CN1225016A - Interleukin-8 as an antiviral and antitumor agent - Google Patents
Interleukin-8 as an antiviral and antitumor agent Download PDFInfo
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- CN1225016A CN1225016A CN 97196176 CN97196176A CN1225016A CN 1225016 A CN1225016 A CN 1225016A CN 97196176 CN97196176 CN 97196176 CN 97196176 A CN97196176 A CN 97196176A CN 1225016 A CN1225016 A CN 1225016A
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Abstract
The present invention relates to the use of an Interleukin-8 (IL-8) agent for the treatment of viral infection, caused by human and animal viruses, and cancer caused by oncoviruses. The IL-8 agent may be an IL-8 protein or an analog thereof having an IL-8 biological activity. The viral infection may be of a DNA virus selected from the group consisting of parvoviridae, papovaviridae, adenoviridae, herpes-viridae, poxviridae and hepadnaviridae, or of a RNA virus selected from the group consisting of retroviridae excluding HIV-1 and HIV-2, picornaviridae, togaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, reoviridae, oncornaviridae and filoviridae.
Description
Background of invention
A) invention field
The present invention relates to the antiviral of interleukin 8 (IL-8) and anti-tumor activity and the application in the treatment of cancer that viral infection that the human and animal virus causes and oncorna virus cause thereof.
B) description of the Prior Art
Some make the mankind suffer painful severe infections disease to be caused that by some fatal virus the plague, variola, poliomyelitis, hepatitis, yellow fever, immunodeficiency and various encephalitis are wherein arranged.Some diseases is arranged, and for example influenza, measles, mumps, chickenpox and respiratory tract-functional gastrointestinal disorder are celebrated with its hyperinfection and acute malaise symptoms.For example rubella and cytomegalovirus can cause congenital anomaly to also have some.Also have some viruses at last, be called oncorna virus, can cause humans and animals tumor and cancer.
In these viruses, herpetoviridae family is the most noticeable.Herpesvirus is propagated and to people's tool height pathogenicity at occurring in nature tool height.For example, Epstein-Barr virus (EBV) is considered to cause children or adolescence or adolescence's infectious monocytosis.The acute infection monocytosis be masked as separating of laryngopharynx swelling and pain, fever, headache, lymphadenopathy, antiadoncus and atypical peripheral blood lymphocyte.Other performance often comprises slight hepatitis, splenomegaly and encephalitis.EBV is also relevant with the cancer of two kinds of forms usually: Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC).Equator African Realm, BL are modal children malignant tumors, account for 80% of child's cancer.NPC is one of modal cancer in SOUTHERN CHINA 25 in 55 years old people, and sickness rate is very general among the Caucasian of North America.As cytomegalovirus, EBV is also relevant with transplanting back lymphadenosis disease, and this disease is a potential fatal chronic immunosuppressant syndrome after solid organs or bone marrow transplantation.
Another kind of herpesvirus is called I herpes simplex virus type (HSV-1), may be the pathogen of gingivostomatitis.Infection shows as fever, laryngopharynx swelling and pain and the damage of oral ulcer sex vesicle shape.The most serious clinical disease that HSV causes is the primary genitalia herpes viral infection.HSV-1 can cause that genital herpes virus infects, and HSV-2 is the main virus relevant with this virus.HSV infects and is accompanied by blister, pustule and the ulcer that makes the genitals damage.Also the urine retention syndrome may take place.People above 80% is HSV-1 and HSV-2 seropositivity, and the probability that studies show that recurrence or viral reactivation is up to 60%.Other disease is also relevant with HSV, comprises skin and eye infection, for example chorioretinitis or keratoconjunctivitis.About 300000 of the case history that the U.S. annual diagnosis eye HSV infects.
Herpes virus hominis 6 (HHV-6) has remarkable preferendum to immune system cell, so the HHV-6 infection may cause immunoreation to change.Known that now HHV-6 is the cause that child's constitutional exanthema infects.Studies show that recently the activatory advantage ratio of seropositive organ transplantation receptor virus provides the immunosuppressant serological evidence of challenge virus reactivate afterwards before transplanting.Having a liking for different in nature negative monocytosis sample disease also infects relevant with activation HHV-6 with non-a non-b hepatitis.HHV-6 is everlasting and isolates in human immunodeficiency virus (HIV-1) infected patient.The fact that HIV and HHV-6 can be retained in the same target cell draws such inference, and both the HHV-6 infection may be the cofactor that HIV seropositivity patient excessively arrives the AIDS syndrome.Research recently shows that also herpes virus hominis and HIV disease are closely related.In fact, Kaposi sarcoma (KS), a kind of tumor that is mainly in the HIV infection population finds to have infection originality.This virus is named as the relevant herpesvirus of KS, the similar HHV-8 of its Official Classification.
In all infectious disease, therapeutic efficacy often depends on host's immunity reflection.Especially true for herpesvirus, in fact, the high incidence rate that the latent infection ability of all herpesviruss causes immunocompromise patient reactivate to infect.To the renal transplantation receptor, 40% to 70% the HSV infection meeting of hiding activates again, and 80% to 100% cmv infection activates again.HIV positive patients (AIDS) is also observed the reactivate phenomenon of this virus.
Today, the medicine that is used for viral infection is relatively limited.For example, be used for the treatment of herpesvirus infection and mainly contain four kinds of chemical compounds: idoxuridine, vidarabine, acyclovir and ganciclovir.Their usefulness is limited and can produce some side effect.Once reported that idoxuridine was used for the treatment of a patient 35% of HSV infection anaphylaxis is arranged.The modal side effect of vidarabine is functional gastrointestinal disorder (15% patient).The major side effects of acyclovir is the change of renal function, because acyclovir is a kind of nucleoside analog, thus can insert the DNA of virus and host cell, thus the proper splitting of host cell influenced.The most important side effect of ganciclovir is that neutrophilic granulocytopenia and thrombocytopenia take place for about 40% patient AIDS.
