Peppermint oil 20 
Tocopherol 5 
Total 285 mg 
______________________________________ 
EXAMPLE 6 
The soft gelatin capsule having the following composition was prepared 
according to the same procedure as Example 1. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
50 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 115 
Castor oil 3.5 
d-.alpha.-Tocopherol 1.5 
Total 270 mg 
______________________________________ 
EXAMPLE 7 
The soft gelatin capsule having the following composition was prepared 
according to the same procedure as Example 1. 
______________________________________ 
Content 
Component (mg/Cap.) 
______________________________________ 
Cyclosporin 25 
Dimethylisosorbide (ARLASOLVE.sup.R DMI) 
100 
LABRAFIL.sup.R M 1944 CS 75 
Refined fish oil 68.5 
d-.alpha.-Tocopherol 1.5 
Total 270 mg 
______________________________________ 
EXAMPLE 8 
The membrane permeation property of dimethylisosorbide over the storage 
period at the room temperature was examined for the soft capsules prepared 
using dimethylisosorbide (ARLASOLVE.RTM. DMI) as a cosurfactant according 
to the present invention and then compared with the membrane permeation 
properties of ethanol, propylene glycol, Transcutol (diethylene glycol 
monoethyl ether) and Glycofurol (tetrahydrofurfuryl alcohol polyethylene 
glycol ether). The content of the cosurfactant in each soft capsule 
preparation was analyzed by gas chromatography. The components and their 
constitutional ratios in the compositions used in this experiment are 
given in the following Table 1 and the experiment results are given in the 
following Table 2. 
TABLE 1 
__________________________________________________________________________ 
Composition of the soft capsules used in the test 
for membrane permeation property 
Content of each component in capsule 
Test Control preparation (cosurfactant) 
preparation B C D 
(dimethyl- 
A (propylene- 
(Trans- 
(Glyco- 
Component isosorbide) 
(ethanol) 
glycol) 
cutol) 
furol 
__________________________________________________________________________ 
Cyclosporin 25 mg 25 mg 25 mg 25 mg 25 mg 
(10.8 wt %) 
(10.8 wt %) 
(10.8 wt %) 
(10.8 wt %) 
(10.8 wt %) 
Cosurfactant 25 mg 25 mg 46 mg 46 mg 46 mg 
(10.8 wt %) 
(10.8 wt %) 
(10.8 wt %) 
(19.8 wt %) 
(19.8 wt %) 
Oil component 105.8 mg 
106.5 mg 
115 mg 
115 mg 
115 mg 
(Soybean oil) (46.0 wt %) 
(46.0 wt %) 
(49.6 wt %) 
(49.6 wt %) 
(49.6 wt %) 
Surfactant 74.52 mg 
75 mg 46 mg 46 mg 46 mg 
(LABRAFIL.sup.R M2125CS) 
(32.4 wt %) 
(32.4 wt %) 
(19.8 wt %) 
(19.8 wt %) 
(19.8 wt %) 
__________________________________________________________________________ 
TABLE 2 
______________________________________ 
Change in the contents of cosurfactants contained in 
soft capsules over the storage period 
Change in contents (per capsule) 
Initial 
Storage period (days) 
Cosurfactant content 2 5 12 14 50 
______________________________________ 
Dimethylisosorbide 
10.8 10.8 10.5 10.4 10.0 9.8 
(Test preparation) 
Ethanol 10.8 7.2 3.8 2.1 1.3 -- 
(Control preparation A) 
Propylene glycol 
19.8 3.6 0.9 -- -- -- 
(Control preparation B) 
Transcutol 19.8 7.3 -- -- 2.7 -- 
(Control preparation C) 
Glycofurol 19.8 8.2 -- -- 3.2 -- 
(Control preparation D) 
______________________________________ 
Note: Conditions of gas chromatography for each cosurfactant 
1) Dimethylisosorbide 
Column: Ultra 2 (Cross-linked 2% phenylmethyl silicone gun phase, 25 
m.times.0.32 mm, thickness 0.52 .mu.m) 
Column temperature: 230.degree. C. (Isothermal) 
Detector: FID (Temperature: 270.degree. C.) 
Injection temperature: 275.degree. C. 
