Pharmaceutical preparations contg macrolide antibiotics - contain, as the carrier, a mixt of a hydrophilic phase, a lipophilic phase and a surfactant

This invention relates to pharmaceutical formulations containing FK506, particularly in the form of a microemulsion pre-concentrate.

FK506 is a macrolide immunosuppressor made from Streptomvces tsukubaensis No. 9993. The structure of FK506 is given in the Appendix of the Merck Index as point A5.

It has now surprisingly been found that stable compositions containing FK506 and offering high absorption efficiency can be obtained by formulating FK506 with certain carriers.

The invention relates to an oral pharmaceutical composition, characterized in that it contains FK506 and a carrier which

i) a hydrophilic phase selected from transcutol, glycofurol and 1,2-propylene glycol, ii) a lipophilic phase selected from triglycerides of medium-length fatty acid chains, mixed mono-, di-, triglycerides and transesterified ethoxylated vegetable oils, and iii) a surfactant, wherein the composition is a microemulsion pre-concentrate.

The pharmaceutical composition is stable and gives a surprisingly high and consistent absorption efficiency when administered orally. Therefore, FK506 can be administered in lower doses, which reduces toxicity problems. For example, animal studies in which the pharmaceutical compositions were administered orally gave high bioavailabilities. The pharmaceutical compositions therefore have very surprising properties which offer great advantages.

A microemulsion pre-concentrate is defined in this specification as a formulation which forms a microemulsion spontaneously in an aqueous medium, for example in water or in the gastric juices after oral administration.

A microemulsion is a non-opaque or substantially non-opaque colloidal dispersion that is formed spontaneously or substantially spontaneously when its components are brought into contact with one another. A microemulsion is thermodynamically stable and contains dispersed particles of a size that is less than approximately 0.2 μm (2000 Å). Microemulsions generally comprise droplets or particles with a diameter less than about 0.15 μίτι (1500 Å); typically from 0.003 to 0.1 µm (30 to 1000 Å). Further features can be found in GB 2 222 770 A.

A microemulsion pre-concentrate is described in this specification as a formulation which spontaneously forms a microemulsion in an aqueous medium, for example in water or in the gastric juices after oral administration. The emulsion formed is opaque, thermodynamically stable and contains dispersed droplets of a size larger than about 100 nm, usually larger than about 200 nm. Bimodal size range distributions are often obtained. The microemulsion preconcentrates are preferably of the type which provide O / W (oil-in-water) microemulsions.

The lipophilic phase can comprise 10 to 85% by weight of the carrier; preferably 15 to 70% by weight, more preferably 20 to 60% by weight, and even more preferably about 25% by weight.

The surfactant can comprise 5 to 80% by weight of the carrier; preferably 10 to 70% by weight, more preferably 20 to 60% by weight, and even more preferably about 40% by weight⁰/».

The hydrophilic phase can be 10 to 50 weight⁰/ of the carrier include; preferably 15 to 40 weight⁰/, more preferably 20 to 35 weight⁰/ and even more preferably about 30 weight⁰/.

FK506 is approximately in an amount of 1 to 15 weight⁰of the composition, more preferably about 2 to 10%.

The hydrophilic phase can be derived from Transcutol (which has the formula C₂H5- [O- (CH₂) 2] 2-OH), glycofurol (also known as tetrahydrofurfuryl alcohol polyethylene glycol ether) and 1,2-propylene glycol or mixtures thereof, and is preferably 1,2-propylene glycol. The hydrophilic phase can include further hydrophilic co-components, for example lower alcohols such as ethanol. These co-components will generally be present as partial substitutes for other components of the hydrophilic phase. It has been found that use of Etha2

AT 409 082 B nol, although not essential in the compositions, has been found to be of particular advantage when the compositions are to be made in a soft, encapsulated gelatin form, particularly improving storage characteristics. The storage life can therefore be extended by using ethanol or another such co-component as an additional component of the hydrophilic phase. The ethanol can comprise 0 to 60% by weight of the hydrophilic phase, preferably 20 to 55% by weight and more preferably about 40 to 50% by weight. Small amounts of liquid polyethylene glycols can also be included in the hydrophilic phase.