Therefore, press for effective Therapeutic Method of research treatment viral infection.
The diseased tissue leukocyte accumulation is thought the sign of inflammatory process.The local chemoattracting cytoking that produces can induce inflammation district leukocyte to recover normal.The protein that shows this character is divided into two subtribes according to the position of preceding two cysteine, perhaps by an aminoacid separately (CXC albumen) or connecting-type (CC albumen).The member of these two subtribes distinguishes summaries such as (, immunology progress, 55:97,1994) Baggiolini according to its chromosome position to the selectivity of target cell and its gene.In these chemoattracting cytokings, interleukin 8 (IL-8) belongs to CXC family, separates at first to obtain from the human blood single cell culture thing supernatant of being excited.IL-8 is a kind of non-glycosylated protein, and it at first is synthesized 99 amino acid whose precursors, is secreted after cutting off one section 20 amino acid whose sequence.The ripe molecule that forms has 79 residues, further in the N-terminal hydrolysis, produces several bioactive N-terminal analog that have, and main two kinds of forms are 72 amino acid patterns and 77 amino acid patterns.The structure of IL-8, sequence and biological property are by summaries such as Baggiolini M. (immunology progress, 55:97,1994).
Many cells can produce IL-8, for example keratinocyte, epithelial cell, synovial cell and hepatocyte.In the peripheral blood leucocyte, find that the main cell source of IL-8 is mononuclear cell and neutrophilic granulocyte rather than lymphocyte.
IL-8 is external in vivo all to produce many biological activitys.The known activity of IL-8 is the threshing ability of its chemotaxis and human neutrophil.IL-8 handles activation and the cytosol Free Ca that neutrophilic granulocyte can observe alteration of form, active system immediately
2+Increase.It is conjugated protein to observe specific granule release Vitamin B12 simultaneously.IL-8 can also cause the release of azurophil granule threshing and elastoser and other hydrolytic enzyme.This threshing is accompanied by the rise of a large amount of adhesion molecules of cell membrane.Threshing also causes the enhancing of I type complement receptors (CR1) and III type complement receptors (CR3) to be expressed.IL-8 also is the especially chemotactic factor of T cell of acidophil and human lymphocyte
The biological action of IL-8 strides diaphragm area by 7 kinds and the G protein binding is receptor-mediated.Describe two kinds of forms of IL-8 receptor, and be defined as A type and Type B.The A receptor of IL-8 has the height affinity to IL-8, but lower to Gro α (having melanoma growth-stimulating activity) affinity, and Type B all has the height affinity to two kinds of cytokines.In the peripheral blood leucocyte, this amphitypy of neutrophilic granulocyte great expression IL-8 receptor, mononuclear cell and CD8
+T lymphocyte expression IL-8 acceptor levels is lower.B cell and CD4
+The T lymphocyte does not detect the IL-8 receptor expression.
Constantly the evidence of accumulation supports that IL-8 plays an important role in many pathology inflammatory process, in fact, IL-8 detects in inflammation tissue and exudate, for example, the Pleural fluid of psoriatic scale extract, rheumatoid arthritis or gout patient's synovial membrane liquid, emphysematic patients and the bronchoalveolar wash of respiratory distress syndrome.And recently find also that the RANTES of C-C chemotactic factor subtribe and MIP-1 α and β have antiviral character, find that they can suppress the external synthetic of HIV-1, HIV-2 and SIV virus protein.
Further reported the antivirus action (Mackewicz and Levy, AIDS research and human reverse transcript virus, 8:1039-1050,1992) of IL-8 recently.Show that IL-8 can suppress HIV and duplicate in that CD4+ is lymphocytic under special condition.In fact, find that IL-8 can influence the virus replication of the CD4+ cell of natural infection, and can not influence the virus replication of actute infection cell.
The antiviral activity mechanism that has several supposition, some of them are perhaps to blocking special virus to having the selectivity of height adhering to of its target cell.Idoxuridine, vidarabine, Ah former times Wei Luohe more former times Wei Luo specificity are used for the treatment of herpesvirus infection, to for example not effect of HIV of other virus, have formed the example of selective antivirus active medicine thus.
The data of the anti-HIV effect of IL-8 that provides according to Mackowicz and Levy can not infer that IL-8 has the activity of anti-all viruses.The great majority treatment medicine for treating viral infections of using is at present very supported this conclusion to the selectivity of special virus.So far, the compellent report that does not have other IL-8 antiviral activity.
At last, the PCT application PCT/US95/12099 of TALMADGE, on March 28th, 1996 published, publication number is WO96/09062, the polypeptide analog of IL-8 has been described, and claim that this analog can be used for the treatment of some pathogenicity diseases, for example viral infection, bacterial infection, fungal infection, yeast, host parasitize.Yet TALMADGE does not provide any data proving this conclusion, and it is based on that this polypeptide has the deduction of these effects or only is hope.
Therefore, press for and develop a kind of antiviral drugs, it has effect preferably and can not show the adverse side effect of known antiviral agents.
Summary of the invention
The invention provides the method for cancer that prevention and treatment viral infection and oncorna virus cause.
According to these methods, take the IL-8 preparation of materia medica and the acceptable treatment effective dose of physiology for the human or animal who needs this treatment.
The more effective ways that an object of the present invention is to provide a kind of antiviral drugs and be used to prevent and treat viral infection, and this method can not produce the adverse side effect of known antiviral drugs.
Another object of the present invention provides a kind of antiviral drugs and is used for prevention and the treatment oncorna virus method for cancer that causes of retrovirus (except HIV-1 and HIV-2), human papillomavirus, adenovirus and herpesvirus for example.
A further object of the present invention provides a kind of antiviral drugs and is used for the prevention of immunosuppressive disease human and animal virus infection and the method for treatment.
