JPH0717677B2 - Novel cyclosporin derivatives with improved "8-amino acids" - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Immune dysregulation is found in a wide range of changes in "autoimmunity" and in chronic inflammatory diseases such as systemic lupus erythematosus, chronic rheumatoid arthritis, type I diabetes, inflammatory bowel disease, biliary cirrhosis, Uveitis, multiple sclerosis and other localized ileitis, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis,
It includes diseases such as ichthyosis and severe eye disorders. The potential etiology for each of these conditions varies considerably, but generally they lead to alterations in autoantibodies and appearance of autoreactive lymphocytes. This autoreactivity lies in part in the lack of homeostatic control that regulates the normal immune system.

Similarly, after bone marrow or organ transplantation, host lymphocytes recognize foreign tissue antigens and start producing antibodies that cause rejection.

As a result of autoimmunity or rejection, it causes tissue destruction by inflammatory cells and their transmigrating mediators. Anti-inflammatory factors such as NSAIDs and corticosteroids primarily block the effects and secretions of these mediators and have no effect on the immunological basal alleviation of the disease.

On the other hand, cytotoxic factors such as cyclophosphamide act in a non-specific manner, shutting off both normal and autoimmune responses. Moreover, patients treated with such non-specific immunosuppressive drugs will lose the infection caused by their autoimmune disease.

Cyclosporine consists of Tolypocladium inflatum and cylindrocarpon.
It is a member of immunosuppressive compounds isolated from the fermentation medium of various fungal species, including rutidam (Cylindrocarpon lucidum).

The general structure of cyclosporins has been established as a cyclic peptide containing 11 amino acids.

For example, the established structure of cyclosporin A is a cyclic peptide, containing some methylated amino acids,
At position 8 this amino acid consists of D-alanine, which is believed to be important for the biological activity of cyclosporine.

Bmt = (4R) -4-[(E) -2-butenyl] -4-methyl-L-threonine Me = methyl Abu = α-aminobutyric acid Val = valine Ala = alanine MeLeu = N-methyl leucine MeVal = methyl Valine Sar = sarcosine Cyclosporine, like cyclosporin A, is not cytotoxic or myelotoxic. It also neither inhibits the migration of mononuclear cells nor does it inhibit the function of granulocytes and macrophages. This effect is specific and has no adverse effect on the main established immune response. However, it has renal cytotoxicity and is known to induce the following unwanted side effects.

(1) Liver dysfunction (2) Hirsutism (3) Gingival hypertrophy (4) Tremor (5) Neurotoxin (6) Hyperesthesia and (7) Digestive discomfort.

Therefore, the object of the present invention is to (1) restore the balance between help and suppression mechanisms of the immune system by acting earlier than anti-inflammatory factors, and
(2) It is intended to provide a novel cyclosporin derivative having lower renal cytotoxicity, which induces a specific long-term transplantation tolerance by a suppressor cell circuit without increasing susceptibility to body infection.

Another object of the present invention is to provide a pharmaceutical composition for administering the active immunosuppressive agent of the present invention to a patient in need of treatment.

Still another object of the present invention is to provide a method for controlling rejection phenomenon, autoimmunity and chronic inflammatory disease by administering a sufficient amount of a novel immunosuppressive agent to a mammalian species in need of such treatment. To do.

Finally, the object of the present invention is also to provide a method for the biosynthesis and isolation of this active compound.

A. Scope of the invention The present invention relates to a cyclosporine derivative in which the 8-position is 3-fluoro-alanine. In the formula, R is 3-fluoro-D-alanine or 2-deutero-3-fluoro-D-alanine. The 3-fluoro-alanine derivative exhibits immunosuppressive properties similar to cyclosporin A and also its renal cytotoxicity is lower than that of cyclosporin A.

B. Biosynthetic Methodology The improved cyclosporin in the present invention is prepared according to the following fermentation method.

Example 1. 8- (2-deutero-3-fluoro-D-alanine)
-Preparation of cyclosporin A Culture: Tolypocladium infratum MF5080, NRRL-8
044 Medium: slant medium A g / malt extract 20.0 Yeast extract 4.0 Agar 20.0 Seed Medium B Malt extract 70.0 Glucose 50.0 culture medium C glucose 40.0 Casein peptone _{10.0 MgSO 4 · 7H 2 O 0.5} KH 2 PO 4 2.0 NaNO 3 3.0 KCl 0.5 FeSO ₄・ 7H ₂ O 0.01 Aseptically open the tube containing the freeze-dried bacteria and spin at 27 ° C for 4 days using seed medium B (20 ml in an Erlenmeyer flask for 250 ml with baffles at 3 places). Grow with shaking (220 rpm).