Carrier gas: He (Partition ratio 80:1) 
Injection volume: 5 .mu.l 
Internal standard: Octyl alcohol 
2) Ethanol 
Column: HP-20M (Carbowax 20M) (25 m.times.0.32 mm, film thickness 0.3 
.mu.m) 
Column temperature: 30.degree. C. (Isothermal) 
Detector: FID (Temperature: 200.degree. C.) 
Injection temperature: 150.degree. C. 
Carrier gas: He (Partition ratio 80:1) 
Injection volume: 5 .mu.l 
Internal standard: Diethyl ether 
3) Propylene glycol 
Column: HP-20M (Carbowax 20M) (25 m.times.0.32 mm, film thickness 0.3 
.mu.m) 
Column temperature: 180.degree. C. (Isothermal) 
Detector: FID (Temperature: 240.degree. C.) 
Injection temperature: 240.degree. C. 
Carrier gas: He (Partition ratio 80:1) 
Injection volume: 5 .mu.l 
Internal standard: Pentadecanoic acid methyl ester 
4) Transcutol 
Column: Ultra 2 (Cross-linked 5% phenylmethyl silicone gun phase, 25 
m.times.0.32 mm, thickness 0.52 .mu.m) 
Column temperature: 230.degree. C. (Isothermal) 
Detector: FID (Temperature: 270.degree. C.) 
Injection temperature: 270.degree. C. 
Carrier gas: He (Partition ratio 80:1) 
Injection volume: 5 .mu.l 
Internal standard: Pentadecanoic acid methyl ester 
5) Glycofurol 
Column: Ultra 2 (Cross-linked 2% phenylmethyl silicone gun phase, 25 
m.times.0.32 mm, thickness 0.52 .mu.m) 
Column temperature: 230.degree. C. (Isothermal) 
Detector: FID (Temperature: 270.degree. C.) p1 Injection temperature: 
270.degree. C. 
Carrier gas: He (Partition ratio 80:1) 
Injection volume: 5 .mu.l 
Internal standard: Dimethylisosorbide 
As can be seen from the results described in the above Table 2, 
dimethylisosorbide used as a cosurfactant according to the present 
invention does not change in its content even after 50 days while other 
cosurfactants used in the prior art were reduced by 50% of the initial 
content after only 2 days. Accordingly, it can be readily determined that 
dimethylisosorbide when used as a cosurfactant according to the present 
invention exhibits substantially no membrane permeation since the amount 
thereof does not significantly change in the composition and therefore is 
most suitable for use in the formulation of soft capsule preparations 
according to the present invention. 
EXAMPLE 9 
The bioavailability of the microemulsion using dimethylisosorbide as a 
cosurfactant according to the present invention was compared with the 
bioavailability of an ethanol-containing preparation according to the 
prior art to estimate the influence of dimethylisosorbide on the 
bioavailability of cyclosporin. In this experiment, rabbits were used as 
the experimental animal. The soft capsules as prepared in Example 1, 
Example 6 and Example 7 and the commercialized product SANDIMMUN.RTM. 
using ethanol as a cosurfactant were used as the test preparations and the 
control preparation, respectively. In this experiment, both of the test 
preparations and the control preparation were administered in an amount of 
300 mg as cyclosporin per kg of rabbit. Rabbits were uniformly fed with 
the conventional rabbit solid feed composition for 4 days or more under 
the same condition in wire cages. When the oral preparations were 
administered, rabbits were fasted for 48 hours in a restraint cage made of 
wood, during which rabbits were allowed to freely take 10% dextrose 
solution. Levin's tube having a diameter of 5 mm was interposed by the 
depth of 30 cm through the esophagus after the surface of the Levin's tube 
was coated with vaseline in order to reduce friction. Each of the test 
preparations and the control preparation was emulsified with 50 ml of 
water and then introduced into a syringe which is attached to the Levin's 
tube. Ear veins of rabbit were dilated using xylene and then blood was 
taken from each rabbit's ear vein at an interval of 5, 15, 30, 60, 90, 150 
and 270 minutes by means of heparin-treated disposable syringe. To 1 ml of 
blood thus obtained were added 0.5 ml of aqueous saturated sodium chloride 
solution and 2 ml of ether, and then the mixture was shaken for 5 minutes 
and centrifuged with 5000 rpm for 10 minutes to separate the supernatant. 
