CN1223588A - Method for treating inflammation - Google Patents

Abstract

A use is provided of a combination of IL-10 and a steroid for the manufacture of a medicament for the treatment or prevention of inflammatory disease, in particular septic shock and inflammatory conditions caused by auto-immune diseases, such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis, and psoriasis. Also provided are pharmaceutical compositions and kits comprising IL-10 plus at least one steroid, e.g., betamethasone or its derivatives.

Description

The method of treatment inflammation

Field of the present invention

The present invention relates to treat inflammation disease or with the disease of inflammation-related, comprise the method for septic shock.

Background of the present invention

One of mechanism of immune system normal regulating self comprises the proteinic generation that is referred to as cytokine.Cytokine mediated panimmunity/inflammatory response.Several cytokines are arranged, as α-Zhong Liuhuaisiyinzi (TNF-α), interleukin-1 (IL-1), gamma interferon (IFN-γ) and interleukin-6 (IL-6) is to be produced by the monocyte/macrophage that stimulates, and with betide infect and tissue injury during relevant with metabolic variation [see as Hart etc., Proc.Natl.Acad.Sci.86:3803 (1989)] of many inflammation, immune, blood.

Septic shock is to produce the example that struvite cytokine is the morbid state of feature.A kind of common fatal diseases that it is normally caused by the Gram-negative bacteremia, although used a large amount of antibiotic and strengthened nursing, sepsis still equally has very high mortality rate [to see as Ziegler etc., New Eng.J.Med.324:429 (1991) with the Gram-negative bacteremia that causes septic shock; Bone etc., New Eng.J.Med.317:653 page or leaf (1987); With Kreger etc., Am.J.Med 68:344 (1980)].It is reported the annual case that the Gram-negative bacteremia that nearly 10-30 ten thousand sepsis cause is arranged approximately, estimate to cause 3 to 100,000 case death [Wolff, New Eng.J.Med.324:486 (1991)].Because the often very fast deterioration of patient status, sepsis need treatment in time.It is the first cause of M ﹠ M among the inpatient.The symptom of septic shock comprise heating or low temperature, tachycardia, rapid breathing, hypotension, the bad or systemic poisoning of periphery perfusion (Ziegler etc., Supra).

The treatment of exploration at present is the antibody that gives facedown tumor necrosis factor (TNF-α), interleukin-1 (IL-1) and lipopolysaccharide (LPS) to septic shock.Unfortunately, in clinical tracking, these treatments are proved disappointing, demonstrate inconsistent or inappreciable result and [see as Gibaldi, Pharmacotherapy (U.S.) 13:302 (1993) and Colletti etc., Crit.Care Nurs.North Am.5:345 (1993)].Therefore, need effectively treat the particularly method of septic shock of inflammation disease.

The present invention can treat or preventible other inflammation disease is the disease that those autoimmune diseasees that show inflammatory symptom cause, as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis, psoriasis or the like.

The present invention's general introduction

The present invention satisfies above-mentioned needs by the method that treatment and prevention inflammation in mammals disease are provided, and described method comprises the effective dose of the compositions that needs the mammalian interleukin of this type of treatment element-10 and at least a steroid.Specifically, the present invention is mammiferous septic shock or autoimmune disease, provide a kind of method as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, uveitis and psoriasis treatment and prevention, this method comprises the effective dose of the combination that needs the mammalian interleukin of this type of treatment element-10 and at least a steroid.The present invention also relates to the purposes of producing in the inflammation in mammals disease therapeuticing medicine that is combined in of interleukin 10 and steroid.Another aspect of the present invention is to comprise the compositions of interleukin 10 and at least a steroid and the Pharmaceutical composition of physiologically acceptable carrier.Of the present invention is the test kit that is used for the treatment of and prevents the inflammation in mammals disease on the one hand again, this test kit comprise with the effective dose of the interleukin 10 of pharmaceutical carrier mixture and with the combination of the blended steroid effective dose of pharmaceutical carrier.