This seed bacterium is inoculated on slope agar (medium A) for more detailed analysis. Incubate in slant medium at 27 ° C for 14 days, then store at 4 ° C until use.

The spores were washed out from the whole slant agar medium with 5 ml of the medium C and taken out to prepare a pre-culture flask (250 ml containing 50 ml of the medium C.
Incubate in an Erlenmeyer flask for l). This preculture is carried out for 5 days at 27 ° C.

5 ml of the pre-culture was added to the production medium (50 ml medium C in an Erlenmeyer flask for 250 ml and 5 mg / ml of 2-
(Including deutero-3-fluoro-D-alanine). Over-sterilized 2-deutero-3-fluoro-D
-Alanine is added after sterilization prior to its culture (final concentration, 5 mg / ml). 27 flasks in total of 2.2 liters of production medium for 14 to 21 days in 44 flasks, 27
Incubate at ℃, stirring (220 rpm). After culturing, it is extracted from the fermentation medium by the method described in item C below.

Example 2 Preparation of 8- (3-Fluoro-alanine) Cyclosporine The pre-culture was supplemented with 5 mg / ml 3-fluoro-alanine instead of 2-deutero-3-fluoro-alanine in a total volume.
A fermentation medium is obtained which is substantially the same as that described in Example 1 except that 400 ml of the production medium is used, and then extraction is performed by the method described in Item C below.

C. Extraction methodology a. Remove cells from medium by centrifugation.

b. Extract the clarified medium 3 times with 25 ml of methylene chloride each.

c. Extract cells 3 times with 25 ml each of acetone.

d. Collect methylene chloride and acetone extract and dry under reduced pressure.

e. Dissolve the residue in methanol, dry over anhydrous Na ₂ SO ₄ , then dry under reduced pressure.

f. Subject the sample to HPLC analysis to determine and isolate derivatives of cyclosporine.

D. HPLC analysis The 8- (2-deutero-3-fluoro-alanine) cyclosporin A crude extract of Example 1 is subjected to HPLC analysis using the following chromatographic system.

Solvent 80/20 v: v Acetonitrile: Water Flow rate 0.6 ml / min Column DuPont Zorbax ODS
4.6mm × 25cm, maintained at 60 ℃ Detector LDS Spectrometer III, 210nm, 0.05AUFS Integrator Spectra Physics SP4100
Computing Integrators The expected concentration of 2-deutero-3-fluoro-D-alanine analog of cyclosporin A with a retention time that is 92% equivalent to that of cyclosporin A is:
It is calculated by dividing the measured area count by the area count of cyclosporin A / mcg obtained from an exogenous standard of cyclosporin A of known concentration.

2.2 From the medium, the extraction residue obtained from 4 fermentation products is collected in 75 ml of methanol and assayed by HPLC chromatography. This sample containing 43.1 mg of crude 8- (2-deutero-3-fluoro-D-alanine) -cyclosporin A is labeled as A. Sample A is concentrated as a slightly oily residue. 6 ml of this residue
1: 1 v: v methylene chloride: soluble in methanol. The solution is loaded onto a 200 ml column consisting of Pharmacia LH-20 which has been equilibrated with methanol in advance, and chromatography is carried out. Chromatography is carried out with methanol at a flow rate of 5 ml / min, and after fractionation of 8 ml each, 40 fractions, 5 ml each are fractionated. Collect fractions 22 to 26 and label as B. The capacity is 25 ml
Is.

Sample B was 33.7 mg 8- (2-deutero-3-fluoro-D-alanine) -cyclosporin-A by HPLC analysis.
Was included.

After concentrating and drying sample B, the residue is dissolved in 1 ml of methanol. This solution was applied to a preparative HPLC using a DuPont Zorbax ODS column 2.1 x 25 cm consisting of 80/20 v: v acetonitrile: water solvent system, flow rate 10 ml / min, 60 ° C.
Chromatograph. The elution trend is followed at a wavelength of 210 nm using a Gilson model 116 U.V. detector set up with a 0.05 mm pathlength cell, 0.32 AUFS. The UV signal was monitored with a Spectra Physics SP4100 Computing Integrator and 15 fractions were obtained based on this UV trace. Fraction 9 was designated as C and fraction 10 was concentrated and dried. The residue was labeled as D.

