Use of leflunomide for inhibiting interleukin 1 beta

N-(4-Trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide is an effective compound for preventing and treating disorders in which interleukin 1 beta is involved. It is used as a pharmaceutical.

Leflunomide (see formula, 
N-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide) is already 
known as a chemical compound (EP 0013376, EP 0217206, U.S. Pat. Nos. 
4,351,841, 4,965,276). 
In addition to its antiinflammatory effects, which have already been 
disclosed, this substance also brings about immunomodulatory effects which 
qualify it for use in the treatment of autoimmune diseases and transplant 
rejection reactions. It is also already known that this metabolite with 
the designation N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide 
(see formula) is responsible for the therapeutic effects of leflunomide. 
##STR1## 
In correspondence with this finding, the pharmacological effects of 
leflunomide cited above can also be obtained by administering this said 
metabolite (Bartlett et al., Agents and Actions, 32 (1991) 10-21). 
Axton et al., J. Chem. Soc. Perkin Trans. 1 (1992) 2203 ff. also describe 
how leflunomide does not represent the active principle and that, instead, 
this primary metabolite exhibits the biological effects. 
It has been possible to demonstrate both in the literature (Bartlett et 
al., Agents and Actions, 32 (1991) 10-21) and in our own experiments that 
the therapeutic effects described in more detail below cannot be obtained 
by administering the leflunomide metabolite. Thus, it was found, in 
accordance with the invention, that leflunomide exerts a strong inhibitory 
effect on the synthesis and liberation of cytokines from human blood 
cells, whereas the leflunomide metabolite does not exhibit this 
advantageous effect. 
Under the experimental conditions employed in accordance with the 
invention, no appreciable metabolism of leflunomide takes place, and the 
inhibitory effect is to be ascribed exclusively to the substance 
leflunomide. 
The cytokines are a class of diverse, biologically highly potent, peptides 
whose structures are already known. It is likewise already known that they 
are induced and synthesized endogenously as transmitter substances. 
The suppression of cytokines in the human or animal body is of great 
medical importance since excessive levels of these cytokines can lead to 
the occurrence or outbreak of numerous disorders. 
Such disorders could be treated with a medicament which inhibits the 
undesirable effect of the cytokine, which might already be present, on the 
organ, cell, tissue and receptor systems of the body; however, it is now a 
further, significant advantage of the present invention that the use of 
leflunomide inhibits the actual synthesis and liberation of the cytokine 
so that the latter never even comes into being and the emergence of the 
disorder can thus be prevented at a very early phase. 
The present invention relates to the use of leflunomide for preparing a 
pharmaceutical for preventing and treating disorders of the human and 
animal body in which the cytokine with the designation interleukin 1 beta 
(IL.beta.) is involved. 
The present invention also further relates to the use of leflunomide for 
treating such disorders. 
The invention also relates to pharmaceuticals which contain an effective 
quantity of leflunomide in addition to pharmaceutically suitable and 
physiologically tolerated excipients, diluents and/or other active 
compounds and auxiliary substances. 
The invention also relates to a process for preparing a pharmaceutical for 
preventing and treating disorders in which interleukin 1 beta is involved, 
wherein leflunomide is brought into a suitable preparation form together 
with pharmaceutically suitable and physiologically acceptable excipients 
and, where appropriate, further suitable active compounds, additives or 
auxiliary substances. 
IL1.beta. and its disease-causing effects are described in detail in 
Ibelgaufts, Lexikon Zytokine (Cytokine Dictionary), Medikon Verlag, Munich 
1992, and in the literature cited therein. 
However, the undesirable effects of IL1.beta. are also referred to in WO 
92/02822 and WO 91/155577, for example, as well as in Thornberry et al., 
Nature 356 (1992) 768-774, Cerretti et al., Science 256 (1992) 97-100 and 
Eastgate, Duff et al., Lancet September 24 (1988) 706-709. Tanaka et al., 
An. Letts 21 (1988) 169-181 described the central role of IL1.beta. in 
septic shock, leukemia and hepatitis. 
In addition to this, IL1.beta. has a broad biological spectrum of 
pathogenic activity in association with, for example, muscle breakdown, 
HIV infection and disorders of brain metabolism, such as Alzheimer's 
disease. 
