CN101262870A

CN101262870A - Methods of reducing the severity of oral and gastrointestinal mucositis

Info

Publication number: CN101262870A
Application number: CNA2006800165614A
Authority: CN
Inventors: T·卡瓦诺; S·科巴亚希; H·希罗塔
Original assignee: Eisai R&D Management Co Ltd
Current assignee: Eisai R&D Management Co Ltd
Priority date: 2005-05-13
Filing date: 2006-05-15
Publication date: 2008-09-10
Also published as: CA2606727A1; KR20080038085A; JP2008540510A; WO2007031879A2; EP1879597A2; AU2006290437A1; WO2007031879A3

Abstract

The invention provides methods of reducing the severity of oral and gastrointestinal mucositis, involving administration of a toll-like receptor 4 antagonist.

Description

Reduce the method for the mouth and the gastrointestinal mucositis order of severity

Background of invention

The present invention relates to reduce the method for the mouth and the gastrointestinal mucositis order of severity.

Mucositis is swollen, is stimulated and uncomfortable disease for being characterised in that mucosa lining (for example lining in gastrointestinal tract and oral cavity and oropharyngeal membrane chamber), and may cause oral cavity and throat pain, diarrhoea, abdominal cramps and tenderness and rectal ulcer.Described disease takes place in half in all cancer patients' pact, and for relating to the common adverse effect of radiation and/or chemotherapeutic treatment of cancer.The target of these treatment of cancer approach is to kill splitted cancerous cell fast, yet unfortunately, has killed other quick splitted cell equally by these treatments, comprises for example cell in gastrointestinal lining zone, produces mucositis thus.Begin the back in treatment of cancer and catarrhal symptom usually occurred in 5-10 days, and in treatment, have no progeny and need 2-4 week to remove.Catarrhal sickness rate with and the order of severity depend on the kind of for example treatment of cancer and the factor of persistent period.For example, in fact mucositis appears among all patients that treat by radiation head and cervical region.It also uses patient's camber of high dose chemotherapy and/or radiation therapy popular for the medullary cell removing in preparing stem cell or bone marrow transplantation.

Mucositis influences cancer patient's quality of the life unfriendly in several modes.For example, catarrhal oral cavity and throat pain can produce tangible pain and make it be difficult to have a meal, drink water even be difficult to take oral drugs.Because it can produce at other protective lining of oral mucosa and gastrointestinal and destroy, the mucositis serious infection risk that also occurs together, it is by multiple microorganism cluster.In addition, the destruction that the work of antagonism mucositis discomfort may cause treatment of cancer changes therapeutic dose, or changes different treatment patterns over to.Serious mucositis also may cause the needs to parenteral absorption or hospitalization.Prevention or to treat the research and development of catarrhal effective means important to the nursing that improves the cancer patient thus.

Summary of the invention

The invention provides the method that in the patient, reduces the mouth or the gastrointestinal mucositis order of severity.These methods comprise to patient's administration and contain (TLR4) active compound compositions of one or more blocking-up Toll sample receptors 4 (Toll-Like receptor4), lipid A analogue for example, and it can be in the following formula scope:

R wherein ¹Be selected from:

Wherein each J, K and Q are straight or branched C1-C15 alkyl independently; L is O, NH or CH ₂M is O or NH; And G is NH, O, S, SO or SO ₂

R ²Be straight or branched C5-C15 alkyl;

R ³Be selected from straight or branched C5-C18 alkyl,

Wherein E is NH, O, S, SO or SO ₂Each A, B and D are straight or branched C1-C15 alkyl independently;

R ⁴Be selected from straight or branched C4-C20 alkyl and

Wherein each U and V are straight or branched C2-C15 alkyl independently, and W is hydrogen or straight or branched C1-C5 alkyl;

R _ABe R ⁵Or R ⁵-O-CH ₂-, R ⁵Be selected from hydrogen, J ' ,-J '-OH ,-J '-O-K ' ,-J '-O-K '-OH and-J '-O-PO (OH) ₂, wherein each J ' and K ' are straight or branched C1-C5 alkyl independently;

R ⁶Be selected from hydroxyl, halogen, C1-C5 alkoxyl and C1-C5 acyloxy;

A ¹And A ²Be independently selected from:

OH，

O-z-CO ₂H

Wherein Z is a straight or branched C1-C10 alkyl; Or its pharmaceutically acceptable salt or its phosphate ester.One aspect of the present invention comprises the phosphate ester of following formula, wherein A ¹Or A ²In at least one hydroxyl can be replaced to form phosphate ester.