Preferred lipophilic phase components are triglycerides of medium-length fatty acid chains, mixed mono-, di-, triglycerides and interesterified ethoxylated vegetable oils. Suitable fatty acid chain glycerides are those which are known and available commercially under the trade names Miglyol, Captex, Myritol, Capmul, Captex, Neobee and Mazol; with Miglyol 812 being most preferred. These triglycerides are found in Fiedler, HP Lexicon of auxiliaries for pharmacy, cosmetics and related areas, Edition Cantor, D-7960 Aulendorf, third improved and expanded edition (1989).

The mixed mono-, di- and triglycerides preferably comprise mixtures of C12-20 fatty acid mono-, di- and triglycerides, in particular mixed Cie-18 * fatty acid mono-, di- and triglycerides. The fatty acid component of the mixed mono-, di- and triglycerides can comprise both saturated and unsaturated fatty acid residues. However, they preferably comprise mainly unsaturated fatty acid residues; especially unsaturated Ci₈-Fatty acid residues. Suitably the mixed mono-, di- and triglycerides comprise at least 60% by weight, preferably at least 75% by weight, more preferably at least 85% by weight of an unsaturated C₁₈-Fatty acids (for example linolenic, linoleic and oleic acid) -mono-, di- and triglyceride. The mixed mono-, di- and triglycerides comprise less than 20% by weight, for example 15% by weight or 10% by weight or less of saturated fatty acids (for example palmitic and stearic acid) mono-, di- and triglycerides.

The mixed mono-, di- and triglycerides preferably mainly comprise mono- and diglycerides; for example, mono- and diglycerides comprise at least 50%, more preferably at least 70%, based on the total weight of the lipophilic phase. The mono- and diglycerides more preferably comprise at least 75% by weight (e.g. about 80% or 85% by weight) of the lipophilic phase.

Preferably, the monoglycerides comprise from about 25 to about 50% of the mixed mono-, di- and triglycerides, based on the total weight of the lipophilic phase. More preferably from about 30 to about 40% (e.g. 35 to 40%) monoglycerides are present.

The diglycerides preferably comprise from about 30 to about 60% of the mixed mono-, di- and triglycerides, based on the total weight of the lipophilic phase. More preferably from about 40 to about 55% (e.g. 48 to 50%) diglycerides are present.

The triglycerides suitably comprise at least 5%, but less than about 25%, of the mixed mono-, di- and triglycerides, based on the total weight of the lipophilic phase. More preferably from about 7.5 to about 15% (e.g. 9 to 12%) triglycerides are present.

The mixed mono-, di- and triglycerides can be prepared by admixing individual mono-, di- and triglycerides in appropriate relative proportions. However, they conveniently include transesterification products of vegetable oils, for example almond oil, ground nut oil, olive oil, persica oil, palm oil or, preferably, corn oil, sunflower oil or safflower oil and in particular corn oil with glycerol.

Such transesterification products are generally obtained by heating the selected vegetable oil with glycerol at a high temperature in the presence of a suitable catalyst in an inert atmosphere with continued stirring (for example in a stainless steel reactor) to effect transesterification or glycerolysis. The transesterification products generally also comprise small amounts of free glycerol in addition to their mono-, di- and triglyceride components. The amount of free glycerol present is preferably less than 10%, more preferably less than 5%, most preferably about 1 or 2% by weight based on the total weight of free glycerol plus mono-, di- and triglycerides.

First, a little of the glycerol is preferably removed so as to substantially reduce it

AT 409 082 B to give a glycerol-free set if soft gelatin capsules are to be produced.