IL1.beta. can be synthesized in many different cells, such as macrophages, 
fibroblasts, endothelial cells and lymphocytes; the synthesis is 
particularly active in peripheral monocytes. It is thus understandable 
that IL1.beta. occupies a central position in particularly serious 
disorders which can currently either not be treated at all or only treated 
inadequately. For this reason too, the effect of leflunomide which has 
been discovered is of great importance. 
The present invention furthermore relates to the use of leflunomide for 
preventing and treating disorders involving increased cartilage 
resorption, meningitis, microbacterial infections, thromboses, 
arteriosclerotic depositions, an elevated fat level or joint destruction 
(Mustafa et al., J. Pediat., 115 (1989) pages 208-213; Kindler et al., 
Cell, 56 (1989) pages 731 ff., Joly et al., Circ. Res., 71 (1992) pages 
331 ff. Ku et al., JBC, 267 (1992) page 14183; Chin et al., Arthritis and 
Rheumatism, 34 (1991) pages 314 ff.). 
Pharmaceutical forms and pharmaceutical preparations of leflunomide, which 
have been prepared in the customary manner, can also, in particular, be 
used for treating these disorders. 
Suitable solid or liquid pharmaceutical preparation forms are, for example, 
granules, powders, coated tablets, tablets, (micro)capsules, 
suppositories, syrups, juices, suspensions, emulsions, drops or injectable 
solutions, as well as preparations having a protracted release of active 
compound, in whose preparation customary adjuvants, such as excipients, 
disintegrants, binding agents, coating agents, swelling agents, glidants, 
lubricants, flavorants, sweeteners or solubilizers are used. Frequently 
used auxiliary substances which may be mentioned are, for example, 
magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, 
talc, milk protein, gelatin, starch, cellulose and its derivatives, animal 
and plant oils, polyethylene glycols, and solvents, such as, for example, 
sterile water and monohydric or polyhydric alcohols, e.g. glycerol. 
In human medicine, dose units of 3 to 5 mg, preferably 10, 25 or 50 rag, 
per patient (70 kg body weight) are administered. If required medically, 
the dose unit can be increased to 100, 200 or 500 mg per patient. Dosing 
can take place once daily to once weekly, preferably up to three or four 
times daily. Administration can be effected orally, peritoneally, 
intravenously, intraarticularly or transdermally in a customary manner. 
The effective doses to be administered within veterinary medicine can also 
be readily calculated from these data. 
Finally, in the preparation of the abovementioned pharmaceutical 
preparation forms, leflunomide can also be formulated together with other 
suitable active compounds, for example antiuricopathic agents, blood 
plateletaggregation inhibitors, analgesics, and other steroidal or 
non-steroidal antiinflammatory agents. 
It was possible to demonstrate the effects of leflunomide experimentally on 
an isolated blood cell fraction (mononuclear cells), which cell fraction 
did not, to any appreciable extent, metabolize the leflunomide to its 
metabolites.

EXAMPLE 1 
The mononuclear cells from freshly isolated human citrate blood were 
enriched in accordance with known standard procedures (see Tiku et al., J. 
Immunol. 136/10 (1986) 3677): 
10 ml of freshly prepared human citrate blood were carefully underlaid with 
15 ml of Lymphoprep.RTM. (Molter GmbH, Heidelberg) and then centrifuged at 
400.times.g for 40 min. at 20.degree. C. The cell fraction which was 
visible as a white ring at the phase boundary was withdrawn with the aid 
of a syringe, diluted 1:1 (v/v) with PM-16 buffer (from Serva 
Feinbiochemica GmbH & Co. KG, Heidelberg) and then centrifuged, as above, 
for 10 min. The supernatant was washed with 10 ml of RPMI 1640 buffer 
(Gibco, Berlin) to which 300 mg/1 L-glutamine had previously been added. 
The washed cell fraction was taken up in 1 ml of RPMI 1640 to which 300 
mg/1 L-glutamine, 25 mmol/1 HEPES (Gibco, Berlin), 0.1 g/ml streptomycin 
and 0.1 g/ml penicillin had previously been added. Using a cell counter 
(type IT, from Coulter Diagnostics, Krefeld), the cell suspension, which 
is composed of about 90% lymphocytes and 10% monocytes, was adjusted to 
about 5 million cells/ml. Cell viability was monitored before and after 
the inhibition experiments using the known lactate dehydrogenase method. 