The example that can join the lipid A analogue in the present composition is the chemical compound with following structure:

Or its pharmaceutically acceptable salt or its phosphate ester.

In example more specifically, this chemical compound has following structure:

Or its pharmaceutically acceptable salt or its phosphate ester.

The patient that can treat according to the present invention comprises those that suffer from mouth or gastrointestinal mucositis.In addition, do not suffer from and but be in those patients that suffer from oral cavity or gastrointestinal mucositis risk and can treat according to the present invention.In the one group of patient in back, this treatment can suppress or prevent catarrhal development.

As herein further as described in or described in the background technology, can impel or make the patient to be in to suffer from mouthful or the example of the treatment of gastrointestinal mucositis risk comprises radiotherapy and chemotherapy.Can for example comprise the cancer patient thus according to the patient of the inventive method treatment, and incite somebody to action recently, in a short time or the current patient who is carrying out head or cervical region radiation therapy, or the patient of stem cell or bone marrow transplantation.

According to the inventive method, being used for compositions of the present invention can be before impelling or making the patient be in to suffer from the combination of the treatment of oral cavity or gastrointestinal mucositis risk or operable these treatments, simultaneously or be administered into the patient afterwards.In one embodiment, said composition and treatment simultaneously, in the 1-4 hour or treating administration on the same day, administration 1-3 (for example 1-2) day then (for example every day 1-2 time).Other example of therapeutic scheme provides as follows.

Said composition can be administered into the patient by any known acceptable manner in this area, comprises part (for example by gel, lotion, lozenge, cream, unguentum or paster), intravenous infusion, oral (for example by tablet, capsule, lozenge, cream, unguentum or paster), rectum (for example by suppository, unguentum or enema) or vagina (for example by cream, unguentum, gel or suppository).As described below, can also treat catarrhal mode with other according to treatment of the present invention and make up and carry out, alternate manner comprises antibiotic and palliative treatment.

Except said method, the present invention also comprises compositions described herein and the application of chemical compound in the medicine of the preparation reduction mouth or the gastrointestinal mucositis order of severity.These medicines can be used for treatment have been suffered from catarrhal patient, be devoted to reduce disease symptoms (partially or completely), prevented that sb.'s illness took a turn for the worse, and/or the level that reduces that sb.'s illness took a turn for the worse.Yet this medicine also can be used for also not suffering from mucositis is in the patient who suffers from the mucositis risk.As described in other places herein, these patients comprise and predeterminedly accept, currently accept, and have perhaps before accepted to relate among the cancer patient of radiation and/or chemotherapeutic treatment of cancer.In these patient for example, the administration that can carry out medicine perhaps prevents the mucositis morbidity to reduce the catarrhal order of severity, to suppress its development.In addition, the chemical compound in these medicines can be other places herein mentioned any those, and the chemical compound in the chemical formula that provides of other places herein.The particular instance of this chemical compound is as follows:

Or its pharmaceutically acceptable salt or its phosphate ester.

The present invention comprise in addition contain chemical compound described herein, the preparation to be used to reduce the compositions of the mucositis order of severity described herein.As following detailed description, these compositionss can comprise the chemical compound in the preparation, gel, lotion, tablet, capsule, chewing gum, lozenge, cream, unguentum, enema, suppository or the paster of described formulation example as being used for topical.

The invention provides several benefits.For example, provide and reduce the mucositis order of severity, for example in the method for the uncomfortable side effect of radiation and chemotherapeutic treatment, the inventive method can impel the patient to maintain a good state when facing these treatments.In addition, the inventive method can reduce infection rate, and it is catarrhal common consequence.In addition, can increase the adaptability of patient, also can help to improve its resume speed its therapeutic scheme for the comfortable of rising, the inventive method are provided to the patient.

Other aspects and advantages of the present invention will be according to following detailed description, accompanying drawing and claim and apparent.