The illustration summary

Behind the figure l explanation mice perfusion spillikin bacillus (C.parvum), recombinant interleukin-1 0 and/or steroid betamethasone phosphate in the body by the influence of the inductive serum interleukin-1 of LPS (lipopolysaccharide).Indicate the double star mark data representation its compare p≤0.05 with PBS/Tween/MSA.

After Fig. 2 illustrates mice perfusion spillikin bacillus, recombinant interleukin-1 0 and/or steroid betamethasone phosphate in the body by the influence of the inductive serum interleukin-6 of LPS.Indicate the double star mark data representation its compare p≤0.01 with PBS/Tween/MSA.

After Fig. 3 illustrates mice perfusion spillikin bacillus, recombinant interleukin-1 0 and/or steroid betamethasone phosphate in the body by the influence of the serum TNF-α of LPS mediation.The data representation that indicates the double star mark is compared with PBS/Tween/MSA, p≤0.01.

After Fig. 4 illustrates mice perfusion spillikin bacillus, recombinant interleukin-1 0 and/or steroid betamethasone phosphate in the body by the influence of the inductive serum I NF-γ of LPS.The data representation that indicates the double star mark is compared with PBS/Tween/MSA, p≤0.01.

Fig. 5 has illustrated another result of experiment, promptly uses betamethasone phosphate and recombinant human interleukin--1's 0 combination (the present invention), single with interleukin-l0 and single with the inhibition meansigma methods of betamethasone phosphate to four kinds of serum cytokines of mice.

The present invention describes in detail

During all references of quoting as proof are attached in full by reference herein.

" interleukin 10 " or " IL-10 " used herein are defined as a kind of protein, this albumen (a) has the biologic activity identical with natural interleukin-10 as plain-10 the aminoacid sequence (as lacking one section secretion targeting sequencing) that is situated between of disclosed eukocyte in No. the 5231012nd, the United States Patent (USP) with (b).Also comprise simultaneously mutain and other albuminoids, comprise the Epstein-Barr virus protein B CRF1 (viral interleukin 10) that remains with the interleukin 10 biologic activity.

The interleukin 10 that is suitable for using among the present invention can obtain from the conditioned medium that can secrete this proteic competent cell, and by the standard method purification.In addition, available standard technique chemosynthesis interleukin 10 as known in the art or its active fragment are (referring to Merrifield, Solid Pllase PeptideSynthesis:A Practical Approach such as Science 233:341 (1986) and Atherton, 1989, I.R.L. publish the Oxford).Also can be) referring to No. the 5231012nd, United States Patent (USP).

Preferred method is that the recombinant technique by the nucleic acid of separated coding interleukin 10 polypeptide obtains described protein or polypeptide.Molecular biological universal method is at the Molecular Cloning as Sambrook etc., A Laboratory Mallual (cold spring port, New York, second edition in 1989) and the Current Protocols in Molecular Biology (Greern/woley of Ausubel etc. (editor), New York, (1987 and supplementary issue)) in all on the books.Utilize standard technique can in genomic library or cDNA library, obtain suitable sequence.Can using polymerase chain reaction (PCR) technology (referring to PCR Protocol:Guide to Methods and Applications, nineteen ninety, Innis etc. (editor), new york academic publishing house, New York).

Gene library constitutes (referring to No. the 5231012nd, United States Patent (USP), it discloses the recombination method of preparation interleukin 10) by extracting nucleotide in suitable cell.Proteinic useful gene order can found as in the various sequence libraries, as GenBank and BMPL or nucleotide and PIR and Swiss-Prot, c/o intelligenetics, Mountain View, Gary Fu Niya, or Genetics Computer Group University Wisconsin BiotechnologyCenter Madison Wisconsin.