Dissolve sample D in 0.5 ml of methanol. This solution is mixed with a solvent system of 80/20 v: v acetonitrile: water at a flow rate of 10 m.
DuPont Zorbax ODS column consisting of l / min, 60 ℃
Subject to preparative HPLC chromatography using 2.1 x 25 cm. The elution trend is followed at a wavelength of 226 nm using an LDC Spectromonitor II instrument with a 1 mm pathlength cell, 0.32 AUFS. The UV signal was monitored with a Spectra Physics SP4100 Computing Integrator and 10 fractions were obtained based on this UV trace. Fractions 4 and 5 were removed and mixed with sample C to a volume of 35 ml. This was labeled as E. This sample was 99% UV pure at 226 nm by HPLC analysis.
The above is shown. It contained 20.1 mg of 8- (2-deutero-3-fluoro-D-alanine) -cyclosporin A. Sample E was concentrated and dried under high vacuum to give 20.2mg
8- (2-deutero-3-fluoro-D-alanine) -cyclosporin A was obtained.

One 8- (3-fluoro-D-alanine) -cyclosporin A of Example 2 The residue extracted from 400 ml of fermentation product was dissolved in 1 ml of methylene chloride and this solution was pre-equilibrated with methanol. Place on a 40 ml column made of Pharmacia LH-20 and chromatograph.
Chromatography is carried out with a flow rate of 2 ml / min of methanol, and after fractionation of 10 ml each, 30 fractions of 1 ml each are fractionated. Fractions 16 to 27 are removed based on the results of the HPLC analysis and combined. The mixed fraction was concentrated and dried. This residue was labeled as F.

Sample F was dissolved in 250 ml of methanol and added to DuPont.
Zorbax ODS column 0.94 x 25 cm, preparative HPLC chromatography maintained at 60 ° C. Chromatography is performed using a 80:20 v: v acetonitrile: water solvent system at a flow rate of 2 ml / min. The elution trend is followed at a wavelength of 220 nm using an LDS spectrum monitor II device with a 1 mm path length cell and 1.28 AUFS setting. The UV signal was monitored by a Spectra Physics SP4100 Coupling Integrator and 11 fractions were obtained based on this UV trace. Fraction 7 by HPLC analysis shows a UV purity at 210 nm of> 99%, 3.2
It contained 5 mg of 8- (3-fluoro-D-alanine) -cyclosporin A. Fraction 7 was concentrated and dried under high pressure to give 3.3 mg of 8- (3-fluoro-
D-alanine) -cyclosporin A was acquired.

Physical properties of E.8- (2-deutero-3-fluoro-D-alanine) -cyclosporin A Mass spectrum: 19 mass units more than the numerical value (1202) (M + M) ⁺ , m / 2 1221 is for cyclosporin A It was found that the alanine residue of cyclosporin A is 2
-Deutero-3-fluoro-D-alanine. ¹ H NMR spectrum: ¹ H NMR data is 2-
Determining uptake of deutero-3-fluoro-D-alanine. ¹³ C NMR Spectra: Spectra are recorded on a Varian XL-400 spectrometer at 100 MHz using CDCl ₃ at constant room temperature. The solvent peak at 77.0 ppm was used as a control and the chemical shift was shown as a correlation to TMS at 0 ppm. 10.0,16.1,17.0,18.1,18.5,18.8,20.
0,20.5,21.2,21.8,22.0,23.6 (2x), 23.9 (2x), 2
4.0,24.2,24.5,24.9,25.0,25.6,29.3,29.88,29.94,29.9
6,31.2,31.4,35.8,36.0,36.2,37.5,39.2,39.6,40.6,48.
75 (2 x), 48.83,50.4,55.3,55.45,55.49,57.6,57.9,5
9.0,74.8,81.9d, (J = 177.2Hz) ^* , 126.2,129.5,169.
8,169.9 (2x), 170.9d ^* , 170.1,171.1,171.53,171.5
6,173.4,173.59,173.61ppm. ^* These resonances of the 3-Fluoro-2-deutero-D-alanine residue are measured in duplicate due to coupling with the ¹⁹ F nuclide. The ¹³ C NMR data for 61 carbons show that the molecular structure C was based on the assumption that the α-carbon of the 3-fluoro-2-deutero-D-alanine residue carrying the deuterium atom was not observed. _{It is the same as 62} H ₁₀₉ DN ₁₁ O ₁₂ F.

F. Utility of the present compounds based on the scope of the present invention The present invention includes administration of a compound represented by the structural formula (I) as an active ingredient to patients suffering from rejection phenomenon after transplantation, autoimmune disease or chronic inflammatory disease. The same applies to methods of treatment.