The accompanying drawing summary

Fig. 1 for expression snout radiation treatment after the chart that changes of C3H/HeOuJ and C3H/HeJ mouse body weight percentage ratio.Animal is weighed every day, calculates body weight percentage ratio from the 0th day and changes, and calculate cell mean peace standard error of mean (SEM) every day.

Fig. 2 is the chart that is used to calculate the area under curve (AUC) that body weight percentage ratio changes of the C3H/HeOuJ of expression snout radiation treatment and C3H/HeJ mouse performance.This calculating uses the trapezoidal rule conversion to carry out.Calculate every cell mean and use the error line of expression SEM to show.Unidirectional Anova test shows the statistically evident difference (P=0.008) between each group.

Fig. 3 is the chart of the average blood plasma IL-6 concentration of the C3H/HeOuJ that at the appointed time puts the snout radiation treatment of measuring by elisa assay and C3H/HeJ mouse.

Fig. 4 is the chart of the average blood plasma TNF-α concentration of the C3H/HeOuJ that at the appointed time puts the snout radiation treatment of measuring by elisa assay and C3H/HeJ mouse.

Fig. 5 is the C3H/HeOuJ of snout radiation treatment and the chart of C3H/HeJ mouse epithelial tissue Histological evaluation.Each sample is estimated the epithelium layer damage with the yardstick of 0-3.

Fig. 6 is the C3H/HeOuJ of snout radiation treatment and the chart of C3H/HeJ mouse connective tissue Histological evaluation.Each sample is estimated connective tissue damage with the yardstick of 0-3.

Fig. 7 is the chart that is illustrated in the inflammatory cell average number of the C3H/HeOuJ of snout radiation treatment of fixed time point measurement and C3H/HeJ mouse.

Fig. 8 is the chart that is illustrated in mitosis average number in the C3H/HeOuJ of snout radiation treatment of fixed time point measurement and the C3H/HeJ mouse epithelium layer.

Fig. 9 is the chart that is illustrated in the average number of the C3H/HeOuJ of snout radiation treatment of fixed time point measurement and the per 10 high power field medium vesselses of C3H/HeJ mouse.

Figure 10 is illustrated in the chart that trunk in the C3H/HeOuJ of snout radiation treatment of fixed time point measurement and per 10 high power fields of C3H/HeJ mouse accounts for the average of total percentage of vessels.This value uses the percentage ratio of observing in these visuals field that accounts for total blood vessel number to represent.

Figure 11 is the chart that the C3H/HeOuJ mice body weight percentage ratio of the eritoran processing of use specified amount after the snout radiation treatment changes.Animal is weighed every day, calculates body weight percentage ratio from the 0th day and changes, and calculate cell mean peace standard error of mean (SEM) every day.

Figure 12 is used to calculate the chart of the area under curve (AUC) of the body weight percentage ratio variation of the C3H/HeOuJ mice performance of snout radiation treatment as shown in figure 11 for expression.This calculating uses the trapezoidal rule conversion to carry out.Calculate every cell mean and use the error line of expression SEM to show.Unidirectional Anova test does not show statistically evident difference (P=0.261) between each group.

Figure 13 is the chart that uses the epithelium layer minimal amount at the C3H/HeOuJ mice tongue back side that the eritoran of specified amount handles after the snout radiation treatment of at the appointed time point measurement.

Figure 14 is the chart that uses the epithelium layer maximum number at the C3H/HeOuJ mice tongue back side that the eritoran of specified amount handles after the snout radiation treatment of at the appointed time point measurement.

Figure 15 is the chart that uses the epithelium layer minimal amount of the C3H/HeOuJ mice tongue outside of belly that the eritoran of specified amount handles after the snout radiation treatment of at the appointed time point measurement.

Figure 16 is the chart that uses the epithelium layer maximum number of the C3H/HeOuJ mice tongue outside of belly that the eritoran of specified amount handles after the snout radiation treatment of at the appointed time point measurement.

The chart of Figure 17 for changing at specified scheme and the weight of animals percentage ratio that do not use eritoran (E5564) to handle in radiation and the placebo group.Animal is weighed every day, calculates body weight percentage ratio from the 0th day and changes, and calculate cell mean and standard error of mean (SEM) every day.