The invention provides the IL-8 medicine as the anti-herpesvirus of antiviral agents including, but not limited to EBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7 and HHV-8, and prevention and treat the method for the infection that these herpesviruss cause.
The present invention also provides the application of IL-8 medicine as anti-other human and animal virus's medicine, these viruses including, but not limited to: belong to the pig enterovirus of Picornaviridae or belong to the bovine diarrhea virus of Togaviridae or belong to the bovine respiratory syncytial virus of Paramyxoviridae.
The present invention also provides the application of IL-8 medicine as antiviral drugs, this application is used for and other antiviral drugs, unites use treatment human and animal virus infection including, but not limited to interferon-' alpha ', interferon-beta, interferon-, tumor necrosis factor, ganciclovir, acyclovir, cytosine arabinoside, idoxuridine and prostaglandin or prostaglandin analogue.
The present invention also provides IL-8 to be used for for example application of the cancer that causes of retrovirus (except HIV-1 and HIV-2), human papillomavirus, adenovirus and herpesvirus of prevention and treatment oncorna virus as antiviral drugs.
The present invention also provide IL-8 as antiviral drugs in the treatment for cancer that anti-oncorna virus causes with other cancer therapy drug, including, but not limited to the use of uniting of AC and methotrexate.
The present invention also provides IL-8 to be used to prevent and treat the application of the viral infection of immunosuppressive disease humans and animals as antiviral drugs.
The immunosuppressant patient comprises organ or tissue's transplant patient and the patient who treats including, but not limited to azathioprine, corticosteroid, AC and methotrexate with immunosuppressant.The immunosuppressant patient also comprise any type of cancer and tumor with or without the patient of anticancer chemotherapy medicine including, but not limited to AC and methotrexate treatment.The immunosuppressant patient also comprises with the inflammation patient of anti-inflammatory drug including, but not limited to corticosteroid, methotrexate, azathioprine and cyclophosphamide treatment.The immunosuppressant patient also comprises the patient of major injury including, but not limited to the calcination damage, or the chronic hemodialysis patient.
The present invention also provide the IL-8 medicine as in the viral infection of antiviral agents under the immunosuppressant of treatment humans and animals with the use of uniting of other antiviral agents.
The present invention also provides a kind of antiviral agents composition formula, comprises the combination of the IL-8 and the medicine acceptable carrier of the acceptable treatment effective dose of pharmacology.
The method that the present invention also provides a kind of prevention and treatment human and animal virus to infect comprises the IL-8 medicine of taking the acceptable treatment effective dose of pharmacology to the human or animal of this treatment of needs.
Brief description
Figure 1A-1F has shown the inhibitory action of IL-8 to EBV inductive SCID Mus tumor growth and splenomegaly.
Fig. 2 A, 2B and 2C have shown that IL-8 and Gro α are to Ca in the inductive human neutrophil of EBV
2+The influence of moving.
Fig. 3 A and 3B have shown that pertussis toxin, PT is to the inductive human neutrophil Ca of EBV
2+The influence (A) of moving and to the synthetic influence of RNA (B).
Preferred embodiment is described with reference to the accompanying drawings
ⅰ) IL-8 medicine
IL-8 medicine of the present invention is 72 amino acid patterns [Ser IL-8] of IL-8
72Or 77 amino acid pattern [AlaIL-8]
77, and [Ser IL-8]
72Or [Ala IL-8]
77Derivant or analog.A small amount of amino acid whose increase of IL-8 known in this field or minimizing may slightly change the activity that promptly strengthens or weaken IL-8.According to this conclusion, the derivant of IL-8 and analog also comprise the peptide of change, and it shows the obvious biological activity that is similar to IL-8 at many biosystems.Tangible biological activity of the present invention includes but not limited to be similar to the antiviral activity of IL-8.Term " IL-8 medicine " comprises [Ser IL-8]
72Or [Ala IL-8]
77This biological activity variant.
Protein modified
[Ser IL-8]
72Or [Ala IL-8]
77Analog or derivant (variant) be confirmed as [Ser IL-8]
72Or [Ala IL-8]
77The molecule that the aminoacid sequence of itself or other character are covalently or non-covalently modified.Therefore, variant has also the molecular weight of about 10KD not (the no Reducing agent for example SDS-PAGE of beta-mercaptoethanol or dithiothreitol, DTT detects).The variant aminoacid sequence not only comprises [Ser IL-8]
72Or [Ala IL-8]
77The relative thing of equipotential, and comprise its predetermined mutant.Usually, the aminoacid sequence of variant and [Ser IL-8]
72Or [Ala IL-8]
77At least 80% homology is arranged itself, more typical at least about 90%.Therefore, term " IL-8 medicine " means or native sequences or variant form, unless understanding is arranged in addition.
Therefore, [the Ala IL-8] that is included in the scope of the present invention
77Has the people [Ala IL-8] who is published on " immunology progress " (immunology progress, 55:97,1994)
77Aminoacid sequence and derive from other kind for example [Ala IL-8] such as cattle, horse, pig, sheep, Canis familiaris L., Mus, cats
77[Ala IL-8]
77Albumen analog etc., and these [Ala IL-8]
77The bioactive amino acid sequences variant of molecule, this variant comprise homotopicly and external syntheticly can show its bioactive [Ala IL-8]
77Proteic covalence derivative.
The biological activity of the derivant of IL-8 and variant amino acid sequence body has determined its treatment effectiveness, just to other place of this paper mention such.