For the treatment of these conditions and diseases, the compound of structural formula (I) is orally administered in a dosage unit formulation which comprises a conventional non-toxic pharmaceutically acceptable carrier, adjuvant and excipient. It is administered topically, parenterally, by inhalation spray, or rectally. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injections or infusion methods.
In addition to treatment of warm-blooded animals such as horses, cattle, sheep, dogs, cats, etc., the compounds of the present invention are effective in treating the human body.

Pharmaceutical compositions containing the active ingredient are in suitable form for oral administration. For example, tablets, troches, candy, aqueous or oily suspensions, easily dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs, etc. Compositions for oral use are in accordance with known techniques for the manufacture of pharmaceutical compositions and in order to provide pharmaceutically refined and palatable preparations, these compositions have sweetening, flavoring, coloring and preservative agents. The composition is prepared by selecting some from the group such as. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients are likewise prepared by known methods. Excipients that can be used include, for example, (1) an inert diluent such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate;
(2) Granule-forming and disintegrating agents such as corn starch or alginic acid, (3) Binders such as starch, gelatin and gum arabic, and (4) magnesium stearate, stearic acid or talc. It is a lubricant forming agent. Although tablets do not require a coating operation, the coating operation may be carried out by known techniques to delay the disintegration of the tablet in the digestive system and the absorption effect, thereby providing a long-lasting effect. For example, glyceryl monostearate or glyceryl distearate is used as the time delay substance. The tablets may also be coated by the techniques described in US Pat. Nos. 4,256,108; 4,106,452; and 4,265,874 which are formed as osmotic therapeutic tablets to control this dissociation.

In some cases, formulations for oral use may take the form of hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent. For example, an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin. It may also take the form of a soft gelatin capsule in which the active ingredient is likewise mixed with water or an oily medium such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are shown below.

(1) Suspension-forming properties such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic.

(2) Dispersing or wetting agent, (a) naturally occurring phospholipid such as lecithin, (b) alkylene oxide condensation reaction product with fatty acid,
For example, polyoxyethylene stearate, (c) an ethylene oxide condensation reaction product with a long-chain aliphatic alcohol, for example, heptadecaethyleneoxycetanol, (d) an ethylene oxide condensation reaction product with a partial ester derived from fatty acid and hexitol oil. , E.g. polyoxyethylene sorbitol monooleate, or (e) ethylene oxide condensation reaction products with partial esters derived from fatty acids and hexitol anhydrides, e.g. polyoxyethylene sorbitan monooleate.

The aqueous suspension may also contain some preservatives. For example, ethyl or n-propyl p-hydroxybenzoate, some colorants, some flavors, and some sweeteners such as sucrose or saccharin.

Oily suspensions are formed by suspending the active ingredient in a vegetable oil. For example, arachis oil, olive oil, sesame oil, coconut oil, or mineral oil such as liquid paraffin. The oily suspensions also contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. A sweetener and a flavoring agent are also included, and it becomes an oral preparation suitable for the mouth. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

Powders or granules which are easy to disperse are suitable for the preparation of aqueous suspensions. They provide the active ingredient as a mixture with dispersing or wetting agents, suspension-forming agents and some preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Excipients as additives, such as sweetening agents, flavoring agents and coloring agents, are also indicated above.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase is formed by a vegetable oil such as olive oil or arachis oil, a mineral oil such as liquid paraffin, or a mixture thereof. Suitable emulsifying agents include (1) naturally occurring gums such as gum arabic and tragacanth, (2) naturally occurring phospholipids such as soybean and lecithin, and (3) sorbitan monoole. An ester or a partial ester derived from a fatty acid and hexitol anhydride such as an ate, (4) a condensation reaction product of the partial ester with ethylene oxide such as polyoxyethylene sorbitan monooleate, and the like.
The emulsion also contains a sweetening agent and a flavoring agent.

Syrups and elixirs are also formed with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations also include demulcents, preservatives and flavoring and coloring agents and the like.

The pharmaceutical composition may take the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated with the appropriate dispersing or wetting agents and suspending agents described above,
It is formed by a known technique. Aseptic injectable preparation means a sterile injectable solution or suspension in a diluent or solvent that is allowed for non-toxic parenteral use such as 1,3-butanediol solution. That is. Suitable excipients and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are also commonly used as solvents or suspension forming media. For this purpose, mild fixed oils such as synthetic mono- or diglycerides have been used. Also, the use of fatty acids, such as olein films, is also found in injectable formulations.