Figure 18 is used for the chart of the area under curve (AUC) that body weight percentage ratio that each animal of Calculation and Study shows changes for expression.This calculating uses the trapezoidal rule conversion to carry out.Calculate every cell mean and use the error line of expression SEM to show.Radiation and significant difference on the statistics between the raying matched group not represented to accept in single asterisk, two asterisks are illustrated in 0-3 days use eritoran to handle and matched group (radiation) between statistically evident difference (P=0.030).

The chart that the weight of animals percentage ratio that Figure 19 handles according to scheme shown in the figure for expression changes.Data only are illustrated in the animal that still survives when research finishes.Animal is weighed every day, calculates body weight percentage ratio from the 0th day and changes, and calculate cell mean peace standard error of mean (SEM) every day.

The chart of the area under curve (AUC) that the body weight percentage ratio that each animal that Figure 20 handles according to scheme shown in the figure for expression is used to calculate shows changes.This calculating uses the trapezoidal rule conversion to carry out.Calculate every cell mean and use the error line of expression SEM to show.Radiation and significant difference on the statistics between the raying matched group not represented to accept in single asterisk, two asterisks are illustrated in 0-3 days use eritoran to handle and matched group (radiation) between statistically evident difference (P=0.041)

Figure 21 is the chart of expression average epithelium evaluation of each designated groups and standard error of mean thereof.

Figure 22 is the chart of expression average connective tissue evaluation of each designated groups and standard error of mean thereof.

Figure 23 is the chart of expression average inflammatory evaluation of each designated groups and standard error of mean thereof.

Figure 24 is the chart of mitosis average and meansigma methods standard error thereof in per 10 high power fields in each designated groups of expression.

Figure 25 is for estimating the chart of ulcer percent and meansigma methods standard error thereof in each designated groups of expression.

Figure 26 is the chart of inflammatory cell average and meansigma methods standard error thereof in per 10 high power fields in each designated groups of expression.

Figure 27 is for being the percentage ratio of infiltration inflammatory cell of neutrophil and the chart of meansigma methods and standard deviation thereof for each sample in each designated groups of expression.

Figure 28 is for being the percentage ratio of lymphocytic infiltration inflammatory cell and the chart of meansigma methods and standard deviation thereof for each sample in each designated groups of expression.

Figure 29 is for being the percentage ratio of infiltration inflammatory cell of mononuclear cell or macrophage and the chart of meansigma methods and standard deviation thereof for each sample in each designated groups of expression.

Figure 30 is the chart of medium and small number of blood vessel of per 10 high power fields in each designated groups of expression and meansigma methods peace standard error of mean thereof.

Figure 31 is the chart of medium vessels number in per 10 high power fields in each designated groups of expression and meansigma methods peace standard error of mean thereof.

Figure 32 is the chart of trunk number in per 10 high power fields in each designated groups of expression and meansigma methods peace standard error of mean thereof.

Figure 33 is the chart of mastocyte number in per 10 high power fields in each designated groups of expression and meansigma methods peace standard error of mean thereof.

Figure 34 measures the serum TNF-alpha levels of measurement and the chart of meansigma methods peace standard error of mean thereof for using ELISA in each designated groups of expression.

Figure 35 measures the chart of the blood serum IL-6 level and the meansigma methods peace standard error of mean thereof of measurement for using ELISA in each designated groups of expression.

Figure 36 measures the chart of the Serum SA A level and the meansigma methods peace standard error of mean thereof of measurement for using ELISA in each designated groups of expression.

In detail explanation

The invention provides the method that reduces mouth or the serious degree of esogastritis and esoenteritis. The method can be used for treating the patient who has suffered from catarrh. In addition, the method also can be used for not suffering from and but is in the patient that suffers from the catarrh risk (for example predeterminedly accept, current accepting or before using radiation and/or chemotherapeutic cancer or other patient). In the rear one group patient who does not also suffer from catarrh, can reduce according to treatment of the present invention the catarrh that is caused by its treatment of cancer serious degree, suppress the development of catarrh, perhaps prevent catarrh.