Transesterification products of corn oil and glycerol provide particularly suitable mixed mono-, di- and triglycerides. An example of a suitably blended glyceride product is the transesterification product which is commercially available under the trade name MAISINE. This product mainly comprises linoleic and oleic acid mono-, di- and triglycerides along with small amounts of palmitic and stearic acid mono-, di- and triglycerides (corn oil itself comprises ingredients of approximately 56% linoleic acid, 30% oleic acid, approximately 10% palmitic acid and approximately 3% stearic acid). The physical characteristics of MAISINE [available from Etablissement Gattefossä, 36, Chemin de Genas, PO Box 603, 69804 Saint-Priest, Cedex (France)] are as follows: up to 10% (typically 3.9 to 4.9% or, in substantially glycerol-free rates, approximately 0.2%) free glycerol; about 35% (typically 30 to 40%, or, in substantially glycerol-free sets, about 32 to 36%, for example about 36%) monoglycerides; about 50% (or, in substantially glycerol-free sets, about 46 to 48%) diglycerides; about 10% (or, in substantially glycerol-free sets, about 12 to 15%) triglycerides; and about 1% free oleic acid.

Further physical characteristics for MAISINE are as follows: a maximum acid value of approximately 2, an iodine number of approximately 85 to 105, a saponification number of approximately 150 to 175 (Fiedler Lexikon der auxiliaries, third improved and expanded edition (1989) Vol. 2, p. 768). The fatty acid content for MAISINE is typically as follows: about 11% palmitic acid, about 2.5% stearic acid; about 29% oleic acid; about 56% linoleic acid; and 1.5% other acids.

It is particularly preferred that the mixed mono-, di- and triglycerides be clear and remain clear for more than 20 days during storage at temperatures of 20 ° C to 25 ° C. A sample of the mixed mono-, di-, and triglycerides that has been stored in a refrigerator at approximately 2 and 8 ° C for 24 hours and then stored at room temperature for 1 hour should also be clear.

The mono-, di- and triglycerides are preferably low in saturated fat. Mixed mono-, di- and triglycerides meeting these requirements can be obtained from commercially available products by known separation methods (for example, freezing methods associated with separation meth ods such as centrifugation) to remove the saturated fatty acid components and the unsaturated ones To improve fatty acid component content. Typically the total saturated fatty acid component content will be less than 15% by weight (e.g. <10% by weight or <5% by weight) based on the total weight of the lipophilic phase. A reduction in the content of the saturated fatty acid component in the monoglyceride fraction can be observed after it has been subjected to the separation process. A suitable method is described in WO 93/09211.

The mixed mono-, di- and triglycerides therefore preferably contain smaller amounts of saturated fatty acids (eg palmitic and stearic acids) and relatively higher amounts of unsaturated fatty acids (eg oleic and linoleic acids) than the starting material.

A suitable example of a mixed mono-, di- and triglyceride product containing minor amounts of saturated fatty acids contains the following: 32 to 36% by weight monoglycerides, 45 to 55% by weight diglycerides and 12 to 20% by weight triglycerides, based on the total weight of the lipophilic phase. Other characteristics include the following:

<td>Fatty acid content (like as a methyl ester Chromatography determined)</td><td>Methyl linoleate 53 to 63% by weight Methyl oleate 24 to 34% by weight Methyl linoleate 0 to 3% by weight Methyl arachate 0 to 3% by weight Methyl palmitate 6 to 12% by weight Methyl stearate 1 to 3% by weight</td>

<td>relativ density</td><td>0.94 to 0.96</td>

<td>Hydroxyl number</td><td>140 to 210</td>

<td>Iodine number</td><td>110 to 20</td>

AT 409 082 B

<td>Peroxide number</td><td> &lt;4,0</td>

<td>Free glycerol</td><td> &lt; 1,0</td>

<td>Stiffening number</td><td>about 150 to 185</td>

<td>Acid value</td><td>max. about 2</td>

Mixed mono-, di- and triglycerides that correspond to these characteristics are used in this

Description referred to as refined glycerol transesterified corn oils. The refined glycerol transesterified corn oils have the advantage that they remain stable for a long time.

The lipophilic phase can, on the other hand, comprise suitable interesterified ethoxylated vegetable oils, such as those obtained by adding various natural vegetable oils (for example corn oil, kernel oil, almond oil, ground nut oil, olive oil, soybean oil, sunflower oil, safflower oil and palm oil, or mixtures thereof) with Polyethylene glycols, which have an average molecular weight of 200 to 800, are reacted in the presence of a suitable catalyst. These methods are known and an example is described in US 3,288,824A. Interesterified ethoxylated corn oil is particularly preferred.

