CN1286086A - Application of leflunomide - Google Patents

Abstract

The leflunomide which is a new immunodepressant and immunoregulator has a new application in treating lapus erythematosus caused by failure in immune system.

Description

A kind of new indication of leflunomide

The present invention relates to immunosuppressant and regulator, more particularly immunosuppressant and regulator leflunomide (Lef) are used in treatment lupus erythematosus medicine.

Because also There are many different versions of a story for the present medical circle of the cause of disease of lupus erythematosus (SLE), think that according to clinical great mass of data the immunology viewpoint explains that the pathogeny of lupus erythematosus is more reasonable, immune disease is because immunodeficiency, not normal and a series of diseases that cause of immunologic function and immunomodulating, the generation of these diseases is all relevant with immunoreation with development, lupus erythematosus is a kind of immune disease, can adopt immunosuppressant, strengthen, regulate, replenish or method such as reconstruction, make the immune state trend normal, impel the state of an illness to alleviate, take a turn for the better, cure or reduce the therapy of recurrence, traditional Therapeutic Method directly acts on pathogen or focus with medicine usually, for example antibiotics directly suppresses or kill bacteria, Chinese traditional treatment is mainly dissimilar at what fall ill from theory of Chinese medical science, the example excessive heat type, yang deficiency of spleen and stomache, liver-kidney yin deficiency, according to dissimilar pin is put in poison again, though medical circle personage has made big quantity research the treatment of lupus erythematosus also there are a lot of medicines and various Therapeutic Method, but do not find a kind of therapeutic effect good yet to this disease up till now, the ideal treatment method that side effect is little.

The object of the invention is sought out a kind of novel immunosuppressant and regulator, utilizes its immunosuppressant and regulating action to use being caused in (system) lupus erythematosus disease medicament by the immune system problem.

Leflunomide is a kind of novel immunosuppressant and immunomodulator, and its chemical constitution is as follows:

Molecular formula: C ₁₂H ₉F ₃N ₂O ₂

Chemical name is the different Evil of α α α three fluoro-5-first bases-N-acyl group-para-totuidine, and it is the white film garment piece.

In intestinal wall and liver, be converted into active metabolite after the leflunomide oral absorption rapidly, it brings into play all main pharmacological actions in vivo, the concentration of leflunomide in blood plasma is very low, in initial leflunomide pharmacokinetic, and the plasma concentration of main detection of active metabolite.Its peak time is at 0.558 ± 0.506 day, 8.79 ± 0.77 days half-life.(20mg, pd) continuous 30 days, its active metabolite blood drug level was near stable state for multiple dose administration.According to reported in literature, in clinical practice, use three days (100mg/ days) loading doses can reach Css fast.The bioavailability 80% that leflunomide is oral, high fat diet can not produce big influence to the active metabolite plasma concentration.Active metabolite distributed density in liver, kidney and skin histology is higher, and content is low in the cerebral tissue.Cf is lower, and extensive and plasma protein combination (99.3%), and conjugated protein amount is linear when treatment concentration.Leflunomide is metabolised to main active metabolite and many micro-metabolite after absorbing, and has only 5-trifluoromethylaniline (TFMA) energy detected in these micro-metabolite, seldom measures parent compound in blood plasma.Do not know the metabolic definite internal organs of leflunomide at present, but in vivo with in vitro tests in, find that liver and gastrointestinal wall have metabolism.The required enzyme of the initial metabolism of leflunomide is also unclear, but the microsome of cytoplasm of liver and cell to be confirmed as be the position of drug metabolism.Active metabolite is drained and is directly drained from liver juice from the kidney liver.43% drains from urine, and 48% drains from feces, and sample analysis shows that initial metabolite in urine is the phenyloxamic acid derivant of glucosiduronate and active metabolite, and the major metabolite of feces is an active metabolite.In two metabolic pathways, initial 96 hours mainly is renal excretion, and later defecate is occupied an leading position.Reported in literature, in the intravenously administrable research, the about 31ml/h of clearance rate.Active carbon or cholestyramine can promote drug metabolism, and the half-life that makes cylinder metabolism-ure, enterohepatic circulation was to cause long principal element of active metabolite half-life from reduce to about 1 day greater than 1 week.Hemodialysis and peritoneal dialysis studies show that active metabolite can not dialyse.

The pharmacological action of leflunomide

Leflunomide is brought into play its immunoregulation effect in vivo by active metabolite, and mechanism of action comprises following several respects:

Suppress kinase whose activity of butyric acid and cell adhesion: leflunomide can suppress the kinase whose activity of butyric acid, and the butyric acid kinases is the key enzyme of the information conduction in cell generation and the division.In addition, leflunomide can obviously suppress activated leukocyte adhesion to blood vessel endothelium, slows down inflammatory cell and enters the joint, and its mechanism of action still imperfectly understands, may be relevant with the information conduction that influences adhesion molecule and cytokine.Therefore, leflunomide can reverse the rejection that has taken place and encircle spore enzyme element and do not have this effect, and this point confirms in animal experiment.