The clone strain that contains coding human interleukin-10 sequence is stored in American TypeCulture Collection (ATCC), Rockville, and Maryland is numbered 68191 and 68192.If the application expression vector to other clone's of containing coding IL-10 INTERLEUKIN-10 sequence evaluation, can maybe can adopt the immunology detection of encoding proteins to finish by nucleic acid hybridization.With U.S. Patent number 5231012 disclosed storage sequences serves as that the oligonucleotide probe that the basis prepares is very useful.Sequence oligonucleotide probe also can obtain from the conserved region of the related gene of other kind.Perhaps can adopt with the synthetic degeneracy probe of the aminoacid sequence of IL-10 INTERLEUKIN-10.

Can use standard method to produce prokaryote, mammal, yeast or the insect cell line of conversion, described cell line can be expressed a large amount of polypeptide.The typical coli strain that is suitable for expressing and clone comprise W3110 (ATCC, Bi, 27325), X1776 (ATCC, No. 31244), X2282 and RR1 (ATCC, Mp/31343).Typical mammal cell line comprise COS-7 cell, mouse Lcell and CHP cell (see Sambrook (1989), supra and Ausubel etc., supplementary issue in 1987, supra).

Can be suitable for multiple expression vector and express the DNA of coding IL-10 INTERLEUKIN-10.Can use the conventional carrier of prokaryotic cell or eukaryotic cell lines expression of recombinant proteins.Preferred carrier comprises that the pcD carrier is set forth in: the Mol.Cell.Biol.8:466 (1988) of the Mol.Cell.Biol.3:280 of Okayama etc. (1993) and Takebe etc.Other mammalian expression vector based on SV40 comprises those carriers that (Mol.Cell.Biol.2:1304 (1982)) such as Kaufaman and United States Patent (USP) are announced for No. 4675285, these carriers based on SV40 are specially adapted to the (ATCC of COS-7 MC system, No. 1651, CRL) and other mammalian cell such as mouse Lcell (see the Cloning Vectors:A Laboratory Manual of (supplementary issues in 1989) such as Pouwels, Elsevier, New York).

IL-10 INTERLEUKIN-10 can produce with soluble form, as transforming or the secreting type product of yeast, insecticide or the mammalian cell of transfection, and then can be with these peptides of standard method purification known in the art.For example, purification step comprises: methods such as ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, electrochromatography, affinity chromatograph (are seen Methodsin EnzymologyPurification Principles and Practices, Springer-Verlag, New York, nineteen eighty-two).

In addition, also can produce IL-10 INTERLEUKIN-10, as polymer or inclusion body with insoluble form.The interleukin 10 of this form can be with standard method purification well known in the art.The example of purification step comprises by centrifugal isolates inclusion body from disruptive host cell, use chaotropic reagent (chaotropic agent) and Reducing agent to dissolve these inclusion bodys then, so that the peptide class becomes the configuration with biologic activity like this.The characteristics of these steps are seen Bio/Technology (3:9923 (1985), the Koths etc. and U.S. Patent number 4569790 of Biochemistry (25:4041 (1986)), the Winkler etc. of Winkler etc.

The application standard technology is modified the nucleotide sequence be used for transfection host cell, can prepare IL-10 INTERLEUKIN-10 or it has the fragment of various purpose characteristics.This adorned IL-10 INTERLEUKIN-10 can change the natural sequence that exists on the primary structure level, for example by changing aminoacid, insert fragment, replacing fragment, deletion fragment and fusion.In a series of reorganization, can these method of modifying of applied in any combination to prepare the protein chain of final modification.

The variant that can prepare aminoacid sequence, these purposes according to different purposes comprise improve serum half-life, be convenient to purification or preparation, raising therapeutic efficacy and reduce to treat in the order of severity or the incidence rate of side effect.Although other variant may be the variant after the translation, the normally natural undiscovered predetermined variant of this amino acid sequence variant.As long as this class variant can keep the biologic activity of IL-10 INTERLEUKIN-10 just can be used for the present invention.

The modification of coded polypeptide sequence can be finished (Gillman etc., Gene 8:81 (1987)) as direct site mutation etc. at an easy rate by various technology.Use appropriate method by conventional screening purpose characteristic, can estimate great majority and modify.For example, U.S. Patent number 5231012 has been described and has been applicable to the active method of external test IL-10 INTERLEUKIN-10 in a large number.