Covalent modification
Comprised in the scope of the invention by the external synthetic covalent modification that carries out the IL-8 medicine easily.This modification can be passed through the target amino acid residue of purification or crude protein and can react with organic derivatization reagent of the residue reaction of selecting side chain and end and introduce molecule.The covalence derivative that produces may have the biological activity and/or the biological effectiveness of change.Therefore, the IL-8 of covalent modification reduces bioactive prodrug, slowly changes into the molecule (original I L-8 medicine) of greater activity after the administration in the body.Variant also may be that the IL-8 metabolism is more stable and more have bioactive analog, this change will cause this chemical compound postpone to distribute (reducing metabolism and/or removing).
Sequence modification
Also can prepare [Ser IL-8]
72Or [Ala IL-8]
77The variant amino acid sequence body.This variant comprises, for example, and [Ser IL-8]
72Or [Ala IL-8]
77The disappearance of self aminoacid sequence residue or insert or substitute.Any disappearance, insertion and alternate combination also can produce desirable active variant.
At gene level, these variants are generally by coding [Ala IL-8]
77The site-directed mutagenesis of the nucleotide of DNA produces, and produces the DNA of coding variant thus, then expresses this DNA in cell mixing is cultivated.This variant typical case performance is with the abiogenous analog biological activity of effect amount equally.
Because [Ala IL-8]
77Can aggregate into dimer, heterodimer and equal dimer are provided in this paper scope, one of them or two subunits are variant, if wherein two subunits all are variants, the change of each subunit chain amino acid sequence can be the same or different.The common transformed host cell of DNA with two subunits of coding can produce heterodimer easily, and if necessary, the heterodimer that purification is required or separate synthetic subunit decomposes subunit, the subunit of mixed decomposition, the subunit of recombinating then.
Term " IL-8 medicine " also comprises the antibody of IL-8 receptor, the anti-idiotype antibody of perhaps anti-IL-8 or one of above-mentioned IL-8 analog and variant antibody, and these antibody induce IL-8 sample biological respinse, for example antivirus action.
Term " IL-8 medicine " also comprise IL-8 receptor (A type and Type B) or can with bonded other receptor of IL-8, perhaps this receptor is selected the corresponding peptide in zone, perhaps virus envelope protein (glycoprotein or lipoprotein), perhaps this albumen is selected the corresponding polypeptide in zone, and its prevention virion combines with IL-8 receptor or other IL-8 bind receptor.
Term " IL-8 medicine " also comprises IL-8 receptor (or with bonded other receptor of IL-8) analog or variant, this receptor is selected corresponding peptide analogues in zone or variant, the analog and the variant of virus envelope protein (glycoprotein or lipoprotein), or this albumen is selected the corresponding peptide in zone.
But term " IL-8 medicine " does not comprise about 17 the amino acid whose polypeptide that meet following general formula:
Glu-Leu-Arg-Cys-Xaa
1-Cys-Xaa
2-Xaa
3-Xaa
4-Xaa
5-Xaa
6-Xaa
7-Xaa
8-Xaa
9-Xaa
10-Xaa
11-Xaa
12
Wherein:
Xaa
1Be Gln, Met, or Val;
Xaa
2Be Ile, or Val;
Xaa
3Be Lys, Gln, or Ser;
Xaa
4Be Thr, or Ile;
Xaa
5Be Tyr, Leu, Met, His, Val, or Thr;
Xaa
6Be Ser, Gln, Thr, or Ala;
Xaa
7Be Lys, Arg, or His;
Xaa
8The disappearance or be Pro, Phe, or Gly;
Xaa
9The disappearance or be Phe, Ile, or Val;
Xaa
10The disappearance or be His, Lys, or Arg;
Xaa
11The disappearance or be Pro, Leu, or Phe;
Xaa
12The disappearance or be Lys, His, or Arg.
ⅱ) viral infection
According to the present invention, the viral infection of available IL-8 Drug therapy is the infection that is caused by the human and animal virus.
" human and animal virus " tends to include but not limited to: common DNA and RNA viruses and remove HIV-1 and the retrovirus of HIV-2.DNA viruses includes but not limited to: Parvoviridae, papovaviridae, Adenoviridae, herpetoviridae, Poxviridae and Hepadnaviridae.RNA viruses includes but not limited to: Retroviridae, Picornaviridae, Togaviridae, orthomyxoviridae family, Paramyxoviridae, coronaviridae, Reoviridae, Oncogenic RNA virus section and filamentous form virus section.
Studied the activity of anti-Epstein-Barr virus (EBV) in the IL-8 body.As everyone knows, the SCID Mus lacks B with it and the T lymphocyte is a feature, mixes the formation B of regular meeting lymph when infecting the peripheral blood lymphocyte of being tried body with EBV.This infection is accompanied by splenomegaly, peritoneal cavity amplification and diarrhoea.Therefore, on behalf of the relevant people of a kind of and viral originality, the SCID Mus cause the lymphadenomatous body inner model that merits attention.Be to estimate the antiviral activity of IL-8, B cell (B95-8 cell line) intraperitoneal that EBV transforms injects the SCID Mus, then according to the different schemes peritoneal injection IL-8 medicine of following table 1.
Research approach in the body that the SCID Mus that table 1 IL-8 infects EBV influences
The 1st group of animal of being untreated
The 2nd group of 0 day injection IL-8
The 3rd group of 0 day injection B95-8 cell
The 4th group of 0 day interval injected B95-8 cell and IL-8 in 30 minutes
The 5th group of 0 day injection B95-8 cell, 0,1,2,3,4,5 day injection IL-8
The 6th group of 0 day injection B95-8 cell, 0,7,15,21,28 day injection IL-8
Group 1-6 put to death animal and postmortem in 49 days
EBV infected B 95-8 cell (60 * 10
6) intraperitoneal (IP) injection SCID Mus.At the appointed time, the IL-8 with reorganization handles Mus.After 49 days, put to death Mus and postmortem.Every group of 3 Mus, IL-8 is mixed with 0.9% the NaCl solution that comprises 0.1%BAS, the IL-8 of every Mus peritoneal injection 25 μ g.