The blocking-up activity that the present invention is based on Toll sample acceptor 4 (TLR4) described herein provides useful result for the treatment of in reducing the serious degree of catarrh. TLR4 is endotoxin or lipopolysaccharides (LPS) acceptor, and its cell membrane from growth or killed bacterial produces and be relevant with bringing out of inflammatory response. According to the present invention, the TLR4 receptor activation produces useful effect by administration TLR4 anti-dose of blocking-up short of money in reducing the serious degree of catarrh. Except the blocking-up endotoxin, (the effect of HSP ' s) of heat shock protein in the treatment according to the present invention catarrh capable of blocking. Especially, this kind pressure inducible protein matter can be brought out during comprising radiotherapy and chemotherapeutic pressure. HSP 60,70 or 90 can be the interior endogenous ligand of TLR4, and works in the catarrh that radiotherapy is brought out thus.

The TLR4 that uses in the inventive method anti-dose of similar thing that for example can be LPS lipid a-quadrant short of money, for example lipid A analogue in the chemical formula in the present invention's general introduction. The example of the lipid A analogue that uses in the present composition is the compound with following structure:

Or its pharmaceutically acceptable salt or its phosphoric acid ester.

In another concrete example, this compound has following structure:

Or its pharmaceutically acceptable salt or its phosphoric acid ester. This compound is known as eritoran (being also known as compd E 5564, compound 1287 and SGEA) and is disclosed in US Patent No. 5,935, in 938.

Other example of the compound that the present invention uses comprises following:

And pharmaceutically acceptable salt or its phosphoric acid ester.

Anti-dose short of money of other TLR4 that can use among the present invention comprises for example compd B 531 (US Patent No. 5,530,113), and disclosed other compound in the following patent: US Patent No. 5,935,938, US 5,612, and 476, US 5,756,718, US 5,843,918, US 5,750, and 664, US 6,235,724, US 6,184,366 and US 5,681,824. The preparation method of these compounds is also disclosed in these files. Other method for preparing these medicines for example is disclosed among the WO 02/94019.

The method according to this invention, TLR4 short of money anti-dose cause mouthful esogastritis and esoenteritis or the patient placed the treatment of suffering from the catarrh risk before, during and/or be administered into afterwards the patient. As mentioned above, this kind treatment comprises radiation and chemotherapy, and the growth of its cell by blocking quick division is worked, for example the upper chrotoplast of cancer cell and stomach, respiratory tract and genitourinary tract surface lining. Can cause the concrete example of the treatment of catarrh to comprise radiation therapy (for example head and/or neck, whole body, target and/or hyperfractionated radiation), and the chemotherapy in the treatment of following disease or as the auxiliary curing of following disease: breast cancer, colon cancer, cancer of the stomach, urogenital device (for example bladder, prostate or testis) cancer, gynaecology (for example cervix, uterus inner membrance, ovary or uterus) cancer, head and neck/cancer of the esophagus, leukaemia, lung (little cell or non-small cell) cancer, lymphoma (Hodgkin ' s or non-Hodgkin ' s), melanoma, Huppert's disease, pancreas cancer and sarcoma.

As well known in the prior art, for example these cancer can use by independent use for example sharp appropriate former times monoclonal antibody, western appropriate former times monoclonal antibody the immunotherapy of bevacizumab or with the method treatment of chemotherapy or radiotherapy combination. In other example, the chemotherapy that can bring out catarrh comprises use (unitary agent or combination preparation use): platinum derivatives for example blocks platinum, suitable platinum and oxaliplatin (oxplatin); The mitosis inhibitor is taxol, docetaxel, Vinorelbine, vincristine and vincaleukoblastinum for example; Topoisomerase enzyme inhibitor for example relies on pool glycosides, Yi Li for health and topotecan; Anti-metabolism medicine is gemcitabine, Ka Peita shore, fludarabine, methotrexate (MTX), 5 FU 5 fluorouracil, gram 2-CdA, spray Si Tading and cytarabine for example; The DNA synthetic inhibitor for example Doxorubicin, epirubicin, Yi Da than star, daunorubicin, bleomycin, mustargen and Mi Tuo anthracene quinone; The alkylation agent is endoxan, different phosphorus acid amides and American and French Lun Kamositing for example; The hormone oncology is estramustine for example; And the Dacarbazine for example of the medicament with other or unknown mechanisms. These and other method for the treatment of cancer is that those skilled in the art are known.