Although also can use viral IL-10 INTERLEUKIN-10, more preferably end user's IL-10 INTERLEUKIN-10 in the mankind's treatment.The IL-10 INTERLEUKIN-10 of most preferably using is recombination human interleukin-10.The preparation of human interleukin-10 is seen and is set forth in United States Patent (USP) No. 5231012.Clone and express viral IL-10 INTERLEUKIN-10 (BCRF1 albumen) and be disclosed among the Science (248:1230 (1990)) from Epstein-Barr virus by Moore etc.

All when mentioning interleukin 10, its active fragment, analog and congener include interior.The active fragment of interleukin 10, similar substance and congener comprise the active protein of those interleukin 10s with one or more different characteristics, polypeptide and peptide class.The active example of interleukin 10 comprises the level that suppresses or significantly reduce interleukin II, lymphotoxin, interleukin 3 or GM-CSF.The interleukin 10 activity comprises that also suppressing activatory macrophage produces cytokine, as interleukin-1, interleukin-6 and TNF-α.

Detect the active method of IL-10 INTERLEUKIN-10 and see United States Patent (USP) No. 5231012 with the detection example.This patent also provides to be had the active protein of IL-10 INTERLEUKIN-10 and comprises reorganization and this albuminoid of synthetic technology production.

Be applicable to that steroid of the present invention comprises glucocorticoid.Preferred glucocorticoid have prednisone, dexamethasone, Fluticasone, doubly his half pine and other steroid with and derivant.In the following embodiments, the steroid of use is the betamethasone phosphate that is dissolved in the normal saline buffer solution solution.The betamethasone derivant is the commercial product of the Schering Corp of the western Kenilworth in knob pool.

In the method for the present invention, preferably interleukin 10 and described steroid are combined in the same compositions.Can obtain the combination that interleukin 10 adds steroid by any method of administering drug combinations.Administering drug combinations can be order or simultaneously." administering drug combinations " is often referred in a specified time interval, treatment that the patient accepts is multiple (two kinds or more than).Generally speaking, if give second kind of medicine in the half-life of first kind of medicine, then these two kinds of medicines promptly are considered to drug combination.The present invention further provides the method for prediction inflammation in mammals disease incidence inducement, it is characterized in that the interleukin 10 level is lower than normal value, this method comprises the level with mammal sampling and measuring LI-10.Be lower than the normal value level comprise detection less than amount, detectable level can with known normal interleukin 10 level relatively.In addition, commercial test kit be can use and inflammatory mediator such as interleukin-1, interleukin-6, TNF-α and IFN-g detected.A kind of excessive generation in these inflammatory mediators all can be pointed out the shortage of interleukin 10 quantity.Blood is preferred sample source.This method can be predicted many inflammation diseases such as inflammatory bowel, rheumatoid arthritis or psoriasic inducement.

For preparation contains the Pharmaceutical composition of interleukin 10 and steroid, with interleukin 10 and steroid with pharmaceutically acceptable carrier or be preferably inert excipients and mix.The medicine carrier can be the compatible innocuous substance that any suitable transmission polypeptide is given patient.The preparation method of this class Pharmaceutical composition is known in the art, referring to Remington ' sPharrmaceutical Sciences and American Pharmacopeia: NF (Mack PublishingCompany, Easton, PA, 1984).

As long as the two all exists with the effective dose of treatment, the ratio of interleukin 10, steroid and additive can change within a large range so.For each dosage, the amount of preferred interleukin 10 in the scope of about 0.5 to 15 microgram of per kilogram of body weight, more preferably 2 to 12 milligrams, most preferably 5 to 12 milligrams.

Compositions can be oral or be injected in the body.Being used for oral preparation includes the protection polypeptide and exempts from and be present in the zymolytic chemical compound of gastrointestinal albumen.Normally intramuscular injection of injection, subcutaneous injection, intradermal injection or intravenous injection.In addition, under suitable situation, intra-articular injection or other approach also may be utilized.