Transesterified ethoxylated vegetable oils are known and are commercially available under the trade name LABRAFIL (H. Fiedler, loc sit, Vol. 2, page 707). Examples are LABRAFIL M 2125 CS (which is obtained from corn oil and has an acid number less than about 2, a saponification number from 155 to 175, an HBL value from 3 to 4, and an iodine value from 90 to 110), and LABRAFIL M 1944 CS (which is obtained from seed oil and has an acid number of approximately 2, a saponification number from 145 to 175, and an iodine number from 60 to 90). LABRAFIL M 2130 CS (which is a transesterification product of a C₁₂.i8 glyceride and polyethylene glycols and which has a melting point of about 35 to 40 ° C, an acid number that is less than about 2, a saponification number of 185 to 200, and an iodine number that is less than about 3) can also be used. The preferred interesterified ethoxylated vegetable oil is LABRAFIL M 2125 CS, which can be obtained, for example, from Gattefossä, Saint-Priest Cedex, France.

Examples of suitable surfactants are as follows:

i) reaction products of a natural or hydrogenated castor oil and ethylene oxide. The natural or hydrogenated castor oil can be reacted with ethylene oxide in a molar ratio of about 1:35 to about 1:60, with optional removal of the polyethylene glycol component from the products. Various such surfactants are commercially available. The polyethylene glycol hydrogenated castor oils available under the trade name CREMOPHOR are particularly suitable. CREMOPHOR RH 40, which has a saponification number of approximately 50 to 60, an acid number which is less than approximately 1, a water content (Fischer) which is less than approximately 2%, an n are particularly suitable_D.⁶⁰ from about 1.453 to 1.457 and an HLB value of about 14 to 16; and CREMOPHOR RH 60, which has a saponification number of about 40 to 50, an acid number that is less than about 1, an iodine number that is less than about 1, a water content (Fischer) of about 4.5 to 5.5% , an nD²⁵ from about 1.453 to 1.457 and an HLB value of about 15 to 17. A particularly preferred product of this class is CREMOPHOR RH40. Polyethylene glycol castor oils available under the trade name CREMOPHOR EL are also suitable, CREMOPHOR EL having a molecular weight (by steam osmometry) of about 1630, a saponification number of about 65 to 70, an acid number of about 2, an iodine number of about 28 to 32 and a nD²⁵ of about 1.471. Similar or identical products, which can also be used, are available under the trade names NIKKOL (e.g. NIKKOL HCO-40 and NIKKOL HCO-60), MAPEG (e.g. MAPEG CO-40h), INCROCAS (e.g. INCROCAS 40), and TAGAT (e.g. TAGAT RH 40) available. These surface-active substances are further in Fiedler loc. cit. described.

ii) Polyoxyethylene sorbitan fatty acid esters, for example mono- and trilauryl-, -palmityl, -stearyl and -oleyl esters of the kind which are known and available commercially under the trade name TWEEN (Fiedler, loc. cit. p. 13001304), including the products TWEEN

20 [polyoxyethylene (20) sorbitan monolaurate],

21 [polyoxyethylene (4) sorbitan monolaurate],

AT 409 082 B

40 [polyoxyethylene (20) sorbitan monopalmitate],

60 [polyoxyethylene (20) sorbitan monostearate],

65 [polyoxyethylene (20) sorbitan tristearate],

80 [polyoxyethylene (20) sorbitan monooleate],

81 [polyoxyethylene (5) sorbitan monooleate],

85 [polyoxyethylene (20) sorbitan trioleate].