In injection 4 weeks of back, the Mus of handling with the B95-8 cell shows tangible intraperitoneal inflammation separately, is characterized as abdominal part enlargement and diarrhoea, is the sign of this model growth of cancers.On the contrary, IL-825 μ g handles Mus week about totally 5 times (the 6th groups) does not observe this symptom.
The Mus (the 5th group) that 25 μ gIL-8 handled in continuous 6 days infects the back and the 4th week observed diarrhoea, but lighter than the degree of the Mus (the 3rd group) of being untreated after infecting.
Only with 25 μ gIL-8 processing Mus (the 2nd group) once, the animal (the 3rd group) of being untreated after observed intraperitoneal inflammation and diarrhoea and the infection is serious equally.
Around the, the animal of group 1,2 and 6 seems very normal, does not show above-mentioned symptom.The animal of group 3 the 5th and the 6th week after infection is dead, and postmortem shows spleen regulating liver-QI massive hemorrhage.The animal that infects all the group survivals of the 7th week of back all is condemned to death and postmortem.
IL-8 processing every Mus of infection animal (the 4th group) is once all found the tumor (showing as Figure 1B) of tangible splenomegaly and the about 0.7cm of diameter.Infect, untreated animal (the 1st group) spleen is normal and do not have tumor (showing as Figure 1A).The animal (the 5th group) that IL-8 handled in continuous 6 days observes splenomegaly and tumor, but compares sx with the 4th treated animal.25 μ g IL-8 handle totally four times animal (the 6th group) week about, do not detect tumor, and spleen (with other organ) is compared no tangible form and distinguished with infection animal not (group 1 and group 2) corresponding organ.
Also studied simultaneously the antiviral activity of anti-EBV in the IL-8 body, this be according to the scheme of following table 2 handle with IL-8 or not the SCID model mouse rebuild of handler's peripheral blood lymphocytes (PBMCs) carry out.
Table 2 IL-8 infects research approach in the body that influences to PBMCS-HU-SCID Mus EBV
The 1st group of NaCl: glucose+0.1%BSA handles Mus (2)
The 2nd group of IL-8 handles Mus (2)
The 3rd group of NaCl: glucose+0.1%BSA handles EBV infected mice (10)
The 4th group of IL-8 handles EBV infected mice (5)
All animal peritoneal injections 40 * 10
6Human PBMC s (among the 0.5ml PBS).After 5 days, the 3rd group and the 4th treated animal infect (every Mus peritoneal injection 0.5 * 10 with EBV
6TFU) all animals were handled with saline or IL-8 at 0,6,13,20 and 27 day then.IL-8 is always NaCl: 25 μ g bolus (peritoneal injection) administrations in glucose+0.1%BSA solution.Handled back 31 days, and put to death Mus.
The result is presented in the following table 3.Infected the back 22-23 days, some animals of group 3 show infection symptoms, rise in 24-25 days, observe fatality rate.25 μ g IL-8 are to handle at interval (peritoneal injection) EBV infected mice 5 times (the 4th group) weekly, and the symptom of appearance is deferred to 26-27 days, and in this group, lethality also postpones, and observe first animal dead on the 28th day.By 31 days, 6 death in the 3rd group of 8 animal, on the contrary, 2 death in the 4th group of 5 animals.The animal of survival in 31 days is condemned to death and postmortem.Two groups surviving animals does not have the little difference of obvious tumor size or quantity and splenomegaly.The 1st group and the 2nd treated animal do not show any symptom.
Table 3 IL-8 brings out the influence of (Indiced) symptom (and fatality rate) to PBMCS-HU-SCID Mus EBV
| Infect related symptoms (1)Mus number/survival Mus the number of influence | |||||
| 22-23 days | 24-25 days | 26-27 days | 28-29 days | 30-31 days | |
| EBV infects (the 3rd group) | ????5/8 (2) | 4/5 (3 death) | 3/4 (4 death) | 3/3 (5 death) | 2/2 (6 death) |
| EBV infection+IL-8 treats (the 4th group) | ????0/5 | ????0/5 | ????3/5 | 4/4 (1 death) | 3/3 (2 death) |
(1) vigor (lackluster) of observed infection related symptoms for causing the abdominal part enlargement owing to tumor growth, become thin and reducing.
(2) this group comprises 10 animals at first, because transplanted with human PBMCs S (IgG and antiviral antibody evaluation occurring by serum) only can consider 8, and two (2) not success in addition.
The Mus (the 1st group and the 2nd group) of using NaCl: glucose+0.1%BSA separately or handling with IL-8 does not separately detect unusual (until 31 days).
Recently report EBV can with the external interaction of human neutrophil, and regulate RNA and albumen is synthetic.IL-8 is considered to induce human neutrophil alteration of form and intercellular Free Ca
2+Concentration ([Ca
2+] i) and of short duration rising.We draw EBV to [Ca from isolating human neutrophil
2+] i generation effect (Ca similar but inequality
2+Cumulative kinetics difference).Therefore, we have studied IL-8 to the inductive neutrophilic granulocyte [Ca of EBV
2+] influence that raises of i.Detected have fluorescent probe FURA-2/AM and with the human neutrophil of handling with IL-8 or Gro α before or after EBV contacts in Ca
2+Move.After neutral granulocyte is with the IL-8 pretreatment, the inductive [Ca of EBV
2+] rising of i is by strong inhibition.On the other hand, EBV pretreatment neutrophilic granulocyte can not suppress the effect (Fig. 2 A, 2B, 2C) of IL-8 or Gro α.Since the IL-8 receptor is the G protein-coupled receptor, the inventor is further by the inductive Ca of pertussis toxin, PT pretreatment neutrophilic granulocyte checking EBV
2+Move and RNA synthetic in the proteic latent effect of G, wherein pertussis toxin, PT is that guanylic acid is in conjunction with the known mortifier of modulin (G albumen).The result who obtains shows that pertussis toxin, PT significantly suppresses the inductive Ca of EBV
2+Move with RNA and synthesize, the inductive result of prompting EBV is protein mediated by G.