When parenteral, said composition can be with the pharmaceutical carrier unit of making injection type (solution, suspension, emulsion).For example, the administration in the aqueous vehicles that contains or do not contain various additives and/or diluent such as water, normal saline or buffer solvent of IL-10 INTERLEUKIN-10 and steroid, the example of suitable solvent comprises normal saline, ringer's solution, glucose solution and Chinese krebs solution.Non-aqueous solvent such as fatty oil and ethyl oleate can be employed.Preferred solvent is glucose/normal saline of 5%.This solvent can contain a small amount of additives that can strengthen isotonicity and chemical stability, as buffer agent and antiseptic.Yet the IL-10 INTERLEUKIN-10 in the said composition is preferably and does not contain polymer or other protein purification form composition substantially.In addition, suspension should be noted,, the suspension that contains polypeptide can be prepared into as zinc suspension liquid.This suspension can be used for subcutaneous injection (SQ) or intramuscular injection (IM).

Terminology used here " treatment effective dose " refers to be enough to improve the dosage of the Sx of inflammation disease.For example, be under the inflammation disease situation, S﹠S comprises the S﹠S of one or more pain, swelling, the rubescent and inflammation that other has been known.

In the septic shock case-specific, S﹠S has hypertension, microthrombus, organ failure, loses owing to vascular permeability increases the blood plasma that causes, and in other known S﹠S of septic shock one or more.

" prevention " of used in addition term inflammation disease (as septic shock) can be defined by following parameters.The individuality of certain condition pretreatment inflammation disease such as septic shock morbidity.The candidate of abdominal operation for example, or any cause enterorrhexis or tear (as rupture of appendix) thus cause microorganism (microflora) in the intestinal to escape to the situation in abdominal cavity.Other example comprises gunshot wound, traffic accident victim of trauma of abdomen or the like is arranged.The individuality of very big inflammation disease morbidity risk rate such as septic shock is arranged for this class, the application interleukin 10 adds a kind of at least steroid before symptom occurs, and will improve these symptoms, prevents the acute attack of all performances of this disease.

The typical mammal of the method treatment among available the present invention comprises Canis familiaris L. and such house pet and the primates of cat, comprises the mankind.Preferred use deutero-interleukin 10 in the kind of the target animal of treatment.According to such as the disease of being treated, the factors such as the order of severity of patient's whole body health situation, medication, approach and dosage and side effect, the effective dose of given patient can have different the variation.Suitable dose is determined with parameter as known in the art by the doctor.Usually, the dosage of beginning little by little increases dosage until reaching that acquisition needs or best effect (generally referring to The Merck Manual, the 269th chapter " Pharmacokinetics and Drug Administration ") then a little less than optimal dose.

The preferable range of interleukin 10 and steroid accumulated dose every day is about 0.5 to 9 milligram of injection steroid, 2.5 to 50 milligrams of oral steroid, and the interleukin 10 of per kilogram of body weight 2 to 15 micrograms.Whether dosage can affect the treatment of expection on time, and can be short-term or long term periodicities.Infusion rate every day of medicine can have different change according to monitoring, cytometry and the curative effect of side reaction (referring to Goodman and Gilman ' the s:The Pharmacological Bases of Therapeutics of (editor) (1990) such as Gilman the 8th edition, Pergamon Press; (1990) Remington ' s Pharmaceutical sciences, the 17th edition, Mack Publishing Co., Easton, Penn; The Pharmaceutical Dosage Forms:Parenteral Medications.Dekker of Avis etc. (editor) (1993), New York; The Pharmaceutical Dosage Forms:Tablets of Lieberman etc. (editor) (1990), Dekker, New York; With (nineteen ninety editor) Pharmaceutical DosageForms:Disperse Systems such as Lieberman, Dekker, New York).