But for example above-mentioned those TLR4 anti-dose of Application standard method short of money administration for example comprises defeated notes the in the local approach and vein. The specific mode and the dosage that use for particular patient depend on several factors, comprise the position of the kind of for example treatment of cancer, any discomfort and patient's general health status. According to these factors, professional doctor can select suitable method.

According to treatment of the present invention can be before treatment of cancer (for example 1-2 days or until front 1 week for the treatment of of cancer before the treatment of cancer), with treatment of cancer simultaneously or basically simultaneously (for example with treatment of cancer simultaneously, in the 1-4 hour, or on the same day), perhaps after treatment of cancer stops soon (for example stop in 1-4 days, and/or before symptom occurs or symptom when occurring) beginning. Then can keep treatment, for example until the risk of this kind symptom is removed or produced to the symptom of catarrh basically spends. Therefore, before treatment of cancer or with treatment of cancer simultaneously or basically simultaneously treatment can keep for example 1-2 days for example 1-3 days. In other embodiments, as those skilled in the art suitable determine like that, treatment is maintenance 1-4 or 2-3 week after treatment of cancer stops. In specific example, only before treatment of cancer, carry out according to treatment of the present invention; Only before treatment of cancer and with treatment of cancer, carry out simultaneously; Before treatment of cancer, simultaneously and carry out afterwards; Only carry out simultaneously with treatment of cancer; Only in treatment of cancer simultaneously and after treatment of cancer stops, carrying out; Only after stopping, treatment of cancer carries out; Perhaps only before treatment of cancer He after stopping, carrying out. In addition, according to the state of any symptom of catarrh, can in the patient, change, stop or restarting according to the treatment of the inventive method. Treatment can be carried out according to the time interval that those skilled in the art determine. For example, administration can every day 1, carry out for 2,3 or 4 times.

Suffering from portacaval mucositis or be among the patient who suffers from portacaval mucositis danger, anti-dose short of money of TLR4 described herein can be applied to the form of gel, paste, spraying, white agent, paste or paster ill in the oral cavity or is in the zone of ill danger. These patients also can be contained by use oral cavity lotion, chewing gum or the lozenge treatment of this medicine. These medicines also can be administered into by the preparation that uses gel, white agent, paste, suspension or suppository form the patient of rectum or vagina site infection. In addition, administration can be used enema. In another embodiment, in the ill patient of nasal cavity, medicine can be by the mode administration of topical described herein, perhaps the inhalation (for example referring to US 6,683,063) by medicine. Following described, in other method, medicine can pass through to inject (for example local injection), or by defeated notes the (vein is interior or artery is interior) administration.

The medical compounds preparation that is used for the above (or other) mode of administration is known in this field, and for example be disclosed in Remington ' s Pharmaceutical Sciences (the 18th edition), A.Gennaro edits, 1990, Mack Publishing Company, Easton, PA (also for example compiles referring to M.J.Rathbone, Oral Mucosal Drug Delivery, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1996; The people such as M.J.Rathbone compile, Modified-Release Drug Delivery Technology, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 2003; The people such as Ghosh compile, Drug Delivery to the Oral Cavity, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 2005; And the people such as Mathiowitz compiles Bioadhesive Drug Delivery Systems, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1999.

All patients, and particularly in body the position ill and be not easy to those patients of topical (perhaps being in ill danger) can be by whole body method infusion of therapeutic in the vein for example. This kind medication is convenient especially for the patient who has catheter in the administration position of chemotherapy or other medicine. The example of this kind method is as follows, and wherein the medicine of administration is that the specified amount of eritoran (referring to above) and medicine is based on 70 kilograms of patients' assumed average body weight. In first example, medicine can be by defeated the notes with than the low dosage administration in the continuous vein. As specific example, medicine during treating with 10-500 (for example 50-400 or 100-200) microgram/hour the continuous administration of speed. In altricious another example of needs of patients, medicine for example 0.1-20 (for example 1-8,2-7,3-6 or 4-5) milligram/hour dosage intermittently (for example every 12-24 hour) administration 2-6 (for example about 4) hour. In the variant of the method, be preservation dose after the initial or load doses, it is less than (for example half) load doses, and perhaps the back is the continuous infusion described in the first example. Those skilled in the art can determine according to the many factors of the observation of the serious degree of for example disease and improvement the duration of this kind treatment. With administration for example the TLR4 of eritoran defeated notes short of money anti-dose use other relevant details and be provided at US-2003-0105033-A1 (bolus injection or intermittent infusion) and WO 00/41703 (continuous infusion), the mode by reference of content is wherein incorporated into herein.