Yet antiviral and/or the Anticancer Effect and Mechanism of the IL-8 that relates to are not clear, can suppose the IL-8 triggering or strengthen natural antiviral mechanism, for example produce tumor lethal factor α (TNF α) or interferon.Perhaps, according to the influence of the IL-8 that obtains to the biological agent of the inductive isolating neutrophilic granulocyte of EBV, the interaction of possible EBV and its target cell relates to IL-8 receptor (maybe can discern the binding site of IL-8), and wherein IL-8 may be by competing (steric effect) and induce receptor internalization to stop the interaction of virus and target cell with the land.Perhaps, may reduce with viral infection relevant bonding, internalization with the target cell of IL-8 contact, duplicate and function of receptors and signal transduction such as cell nature antiviral mechanism inhibition.Nearest report has been supported these hypothesis: chemical kinetins receptor associated protein(RAP) is expressed by various encoding virals and on the plasma membrane of infection cell.The EBV system is thought the BLR-1 receptor (may be equal to EBI-2) and the BLR-2 receptor (being equal to EBI-1) of virus induction transformant.They are and the homologous G protein-coupled receptor of IL-8 receptor height.Expression with these cytokine receptor of IL-8 receptor homolog in the EBV infection cell can make the IL-8 antiviral activity sensitivity of the B cell of infection cell, non-loaded IL-8 receptor to supposition.These hypothesis are for the understanding that some IL-8 antiviral activities supposition mechanism are provided and should not limit the scope of the invention.
These results show that IL-8 can be used for the treatment of the cancer that viral infection and oncorna virus cause.
ⅲ) dosage range
The therapeutic dose of interleukin 8 medicine will change along with the character that should handle symptom and the order of severity, special IL-8 medicine, other reactive compound of uniting use and its route of administration, and determine based on clinical diagnosis.Dosage also changes along with age, body weight and the reaction of individual patient.Therefore the effective dose of IL-8 medicine can be passed through CLINICAL PHARMACOKINETIS STUDY ON, considers all conditions and uses the best of patients ' behavior to judge to determine.Usually, effective dose is about in the blood 1 to 100nM.In view of the above, the clinicist will use the dosage that can produce this blood drug level.
ⅳ) pharmaceutical composition
Can use any suitable route of administration to offer mammal, especially the IL-8 medicine of the present invention of people's effective dose.For example, can be taken orally, administration in the parenterai administration, topical, artery administration, intraperitoneal administration, intravenously administrable, pleura, eye drops, drug administration by injection or subcutaneous administration etc.Injection comprises perfusion and continuous drip.Dosage form comprises tablet, capsule, powder, solution, dispersant, suspending agent, extrudes agent, ointment and aerosol.
Pharmaceutical composition of the present invention comprises as the IL-8 medicine of active component and medicine acceptable carrier and other optional therapeutic component.
Said composition comprises the compositions of suitable oral or parenterai administration.Made unit dosage form easily and made with the pharmaceutical field known method.Unit dosage form can be a kind of slow release unit dosage form.
In the practical application, the IL-8 medicine can be used as active component thoroughly to be mixed according to the medicament mixed technology of routine with pharmaceutical carrier.Can in relative broad range, select carrier according to required administering mode.The preparation oral formulations can use any useful drug matrices, for example, oral liquids such as water, glycerol, oil, ethanol, flavoring agent, antiseptic, stain are the composition in suspending agent, elixir and the solution for example, perhaps oral solid formulation for example starch, sugar, microcrystalline Cellulose, diluent, granulating agent, lubricant, binding agent, disintegrating agent etc. of the carrier in powder, capsule and the tablet for example.Use because tablet and capsule are convenient,, wherein used solid pharmaceutical carriers so they have represented best peroral dosage form unit.If desired, tablet can have water or anhydrous technology coatings with standard.
Be suitable for oral pharmaceutical composition of the present invention and can be discrete units for example capsule, cachet or tablet, per unit comprises the IL-8 medicine of scheduled volume, also can be powder or granule, the perhaps solution of aqueous solution or non-aqueous solution or suspending agent, perhaps oil-in-water emulsion, perhaps water-in-oil emulsion.These preparations can be made by some practice of pharmacy, comprise that the IL-8 medicine mixes with the carrier of one or both neccessary compositions.Usually, at first with IL-8 medicine and liquid-carrier or the smart solid carrier that divides or with the mixing of both uniform and complete, then, if necessary, the shape that need provide is provided product.For example, tablet can select one or more auxiliary elements by tabletting or mold preparation.Compressed tablet can be by for example powder or granule and preferred adhesive, lubricant, inert diluent, surfactant or dispersant form with the suitable machine compacting with non-current attitude active component.Molded tablet can be molded as the powder compounds mixture with the inert liquid diluent humidity with suitable machine.
Should be appreciated that the IL-8 medicine is that this dosage is interpreted as patient harmless with pharmacology and the acceptable dosed administration of physiology, or the harmful side effect of individual patients can be surpassed by the beneficial effect of this medicine.Equally, the IL-8 medicine is that this dosage should be understood that to satisfy the object of planned treatment and produce the amount of beneficial effect after taking the IL-8 medicine with the administration of treatment effective dose.