Particularly preferred products in this class are TWEEN 40 and TWEEN 80.

iii) Polyoxyethylene fatty acid esters, for example polyoxyethylene stearic acid esters of the type that im

Trade under the trade name MYRJ (Fiedler, loc. Cit., 2. pp. 834-835) are known and available. A particularly preferred product in this class is MYJR 52 with a D²⁵ of about 1.1, a melting point of about 40 to 44 ° C, an HLB value of about 16.9, an acid value of about 0 to 1, and a saponification number of about 25 to 35.

iv) Polyoxyethylene-polyoxypropylene copolymers and block copolymers, for example of the type known and available under the trade names PLURONIC, EMKALYX and POLOXAMER (Fiedler, loc. cit., 2, p. 959). A particularly preferred product of this class is PLURONIC F68, with a melting point of about 52 ° C and a molecular weight of about 6800 to 8975. Another preferred product of this class is POLOXAMER 188.

v) Dioctyl sulfosuccinate or di- [2-ethylhexyl] succinate (Fiedler, loc. cit., 1. pp. 107-108).

vi) Phospholipids, especially lecithins (Fiedler, loc. Cit., 2, pp. 943-944). Suitable lecithins include, in particular, soy lecithins.

vii) Propylene glycol mono- and difatty acid esters such as propylene glycol dicaprylate (also known and available commercially under the trade name MIGLYOL 840), propylene glycol dilaurate, propylene glycol hydroxystearate, propylene glycol isostearate, propylene glycol laurate, propylene glycol laurate, propylene glycol and so on 808 -809).

The components of the carrier may contain unreacted starting materials such as polyethylene glycol.

The surfactant selected preferably has an HLB of at least 10.

The relative proportions of the hydrophilic phase component (s), the lipophilic phase and the surfactant lie within the microemulsion area on a normal three-way diagram. The compositions obtained in this way are microemulsion preconcentrates with high stability which, after the addition of water, are capable of producing microemulsions which have an average particle size of &lt;1500 Å and which are stable for 24 hours.

The microemulsion preconcentrate compositions show good stability characteristics, as shown by normal stability tests, for example with a shelf life of up to three years and even longer.

The pharmaceutical composition can also include other additives or ingredients, for example antioxidants (such as ascorbyl palmitate, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT) and tocopherols) and / or preservatives. These additions or ingredients can comprise about 0.05 to 1% by weight of the total weight of the composition. The pharmaceutical composition can also include sweetening or flavoring agents in an amount of up to 2.5 or 5% by weight based on the total weight of the composition. The antioxidant is preferably α-tocopherol (vitamin E).

The pharmaceutical composition exhibits particularly advantageous properties when administered orally; for example in terms of persistence and a high level of bioavailability obtained in normal bioavailability tests, e.g. 2 to 4 times higher than emulsions. These experiments are performed on animals or healthy volunteers using HPLC or specific or non-specific monoclonal equipment to determine the level of FK506 in the blood.

Pharmacokinetic parameters such as absorption and blood levels also become surprisingly more predictable and administration problems with erratic absorption can be eliminated or reduced. In addition, the pharmaceutical composition is effective with surfactant materials, for example bile salts, which are present in the gastrointestinal tract. That is, the pharmaceutical composition is completely soluble in aqueous systems comprising such natural surfactants and can therefore be microemulsions6

AT 409 082 B

Provide systems in situ that are stable and do not show precipitation of the active ingredient or other fine particle structure disruption. The function of the pharmaceutical composition after oral administration remains essentially independent and / or unaffected by the relative presence or absence of bile salts at any particular time or for any particular person.

The pharmaceutical composition is preferably associated in unit dosage form, for example by filling it into orally administrable capsule shells. The capsule shells can be soft or hard gelatin capsule shells. Where the pharmaceutical composition is in unit dosage form, each unit dosage will suitably contain between 10 and 100 mg of FK506, more preferably between 10 and 50 mg; for example 15, 20, 25 or 50 mg of FK506. Such unit dosage forms are suitable for administration from 1 to 5 times daily, depending on the particular therapeutic purpose, phase of therapy and the like.

However, if desired, the pharmaceutical composition may be in beverage solution form and may include water or some other aqueous system to provide microemulsion systems suitable for drinking.

Usage of the pharmaceutical composition can be observed in normal clinical tests in, for example, known indications of FK506 dosages which give equivalent blood levels of FK506; for example, using dosages in the range of 2.5 mg to 1000 mg of FK506 per day for an adult weighing 75 kilograms, and in normal animal models. The increased bioavailability of the FK506 provided by the compositions can be observed in normal animal studies and in clinical trials.