The de novo synthesis that suppresses pyrimidine: leflunomide is by suppressing the activity of dihydroorate dehydrogenase (DHODH), the de novo synthesis of blocking-up pyrimidine.Leflunomide depends on that to different types of cell inhibiting effect this cell utilizes the ability of the synthetic pyrimidine of salvage pathway, rely on the cell (cell of active proliferation of de novo synthesis, as activated lymphocytes, bone marrow structure, hepatocyte, epithelial cell etc.) be the main target cell of leflunomide effect.The activity of dihydroorate dehydrogenase is suppressed by active metabolite, prevent the excessive generation of pyrimidine, a kind of factor (P53) that the tumor suppressor protein cell is translated in transcribing increases, activatory P53 scalable P21WAF-1 transcribes, suppress the activity of cell cycle dependent protein enzyme (cdk (cyclin-dependent-kinase)), prevent that the high phosphorylation of retinoblastoma protein (Rb (retinoblastoma protein)) from closing inactivation.Rb is the negativity regulator of transcription factor E2F, and E2F is the necessary factor of S phase expression of specific gene, thereby influences the synthetic of DNA and RNA, makes activatory cell dormancy G1S intersection resting stage or S phase presynthetic phase of the DNA of cell cycle.The inhibitory action of active metabolite pair cell is reversible, can not cause apoptosis.After inhibitory action was removed, the cell of dormancy can recover normally, and this inhibitory action can be reversed by ectogenic pyrimidine.

Suppress the generation of antibody (immunoglobulin), leflunomide suppresses the synthetic of pyrimidine, can block activatory B cell proliferation, thereby reduces the generation of antibody; In addition, leflunomide also can directly suppress the secretion of antibody.

Leflunomide can suppress nonspecific immunity, humoral immunization, cellular immunization, lymphokine secretion, the regeneration of inhibition lymphocyte, local connective tissue proliferation, local inflammation and arthritis general reaction.The active metabolite of leflunomide has obvious inhibitory action to the generation of rat pleuritis transudate emiocytosis leukotriene B4, non-specific (the inductive mouse boosting cell of concanavalin A, Con A (Con-A)) t helper cell (Th cell) is had obvious inhibitory action, and mice hot plate method and the test of radiation tail-flick method show the obvious anti-inflammatory and anti analgesic activity.Utilize and suffer from congenital systemic lupus erythematosus (sle) (SLE) MRL/lpr mouse model, leflunomide can suppress the progress of SLE, prevents brightic generation, reduces the level of anti-bispin dna antibody and rheumatoid factor (RF).Leflunomide energy prophylactic treatment SLE and concurrent glomerulonephritis are observed after the drug withdrawal, and SLE and glomerulonephritis all do not have recurrence.Leflunomide effectively suppresses the acute and chronic rejection of the multiple organs of animal such as rat, Canis familiaris L., monkey in the organ transplantation test, and has overcome the xenotransplantation rejection, has successfully prolonged hamster to the rat implantation heart time-to-live.

The toxicological effect of leflunomide

Leflunomide mice single is irritated stomach or intraperitoneal injection, observes to record LD50 in 14 days and be respectively 258.9mg/kg and 166.9mg/kg.6 months oral leflunomides of SD rat (1.8,3.5,6.6mg/kg) long term toxicity test shows that high dose group has anorexia, phenomenon loses weight, female rats alanine aminotransferase (ALT) and aspartic transaminase (AST) have the phenomenon of increasing, and 7 (23%) die from infection in the administration phase in the high dose group.Low, middle dosage group does not have animal dead.Use transplantation model, oral high dose leflunomide 20m/kg and 30mg/kg treatment rat 112 days, show its target organ for regeneration active organ as bone marrow, spleen, mucous membrane of small intestine, and immune organ coincide with foreign data as thymus and spleen.Liver function injury and bone marrow depression appear in the Lewis rat.6 months oral leflunomides of Beagle Canis familiaris L. (2,10,16,20mg/kg) long term toxicity test shows, only high dose group slightly descends the erythroid cells sum and hematocrit reduces.The microorganism reverse mutation test of leflunomide, mammal cultured cell chromosomal aberration test, and rodent micronucleus test result does not all have mutagenic action.Leflunomide has teratogenesis in middle and high dosage 10,20mg/kg.

Embodiment 1

Leflunomide is to the inhibitory action of the spontaneous system lupus erythematosus of MRL mice

Adopt MRL/lpr mice (providing) body weight 25-30g/ only by U.S.'s Jackson laboratory, Mus 10-20 in age week each treated animal number of female Mus is 8, be suspended in 1% carboxymethyl cellulose for content greater than 99% leflunomide particulate powders, dosage is 20,35,65mk/kg; Ciclosporin 50mg/kg group, 63 days administration cycles, administration is gathered blood system from serum 0.3ml after 63 days, detect anti-double-chain DNA (ds DNA) antibody then, can control non-T cell dependent form B cell function effectively from detected data declaration leflunomide, reduce the spontaneous high-level anti-ds dna antibody that produces of MRL/lpr mice and controlled the deposition of immune complex in glomerule that IgG mediates effectively, the further effective anti-B cell function of checking leflunomide effect, therefore leflunomide has more slow Jie's effect to the spontaneous systemic lupus erythematosus (sle) of MRL/lpr mice, the generation of antagonism ds dna antibody has stronger inhibitory action, concurrent lymphadenectasis can slowly be situated between, can obviously alleviate brightic pathological manifestations, reduce the deposition of lgG immune complex in glomerule.