Embodiment
Research in the body
Embodiment 1
The SCID Mus interior resisting virus effect that IL-8 infects the EBV transformant
This research used 8 ages in week female SCID-CB17 Mus (Charles River, St.Constant, Canada).Mus is divided into six groups every group three, and the EBV transformant infects, then with or handle as above table 1 described (the 3rd to the 6th group) without IL-8.The 0th day, B95-8 cell (60 * 10
6) intraperitoneal injection SCID Mus.As indicated, Mus also by one or many injection (intraperitoneal) IL-8 (the 2nd, 4 to 6 groups) (every Mus per injection 25 μ g) (Peprotech Inc., Rocky Hid, NJ).The IL-8 that uses is 72 amino acid patterns, and uses in comprising 0.9% the NaCl solution of 0.1%BAS.After handling for 7 weeks, Mus is put to death and postmortem by cervical dislocation.Tumor and other interested tissue are taken a picture, and (Figure 1A-1F), dissection and freezing preservation are so that carry out immunofluorescence research and the viral DNA analysis.Figure 1A has shown the non-processing of non-infection Mus (group 1).Figure 1B and 1C shown behind the B95-8 cell infection 30 minutes shot IL-8 (the 4th group) the EBV infected mice.Figure 1B and 1C show the intraperitoneal of same animal, and wherein Fig. 1 C is closeup photograph (the 4th group).Rose weekly with IL-8 continuous 4 all EBV infected mices of handling (the 6th group) in 30 minutes behind Fig. 1 D and the 1E demonstration B95-8 cell infection.Fig. 1 F shows that IL-8 handles the intraperitoneal (left side) of EBV infected mice once, IL-8 handle 4 Mus (in) and (right side) of the Mus of being untreated.
Embodiment 2
IL-8 infects antiviral effect to the EBV of the SCID Mus (PBMCs-hu-SCID) that the human peripheral blood mononuclear cell rebuilds
Healthy human volunteer is carried out lymphocyte to be extracted.Collecting cell and carry out Ficoll gradient centrifugation (750 * g) 25 minutes purification in 1 hour.(every Mus injection 0.5ml contains 40 * 10 to wash PBMCs and peritoneal injection (i.p.) then in PBS
6The PBS of individual cell) to C.B.-17/SCID Mus (4-5 age in week).After 5 days, Mus is divided into 4 groups, and animal is used EBV (50 μ l 10
7TFU/ml/ Mus) peritoneal injection infect and with or without the IL-8 processing, as shown in table 2.IL-8 is mixed with the sodium chloride that comprises 0.1%BSA: glucose solution uses.After handling for 4 weeks, Mus is put to death and postmortem by cervical dislocation.Interested tissue is removed also freezing preservation so that carry out analysis of molecules.
In vitro study
Embodiment 3
The inhibitory action that IL-8 (with Gro α) moves calcium ion in the inductive isolating human neutrophil of EBV
Neutrophilic granulocyte suspension (10 * 10
6Individual cells/ml) (molecular probe, Eugene OR) cultivate altogether (1 μ M 37 ℃ 30 minutes) with fluorescent probe Fura-2/ methyl acetate.Cell is washed till the outer probe of no born of the same parents and is suspended in Hank ' the s balanced salt solution (HBSS) of the HEPES that comprises the 1.6mM calcium ion and add 10mM once more.The suspension that comprises the Fura-2 cell is with different order EBV (10
6Individual conversion unit [TFU]/milliliter) and IL-8 or Gro α (100nM) handle.Use Aminco-Bowman, (SLM-Aminco, Rochester NY) detect fluorescence (excitation wavelength and absorbing wavelength are respectively 340nm and 510nm) (Fig. 2 A, 2B and 2C) to series 2 devices.The EBV strain is that the preparation of B95-8 was undertaken by former described method.Briefly, the B95-8 that does not detect mycoplasma is grown in the RPMI1640 culture medium of adding 10% hot deactivation FBS liquid.When finding that cell viability is lower than 20%, to gather in the crops acellular culture medium supernatant and filter with the filter in 0.45mm aperture, virion is with super centrifugal being further purified.Virion is suspended in the sodium radio-phosphate,P-32 solution of 5mM (pH7.5) and with 10-30% (wt/vol) glucosan gradient centrifugation purification.Spissated viral liquid resuspending is in RPMI1640, and-80 ℃ of preservations are until use behind the five equilibrium.Virus titer is found to be 1 * 10
7Transform unit (TFU)/milliliter.Fig. 2 A, 2B and 2C result displayed are further made description below:
Fig. 2 A:a:Gro α (100nM); B:EBV; C:IL-8 (100nM)
Fig. 2 B:a:Gro α (100nM); B:IL-8 (100nM); C:EBV
Fig. 2 C:a:EBV1/10 dilution, 10
5TFU/ml; B:EBV; C:Gro α (100nM); D:IL-8 (100nM)
Embodiment 4
Pertussis toxin, PT is to Ca in the inductive human neutrophil of EBV
2+Move influence with the RNA de novo synthesis
Human neutrophil (10 * 10
6Individual cells/ml) adding 0.5mg/ml pertussis toxin, PT (the proteic ADP-of catalysis G-riboseization and the protein mediated reaction of inhibition G) is cultivated 2 hours in advance afterwards with EBV (10 for 37 ℃
6TFU/ml) or IL-8 (100nM) stimulate.According to moving of embodiment 2 described measurement calcium ions.Fig. 3 has shown the result, wherein:
A:IL-8 stimulates
B:EBV stimulates
C: IL-8 stimulates after the pertussis toxin, PT pretreatment
D: EBV stimulates after the pertussis toxin, PT pretreatment
Mix [the 5-of full RNA by measurement
3H] to have studied the inductive RNA of EBV synthetic for uridnine.Pertussis toxin, PT (0.5mg/ml) pretreatment or the neutrophilic granulocyte of not handling (5 * 10
6Individual cells/ml) is suspended in the HBSS buffer of the autologous plasma that adds 1% hot deactivation (1 hour, 56 ℃).Every part of [5-that adds 1mCi of 100 milliliters five equilibrium cell suspension
3H] uridnine cultivates the GM-CSF (positive control) that uses 3nM subsequently or with infective EBV (10 on the microdroplet flat board in 96 holes
6TFU/ml) handle.Flat board is containing 5%CO
2Humid air in 37 ℃ cultivated 5 hours.Subsequently, glass mat filters collecting cell, liquid flashing counter measuring radioactivity.(St.Louis MO), is formulated in during use in the 0.9%NaCl solution that comprises 0.01%BSA pertussis toxin, PT from Sigma chemical company.Gro α uses with the manner of formulation that above-mentioned pertussis toxin, PT is same from Peprotech.