In this case, no change in viability was observed. 
The synthesis and liberation of cellular IL1.beta. was induced by adding a 
solution of 500 ng of lipopolysaccharide (Salmonella abortus equi, Sigma 
GmbH, Deisenhofen) in 0.01 ml of dimethyl sulfoxide/water (1:10, v/v) to 
0.48 ml of the above-described cell fraction. At the same time, a solution 
of leflunomide or leflunomide metabolite in 0.01 ml of dimethyl sulfoxide 
(for the final concentration in each case, see Tab. 1) was added to the 
cell fraction and the mixture was left at 37.degree. C. for 20 h in a 
commercially available incubator. After cooling down to 0.degree. C., the 
samples were centrifuged for 1 min. in a bench centrifuge and in each case 
0.025 ml aliquots of the supernatant were examined for their IL1.beta. 
content using a "sandwich" enzyme-immuno test kit (from Biermann GmbH, Bad 
Nauheim) in accordance with the manufacturer's instructions. The control 
values were determined without the addition of leflunomide or metabolite 
and set at 100%. In particular, any possible influence of dimethyl 
sulfoxide on the IL1.beta. level was excluded by appropriate comparative 
measurements. 
In addition, aliquots of the test sample containing leflunomide were 
removed in a time-dependent manner and tested for their content of 
leflunomide or leflunomide metabolite using the high pressure liquid 
chromatography (C-18 column 3.9.times.150 mm, Waters GmbH, Eschborn, 
eluent: 600 ml of methanol/350 ml of water/50 ml of tetrahydrofuran/1 ml 
of phosphoric acid; flow rate 0.7 ml/min. at 2000 pounds per square inch 
(psi); detection in the ultraviolet range at 273 nm). It was found that, 
under the conditions employed, leflunomide is metabolized only very 
slowly, with a half life of about 10 hours. 
TABLE 1 
______________________________________ 
ILl.beta. in the 
Substance 
Concentration 
supernatant 
under in experiments 
% +/- standard 
Number 
examination 
mmol/l deviation n = 
______________________________________ 
Leflunomide 
0.1 17 +/- 4 6 
0.05 27 +/- 6 3 
0.01 70 +/- 8 6 
0.005 68 2 
0.0001 86 +/- 11 4 
Leflunomide 
0.1 99 +/- 12 3 
metabolite 
0.01 100 +/- 3 4 
Without 0 100 
either 
______________________________________ 
The abovementioned experiments demonstrate that the leflunomide metabolite 
has practically no effect on the IL1.beta. level, whereas the IL1.beta. 
level is clearly lowered following the addition of leflunomide. 
EXAMPLE 2 
Preparation of N-(4-trifluoromethylphenyl)-5-methylisoxa-zole-4-carboxamide 
##STR2## 
A solution of 0.05 mol of 4-methylisoxazole-4-carbonyl chloride (7.3 g) in 
20 ml of acetonitrile is added dropwise, at room temperature, to a 
solution of 0.1 mol of 4-trifluoromethylaniline (16.1 g) in 150 ml of 
acetonitrile. After stirring for 20 minutes, the precipitated 
4-trifluoromethylaniline hydrochloride is filtered off with suction and 
washed twice with 20 ml of acetonitrile on each occasion, and the combined 
filtrates are concentrated under reduced pressure. Yield: 12.8 g of white, 
crystalline N-(4-trifluoromethylphenyl)-5-methylisoxa-zole-4-catboxamide 
(leflunomide). 
EXAMPLE 3 
Acute toxicity following intraperitoneal administration 
The acute toxicity following intraperitoneal administration of the test 
substance was carried out using NMRI mice (20 to 25 g) and SD rats (120 to 
195 g). The test substance was suspended in a 1% strength solution of 
sodium carboxymethyl cellulose. The different doses of the test substance 
were administered to the mice in a volume of 10 ml/kg of body weight and 
to the rats in a volume of 5 ml/kg of body weight. 10 animals were used 
per dose. After 3 weeks, the acute toxicity was determined by the method 
of Litchfield and Wilcoxon. The results are summarized in Table 2. 
TABLE 2 
______________________________________ 
Leflunomide acute toxicity 
intraperitoneally LD.sub.50 (mg/kg) 
______________________________________ 
NMRI mouse 185 (163-210) 
SD rat 170 (153-189) 
______________________________________