The unit dose that effective therapeutic dose preferably provides in ampoule.In addition, effectively the therapeutic dose glass tube vial that can contain a plurality of dosage provides or can other form provide.Every day, accumulated dose can give by a shot, seriality infusion, perhaps can be divided into several low doses for intravenously administrable or adopt some other approach such as administered intramuscular.Compositions of the present invention can also by implant or the drug delivery system of injection be injected in the patient body (as Urquhart etc., Ann.Rev.Pharmacol.Toxicol.24:199 (1984); The Controlled Release of Pesticides and Pharmaceuticals (Plenum Press, New York, 1981) of Lewis (editor); No. the 3270960th, United States Patent (USP), or the like).

Under suitable situation, multiple medicine can administering drug combinations.For example, with the combination of interleukin 10 and steroid and the medication combined administration of therapeutic activity of one or more other treatment effective doses.

With reference to the following example scope that the present invention may be better understood, these embodiment are not used in and limit particular of the present invention.

Embodiment

Every group of 6 mices (employing intravenous injection) are injected 0.5 milligram of lethal spillikin bacillus of heat to attack in contrast, are attacking last hour for one group, handle with phosphate buffer (PBS) and 0.5%Tween 20 (trade mark or trade name).As further contrast, this group is handled with mice serum albumin (MSA, a kind of albumen placebo) when attacking.Attacking 0.1 milligram of/kilogram betamethasone phosphate treated that is dissolved in the normal saline buffer solution of orally give last hour for second group.The 3rd group of lumbar injection 1 microgram recombinant human interleukin--1 0 (rhu IL-10) processing when attacking.According to the present invention, another group is dissolved in the normal saline buffer solution 0.1 milligram/kilogram of betamethasone phosphate attacking last hour orally give, and lumbar injection 1 microgram rHulI-10 handles when attacking.

Attack after 90 minutes, from the concentration of every mice blood drawing and mensuration TNF-α, interleukin-1 and interleukin-6.Attack after 3 hours, from the blood drawing of every mice and measure its IFN-γ.The results are shown in Fig. 1 to Fig. 4.

Combination that it should be noted that interleukin 10 and steroid in all situations with steroid or interleukin 10, can reduce the quantity of inflammatory factor than single more.And in this experiment, the interleukin 10 of associating and steroid show synergism in the concentration of at least a inflammatory factor TNF-α reduces.

Similar experiment the results are shown in Figure 5.

In sum, think to the invention provides treatment or prevention of inflammation comprises the method for toxic shock, this method than single with steroid or single more effective with interleukin 10.

For obtaining other or in addition more effectively treatment, expection the present invention has elimination or reduces the advantage of the well-known side effect of steroid, and these side effect comprise hepatic injury, renal damage and increase susceptibility to infecting.This advantage is united the addition that provides and/or cooperative effect by interleukin 10 and steroid and is caused using steroid in a small amount or short course of treatment and produce.

Claims (10)

1.IL-10 and steroid is combined in the purposes in the medicine of production for treating or prevention inflammation in mammals disease.

2. the purposes of claim 1, wherein said diseases associated with inflammation is septic shock or autoimmune disease.

3. claim 1 or 2 purposes, wherein said diseases associated with inflammation is a septic shock.

4. each purposes among the claim 1-3, wherein IL-10 is people IL-10.

5. each purposes in the claim 1-4 item, wherein said steroid is a glucocorticoid.

6. each purposes in the claim 1-5 item, wherein said glucocorticoid is the betamethasone or derivatives thereof.

7. each purposes among the claim 1-6, wherein IL-10 dosage every day at 0.5 to 15 microgram/kilogram and steroid dosage every day at 0.5 to 50 milligram.

8. each purposes in the claim 1-7 item is wherein injected with the daily dose of 0.5-9mg and is given described steroid.

9. each purposes in the claim 1-8 item is wherein with the described steroid of daily dose orally give of 2.5-50mg.

10. be used for the treatment of or prevent the test kit of inflammation in mammals disease, it comprise by with the effective dose of the blended IL-10 of pharmaceutical carrier and with the combination of the effective dose of the blended steroid of pharmaceutically acceptable carrier.

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