Fig. 3 B result displayed is represented six different experiments (GM-CSF, granulocyte-macrophage colony stimutaing factor).
This paper has described the present invention and relevant specific embodiment thereof, be to be understood that the present invention can further revise, the application makes every effort to cover any change, application or the adjustment that comes from principle of the present invention, the present invention includes those are in known or conventional techniques from of the present invention association area different with disclosure text, with be to use above the inner characteristic of setting forth and in the additional claim scope of the present invention.
Claims (17)
1. the application of interleukin 8 medicine in treatment human or animal viral infection.
2. according to the application of claim 1, wherein said medicine is that interleukin 8 albumen or its have the bioactive analog of interleukin 8.
3. according to the application of claim 1 or 2, wherein said viral infection is DNA viruses or RNA viruses.
4. according to the application of claim 3, wherein said viral infection is the DNA viruses that is selected from Parvoviridae, papovaviridae, Adenoviridae, herpetoviridae, Poxviridae and Hepadnaviridae.
5. according to the application of claim 3, wherein said viral infection is the RNA viruses that is selected from Retroviridae, Picornaviridae, Togaviridae, orthomyxoviridae family, Paramyxoviridae, coronaviridae, Reoviridae, Oncogenic RNA virus section and filamentous form virus section.
6. according to the application of claim 1, wherein said medicine is 77 amino acid whose interleukin 8 albumen.
7. according to the application of claim 1, wherein said medicine is 72 amino acid whose interleukin 8 albumen.
8. according to the application of claim 4, wherein said viral infection is the herpesvirus coe virus that is selected from EBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7 and HHV-8.
9. according to the application of claim 5, wherein said viral infection is from the bovine diarrhea virus of Togaviridae.
10. according to the application of claim 5, wherein said viral infection is from the pig enterovirus of Picornaviridae.
11. according to the application of claim 5, wherein said viral infection is from the bovine respiratory syncytial virus of Paramyxoviridae.
12. according to the application of claim 1, wherein said medicine be selected from interferon-' alpha ' ,-β ,-the antiviral agents administering drug combinations of γ, tumor lethal factor-α, ganciclovir, acyclovir, vidarabine, idoxuridine, general Xi Luowei, prostaglandin and prostaglandin analogue.
13. according to the application of claim 1, wherein said human or animal is the immunosuppressant human or animal, perhaps uses the human or animal of the drug treating of known increase viral infection incidence rate.
14. according to the application of claim 13, wherein said medicine is selected from azathioprine, corticosteroid, amycin and methotrexate.
15. the application of interleukin 8 medicine in human or animal's treatment of cancer that oncorna virus causes.
16. according to the application of claim 15, wherein said medicine is united use with the cancer therapy drug that is selected from AC and methotrexate.
17. an antiviral drugs preparation comprises the interleukin 8 medicine of the acceptable treatment effective dose of pharmacology and medicine acceptable carrier.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97196176 CN1225016A (en) | 1996-07-05 | 1997-07-04 | Interleukin-8 as an antiviral and antitumor agent |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/674,633 | 1996-07-05 | ||
| CN 97196176 CN1225016A (en) | 1996-07-05 | 1997-07-04 | Interleukin-8 as an antiviral and antitumor agent |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102727869A (en) * | 2012-07-11 | 2012-10-17 | 武汉大学 | Application of soluble interleukin 6 receptor in antiviral medicine preparation |
| CN105944086A (en) * | 2016-06-13 | 2016-09-21 | 浙江生创精准医疗科技有限公司 | Application of IL-8 or IL-8 and other cytokines to treatment of hepatic fibrosis |
| CN107427555A (en) * | 2015-01-05 | 2017-12-01 | 康奈尔大学 | Healthy reproduction and suppress the composition and method of hyperketonemia using IL 8 to increase the output of milk and improve in mammal |
| US11464831B2 (en) | 2015-01-05 | 2022-10-11 | Cornell University | Compositions and methods using IL-8 for improving health of mammals |
-
1997
- 1997-07-04 CN CN 97196176 patent/CN1225016A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102727869A (en) * | 2012-07-11 | 2012-10-17 | 武汉大学 | Application of soluble interleukin 6 receptor in antiviral medicine preparation |
| CN102727869B (en) * | 2012-07-11 | 2013-09-18 | 武汉大学 | Application of soluble interleukin 6 receptor in antiviral medicine preparation |
| CN107427555A (en) * | 2015-01-05 | 2017-12-01 | 康奈尔大学 | Healthy reproduction and suppress the composition and method of hyperketonemia using IL 8 to increase the output of milk and improve in mammal |
| US11406689B2 (en) | 2015-01-05 | 2022-08-09 | Cornell University | Compositions and methods using IL-8 to improve milk production and reproductive health in mammals |
| US11464831B2 (en) | 2015-01-05 | 2022-10-11 | Cornell University | Compositions and methods using IL-8 for improving health of mammals |
| CN105944086A (en) * | 2016-06-13 | 2016-09-21 | 浙江生创精准医疗科技有限公司 | Application of IL-8 or IL-8 and other cytokines to treatment of hepatic fibrosis |
| CN105944086B (en) * | 2016-06-13 | 2017-08-25 | 浙江生创精准医疗科技有限公司 | IL 8 is independent or combines the purposes in treatment liver fibrosis with other cell factors |
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