FK506 is particularly useful for the following conditions:

a) Treatment and prevention of organ transplant rejection, for example for the treatment of recipients of heart, lung, combined heart-lung, liver, kidney, pancreas, skin or cornea transplants. The pharmaceutical compositions are also indicated for the prevention of graft versus recipient disease, which sometimes occurs after bone marrow transplants.

b) Treatment and prevention of autoimmune diseases and inflammatory conditions, particularly inflammatory conditions with an etiology that include an autoimmune component such as arthritis (e.g. rheumatoid arthritis, chronica progressive arthritis and arthritis deformans) and rheumatic diseases. Specific autoimmune diseases for which the pharmaceutical compositions can be used include autoimmune hematological diseases (including e.g. hematological anemia, aplastic anemia, red blood cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, auto ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Greaves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal arthritis, pulmonary fibrosis, psoriasis interstitial keratoconjunctivitis Glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy) and adolescent dermatomyositis.

c) Treatment and prevention of asthma.

d) Treatment of multi-drug resistance (MDR = resistance to a variety of chemical cytostatics. FK506 suppresses P-glucoproteins (Pgp), which are membrane transport molecules that are assigned to MDR. MDR is particularly problematic in cancer patients and AIDS patients who do not respond to conventional Chemotherapy respond as the medication is pumped out of the cells by Pgp. Therefore, FK506 is useful to improve the effectiveness of other chemotherapeutic agents in the treatment and control of multidrug resistant conditions, such as multidrug resistant cancer or AIDS.

An oral pharmaceutical composition according to the present invention can be prepared by a

Process are produced that includes

i) a hydrophilic phase selected from transcutol, glycofurol and 1,2-propylene glycol, ii) a lipophilic phase selected from triglycerides of medium-length fatty acid chains, mixed 7

AT 409 082 B ten mono-, di-, triglycerides and interesterified ethoxylated vegetable oils, and iii) a surface-active substance, to be brought into close admixture and to add FK506. If desired, the composition can be combined in unit dosage form, for example filling the composition into gelatin capsules.

Optionally, further components or additions, in particular a co-component of the hydrophilic phase, for example ethanol, can be mixed with components (1), (2) and (3).

The composition can be combined with enough water or a sufficient amount of an aqueous solvent that a microemulsion is obtained.

The following examples illustrate compositions in unit dosage form suitable for use, for example, in the prevention of graft rejection or in the treatment of autoimmune disease, when administered from 1 to 5 unit doses / day.

EXAMPLE 1: Refined, glycerol interesterified corn oil is prepared as follows:

Corn oil transesterified with glycerol, which is essentially free of glycerol, is slowly cooled to a temperature of + 20 ° C. and stored at this temperature for one night. The grain oil is centrifuged at an acceleration of 12,000 G and a flow rate of 103 kg / h in a continuous flow centrifuge to give a liquid phase (62 kg / h) and a sediment-containing phase (41 kg / h). The liquid phase is slowly cooled to + 8 ° C and kept at this temperature for one night. The liquid phase is then centrifuged at an acceleration of 12,000 G and a flow rate of 112 kg / h to give a liquid phase (76.2 kg / h) and a sediment-containing phase (35.8 kg / h). The liquid phase is refined, glycerol-transesterified corn oil. On the other hand, an improved product can be obtained by effecting centrifugation in three steps, for example at + 20 ° C, + 10 ° C and + 5 ° C.

The process is characterized by a small percentage reduction in the monoglyceride component in the refined glycerol interesterified corn oil compared to the starting material (eg 35.6% compared to 38.3%).