The compounds of (I) are sometimes administered in the form of suppositories for rectal use of the drug. These compositions are prepared by mixing the agent with a suitable non-irritating excipient which is solid at normal temperatures but liquid at rectal temperatures. It causes melting in the rectum and release of the drug. Such materials include cocoa butter and polyethylene glycol.

For topical use, cream, ointment, jelly, solution or suspension containing the immunomodulator is used. The volume of active ingredient in admixture with a carrier material, prepared as a single dose, is very dependent on the side to be treated and on the particular mode of administration. For example, a preparation for oral administration to humans may have a dosage of about 5 to about
It contains a therapeutically sufficient volume of the active ingredient in admixture with suitable and conventional amounts of carrier material which may be varied between 95%.

Specific dose levels for a particular patient will include the activity of the specific compounds available, the patient's age, weight, physical condition, sex,
It depends on various factors such as the content of the meal, the administration time, the administration route, the rate of excretion from the body, the combination with other drugs, and the degree of a specific disease during treatment.

G. Biological evidence supporting the utility of the present compounds within the scope of the present invention The compound of structural formula (I) has immunosuppressive activity and is thus useful in the treatment of various "autoimmune" and chronic inflammation patients. It is known to be effective. The compounds are also effective in terms of graft rejection when undergoing transplant surgery or in blocking rejection of "donor" organs. The following table illustrates and supports the utility of the compounds of this invention.

In this renal cytotoxicity assay in vitro, 8-
(2-deutero-3-fluoro-D-alanine) -cyclosporin A is more potent than cyclosporin A over a relatively limited dose range in which experimental results can be analyzed.
Its toxicity was low.

In Vitro Renal Cytotoxicity Assay Sample 2 was prepared using an in vitro renal cytotoxicity assay that utilizes changes in uptake of ³ H-leucine as a toxicity parameter, using freshly procured proximal convoluted tubules from rabbits as target tissues. -Deutero-3-fluoro-D-alanine was evaluated. The purpose of this assay is to determine the toxicity of test samples to cyclosporin A. Confirmation of previous assays using cephalosporin antibiotics indicates that this assay can accurately predict correlation, intrinsic toxicity potential at the cellular level. The only assumption to be added is that the pharmacokinetic / drug distribution parameters are not substantially different. The utility of this methodology has been further demonstrated by comparison of in vivo and in vitro data for tunicamycin analogs versus control compounds. It is this in vitro assay that provides a prediction of in vivo renal cytotoxicity with at least 90% accuracy.

Capillary suspension and test compound of appropriate concentration are mixed and allowed to react for a total of 23 hours, and in the last 3 hours, 3 H-leucine, which gives a pulse, is added. Leucine uptake was measured as a numerical value for 1 μg protein and the specific activity at each test point is graphed as a percentage of the control specific activity. In this experiment, the amount of drug level of the highest possible volume released in solution (which gives a compound that can be dissolved and equilibrated during the 23 hour reaction) was determined. As shown in the table below, compound 8- (2
-Deutero-3-fluoro-D-alanine) cyclosporin A showed lower toxicity than cyclosporin A at a dose of 30 μg / ml.

Due to the limited data in the study range and the assumption that the pharmacokinetic factors are equivalent, 8-
(2-deutero-3-fluoro-D-alanine) -cyclosporin A is expected to have lower renal cytotoxicity than cyclosporin A.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 38/00 ABL ACJ ACS ADA ADP C12P 21/04 9282-4B // (C12P 21/04 C12R 1 : 645) C07K 99:00 A61K 37/02 ADP ACJ ACS ADA ABL (72) Inventor Robert Ta. The Yogilman USA, 07036 New Jersey, Linden, Academy Terrace 437

Claims (5)

[Claims] 1. A compound having the following structural formula: (In the formula, R is 3-fluoro-D-alanine or 2-
Deutero-3-fluoro-D-alanine) 2. R is 2-deutero-3-fluoro-D-
The compound according to claim 1, which is alanine. 3. A pharmaceutical carrier and the following structural formula (I):
A pharmaceutical composition for preventing, controlling or treating an immune dysregulation or disease, which comprises a therapeutically effective amount of the compound. (In the formula, R is 3-fluoro-D-alanine or 2-
Deutero-3-fluoro-D-alanine) 4. R is 2-deutero-3-fluoro-D-
The composition of claim 3 which is alanine. 5. (a) Incubating trypocladium infratum MF5080 with 3-fluoro-D-alanine or 2-deutero-3-fluoro-D-alanine in a culture medium, and (b) step ( The method for preparing the compound of structural formula (I) according to claim 1, which comprises extracting and isolating the product from the fermentation medium obtained in a).