Use the integrated enzyme reaction method and detect anti-dsDNA antibody, acyl connection reaction method flat board with the negative contrast of 10 μ g/ml poly glutaminols.Cultivated 20 hours in 4 ℃ with calf thymus ds DNA 10 μ g/ml.Detect serum and begin from 1: 1000 dilution factor, double dilution is inserted in the integrated enzyme reaction method flat board and is cultivated, and bonded antibody detects through integrated enzyme reaction method detector to be associated with oxidasic anti-mice lgG of Radix Cochleariae officinalis or lgM, sees Table 1A table 1B.

Table 1A, MRL/lpr mice ds DNA lgM level

Dilution factor	Matched group	????Lef20	????Lef35	????Lef65	????CsA50
						?1000	1.263±0.167	?0.927±0.153	?0.556±0.211 *	0．319±0.057 *	1.297±0.186
?2000	0.874±0.062	?0.661±0.130	?0．342±0.125 *	0.180±0.023 *	0.944±0.152
						?4000	0.553±0.053	?0.407±0.101	?0.215±0.070 *	0.108±0.022 *	0.641±0.119
?8000	0.287±0.034	?0.200±0.059	?0.115±0.035 *	0.050±0.010 *	0.345±0.063
						?16000	0.137±0.013	?0.087±0.027 *	0.046±0.017 *	0.015±0.006 *	0.180±0.038
?32000	0.071±0.011	?0.040±0.008 *	0.019±0.011 *	0.003±0.005 *	0.088±0.017
						?64000	0.065±0.007	?0.027±0.004 *	0.014±0.008 *	0.015±0.007 *	0.053±0.009

^*Compare with matched group, there were significant differences.

Table 1B, MRL/lpr mice dsDNA lgG level

Dilution factor	Matched group	????Lef20	????Lef35	????Lef65	????CsA50
						?1000	1.579±0.109	?1.279±0.126	1．080±0.184 *	0.997±0.095 *????	1.809±0.072
?2000	0.978±0.093	?0.767±0.099	0.666±0.093 *	0.607±0.045 *	1.281±0.138
						?4000	0.543±0.050	?0.459±0.049	0.354±0.048 *	0.312±0.023 *	0.831±0.114
?8000	0.318±0.048	?0.220±0.025	0.166±0.024 *	0.175±0.015 *	0.459±0.087
						?16000	0.186±0.038	?0.130±0.044 *	0.075±0.009 *	0.107±0.020 *	0.257±0.042
?32000	0.106±0.013	?0.076±0.038 *	0.027±0.004 *	0.055±0.018 *	0.154±0.031
						?64000	0.096±0.009	?0.055±0.028 *	0.039±0.005 *	0.054±0.014 *	0.101±0.012

^*Compare with matched group, there were significant differences.

After the administration 63 days, animal is put to death down in anesthesia, get all direct-views down findable lymph node go out all visible a large amount of enlarged lymph nodes of weight, not treatment group and ciclosporin in treating group with the electronic scale scale, be distributed in that back, cervical region and intraperitoneal, lymph node gross weight relatively see Table 2 tables 2, leflunomide suppresses MRL/lpr mouse lymph knot proliferative effect

The treatment group	Lymph node weight (gram)
		Not treatment contrast	????2.55±0.18
????Lef20	????1.90±0.41 *
		????Lef35	????0.60±0.13 *
????Lef65	????0.35±0.15 *
		????CsA50	????2.12±0.39

^*Compare P＜0.05 with matched group.

Pathological examination results:

Leflunomide 35 and 65mg/kg dosage obviously reverse the inflammation infringement of the glomerule of getting involved to the obvious quantity that reduces the glomerule of getting involved in 9 week of MRL/lpr mice treatment back.Deposition in the LgG immune complex glomerule obviously alleviates, and ciclosporin 50mg/kg and leflunomide 20mg/kg fail obviously to slow down infringement of glomerule inflammation and 1gG deposition.The no significant change of lgM deposition in each group.

Lef (35mg/kg/ days) is to effect, histology and the immunohistochemistry of MRL/lpr mice autoimmunity kidney disease.

A) not treatment group shows proliferative glomerulonephritis: glomerule increases, high cellularity (big amount lymphocyte, neutrophilic infiltration are arranged)

B) CsA (50mg/kg/ days) treatment does not obviously alleviate the nephritis pathological changes;

C) most glomerule histologys are normal for group in Lef treatment (obviously reduce the numbers of glomeruli of getting involved, obviously reverse the glomerule infringement of getting involved);

D) not treatment group: significant lgG is deposited on the glomerule of getting involved;

Claims (1)

1, a kind of new indication of leflunomide is characterized in that using in treatment lupus erythematosus medicine.