EXAMPLE 2:

The refined glycerol interesterified corn oil obtained as described in Example 1 is used in the preparation of the following oral unit dosage form:

<td>COMPONENT</td><td>QUANTITY (mg / capsule)</td>

<td>FK506</td><td> 20,0</td>

<td>1) ethanol</td><td> 75,0</td>

<td>2) 1,2 propylene glycol</td><td> 81,0</td>

<td>3) refined oil</td><td> 121,5</td>

<td>4) Cremophor RH40</td><td> 202,5</td>

<td>Total</td><td> 500,0</td>

The FK506 is suspended in (1) with stirring at room temperature, and (2), (3) and (4) are added to the resulting solution while stirring. The mixture obtained is filled into hard gelatin capsules of size 0 and sealed using the quasi-seal method.

EXAMPLE 3: Pharmokinetics

Two formulations prepared as in Example 2 are used:

AT 409 082 B

<td>formulation</td><td>component</td><td>Amount weight%</td>

<td>A.</td><td>Tween 80 Maisine Propylene glycol Ethanol FK506</td><td>41.5% by weight 24.9% by weight 16.6% by weight 15.0 weight% 2.0 weight%</td>

<td>B.</td><td>Cremophor RH40 Maisine Propylene glycol Ethanol FK506</td><td>41.5% by weight 24.9% by weight 16.6% by weight 15.0% by weight 2.0 weight%</td>

Formulation A is an emulsion pre-concentrate and Formulation B is a microemulsion pre-concentrate. 6 male Wistar rats with an average body weight of 300 g are used per shape. One day before treatment, the rats are deprived of food, but the rats are allowed free access to water. The rats are then anesthetized by injecting 2 x 1 ml of 20% urethane intraperitoneally and a permanent catheter is inserted into the right jugular vein to allow blood to be drawn. 500 ml / animal of the formulation is administered by gastric introduction 20 hours after the operation. A total dose of 10 mg of the drug per animal is administered. 0.7 ml blood samples are taken from the juguiar catheter of each animal 15 minutes before drug administration and then 0.17, 0.5, 1, 1.5, 2, 3, 5 and 8 hours after drug administration . The samples are kept in heparinized tubes and analyzed by ELISA using microtiter plates coated with specific antibodies from FK506. The animals are killed immediately after the last blood sample has been taken. The results are given in the following table:

<td>For m</td><td>AUC (0-8 hours) [ng.h / ml]</td><td>CV [%]</td><td>Cmax [ng / ml]</td><td>CV [%]</td><td>Imax [Hours]</td><td>CV [%]</td>

<td>A.</td><td> 11951</td><td> 44</td><td> 2671</td><td> 42</td><td> 3,8</td><td> 29*</td>

<td>B.</td><td> 13826</td><td> 13</td><td> 3405</td><td> 30</td><td> 4,0</td><td> 35+</td>

*) n = 5

+) n = 2 due to difficulties in drawing blood

The results indicate that FK506 is well absorbed.

EXAMPLE 4: Comparison

Formulations A and B are compared to a formulation comprising 38.6% corn oil, 41.6% Labrafil M21 / 25C, 17.8% ethanol and 2% FK506 (formulation C). The same procedure is used as in Example 3, except that the animals each receive a total dosage of 0.5 mg of the formulation.

The results are given in the following table:

AT 409 082 B

<td>For m</td><td>AUC (0-8 hours) [ng.h / ml]</td><td>CV [%]</td><td>Cmax [ng / ml]</td><td>CV [%]</td><td>tmax [Hours]</td><td>CV [%]</td>

<td>A.</td><td> 105,8</td><td> 28</td><td> 31,22</td><td> 35</td><td> 1,6</td><td> 51*</td>

<td>B.</td><td> 96,6</td><td> 32</td><td> 36,13</td><td> 60</td><td> 0,4</td><td> 30</td>

<td>C.</td><td> 36,2</td><td> 31</td><td> 7,83</td><td> 27</td><td> 3,0</td><td> 78</td>

*) n = 4

The results indicate that Formulations A and B provide much better pharmacokinetic properties than Formulation C.

EXAMPLE 5:

FK506 is made into a microemulsion pre-concentrate with the following composition in% by weight: 2% active compound, 44% Cremophor RH40, 26.4% corn oil mono-, di-, and triglycerides, 17.6% 1,2-propylene glycol and 10% Ethanol.