1 ml of the supernatant was collected and then developed in an activated 
silica SEP-PAK.RTM. (Waters). The developed SEP-PAK.RTM. was washed with 5 
ml of n-hexane and eluated with 2 ml of methanol. The eluate was 
evaporated to dryness in nitrogen gas under reduced pressure. The residue 
was analyzed by means of HPLC (High Performance Liquid Chromatography) 
[HPLC condition: Column .mu.-BONDAPAK.RTM. C.sub.18 (Waters), Mobile phase 
CH.sub.3 CN: MeOH:H.sub.2 O=55:15:30, Detection 210 nm, Flow rate 1.0 
ml/min., Column temperature 70.degree. C., Sensitivity 0.01 Aufs, 
Injection volume 100 .mu.l]. 
The results are illustrated at FIGS. 1, 2 and 3 for Examples 1, 6 and 7, 
respectively. As can be seen from FIGS. 1, 2 and 3, since the soft capsule 
preparation according to the present invention shows substantially the 
same blood level as that of the ethanol-containing soft capsule 
preparation according to the prior art, it is apparent that 
dimethylisosorbide used in the present invention exhibits a superior 
effect as to the stability of soft capsule formulation without influencing 
the bioavailability of cyclosporin. 
EXAMPLE 10 
The bioavailability of the microemulsion using dimethylisosorbide as a 
cosurfactant according to the present invention was compared with the 
bioavailability of an ethanol-containing preparation according to the 
prior art to estimate the influence of dimethylisosorbide on the 
bioavailability of cyclosporin in human body. In this experiment, 16 
healthy volunteeres aging 20 to 25 in average were involved and the soft 
capsules as prepared in Example 6 and the commercialized product 
SANDIMMUN.RTM. using ethanol as a cosurfactant were used as the test 
preparation and the control preparation, respectively. In this experiment, 
both of the test preparation and the control preparation were administered 
in an amount of 400 mg as cyclosporin per human body to examine the 
biological equivalency between two preparations. The test was conducted by 
dividing the 16 volunteeres into two groups as described in the following 
and then subjecting them to the cross experiment according to a Latin 
square cross over design. The interval between Period I and Period II was 
eight days. 
______________________________________ 
Subject Period I Period II 
______________________________________ 
Group I Control Test 
Preparation 
Preparation 
Group II Test Control 
Preparation 
Preparation 
______________________________________ 
Blood was taken from the subjects before and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 
and 24 hours after the administration of each preparation in an amount of 
8 ml each time and analyzed by means of a cyclosporin monoclonal analysis 
kit using monoclonal antibody to determine the concentration of 
cyclosporin in the whole blood and plasma. To 150 .mu.l of whole blood or 
plasma which was treated with heparin as an anticoagulant were added 50 
.mu.l of a solubilization reagent and 300 .mu.l of a precipitation reagent 
for whole blood and then the mixture was agitated for 10 seconds to 
precipitate the protein and centrifuged with 9500xg for 5 minutes to 
obtain the supernatant which was then analyzed with TDX system. 
The results are illustrated at FIG. 4. As can be seen from FIG. 4, since 
the soft capsule preparation according to the present invention shows 
substantially the same blood level as that of the ethanol-containing soft 
capsule preparation according to the prior art in human body, it is 
apparent that dimethylisosorbide used in the present invention has no 
effect on the bioavailability of cyclosporin. 
Accordingly, the use of dimethylisosorbide as a cosurfactant in the 
cyclosporin-containing soft capsule according to the present invention 
effectively prolongs the shelf life of the capsules without adversly 
affecting bioavailability. Further, the cyclosporin pharmaceutical 
preparation according to the present invention overcomes a major problem 
in the soft capsules containing conventionally used cosurfactants, such as 
ethanol, propylene glycol, transcutol, glycofurol and the like. That is, 
the present invention alleviates membrane permeation of cosurfactant and 
the resulting change in the composition contained in the soft capsule 
which may lead to the crystallization of cyclosporin. 
Although this invention has been described in its preferred form with a 
certain degree of particularity, it is appreciated by those skilled in the 
art that the present disclosure of the preferred form has been made only 
by way of example and that numerous changes in the details of the 
construction, combination and arrangement of parts may be resorted to 
without departing from the spirit and scope of the invention.