In passing through vein, fail when annotating administration Verbindung ritoran, the preferred device compatible with medicine and the equipment (catheter for example that uses, for example central authorities or systemic vein conduit, conduit, sedimentation basin (drip chamber), flashback bulbs, injection Y sites, cock valve and infusion bag). Especially, have been found that the size division of the medicine micelle that will form during the catheter with the own fixed basic antimicrobial coating of chlorine will be prepared, cause inadequate concentration in the blood. Therefore preferred the use has oneself fixed basic antimicrobial coating of non-chlorine for example, for example contains one or more other antibiotic for example device and the equipment of the antimicrobial coating of rifampin or minicyclin.

The present invention also comprises the reagent box that contains anti-dose short of money of one or more TLR4 (for example as mentioned above lipid A analogue, for example Verbindung ritoran) and use the explanation of medicine in method described herein. Also can choose wantonly in the reagent box and contain device or the equipment (oneself decides the catheter of coating for example not have chlorine) that is useful on administration and/or the solution that is used for administration, for example 5% dextrorotation sugar (for example glucose) solution.

The inventive method can be used separately or reduce collaborative use of method of the serious degree of catarrh with other. For example, the inventive method can be carried out with antibiotic or antimycotic therapy combination, for example relates to for example treatment of sharp mould element, both sexes mycin, acyclovir, Valaciclovir, clotrimazole and Fluconazole of administration antibiotic. As the specific example of this kind treatment, suffer from head and neck cancer and accept radiotherapeutic patient has gramnegative bacterium at the oropharynx position cluster. Use the selective cleaning in the oral cavity of Antimicrobial agent to have the benefit that reduces the portacaval mucositis relevant with radiotherapy, yet may limit the useful effect of this kind treatment. Antimicrobial therapy can killing bacteria, yet can not reduce endotoxin, and in fact may increase endotoxin. Because endotoxin is the Effective medium of inflammation, it may help the deterioration of catarrh, and therefore according to the present invention, use anti-endotoxin compounds (lipid A analogue for example, eritoran for example) and the common treatment of antibiotic can be used as more effectively means, in this kind patient, to treat portacaval mucositis.

The inventive method also can be used with palliative treatment is collaborative, comprises and uses local lotion, gel agent or the paste that contains lidocaine, Articaine and/or morphine and other anodyne or antiphlogistic. Can comprise following according to the specific example of the inventive method and TLR4 anti-dose of other medicine that is used in combination short of money and method: (restructuring cell cutin forms cell growth factor to Pa Lifuming (palifermin); RHuKGF; Kepivance^TM Amgen) and AES-14 (absorb strengthen Glu suspension) (Peterson, J.Support Oncol.4 (2Suppl.1) 9-13,2006); Oneself is fixed for the freezing therapy in oral cavity, Low level laser therapy, chlorine, Amifostine, hematology the growth factor, PTX and glutamine (Saadeh, Pharmacotherapy 25 (4): 540-554,2005); That Amifostine, antibiotic paste or lozenge, hydrolysis enzyme, borneol, benzyl reach is bright, calcium phosphate, honey, oral care regimen, poly-dimension ketone and the zinc sulfate (people such as Worthington, Cochrane Database Syst. Rev.2:CD000978,2006); Flurbiprofen (for example, pastes administration as tooth; The people such as Stokman, Support Care Cancer 13 (1): 42-48,2005); Diphenhydramine, hydroxide magnesium/aluminium hydroxide, sharp mould element and corticosteroid (people such as Chan, J.Oncol.Pharm.Pract. 11 (4): 139-143,2005); The oral cavity transforms mucus matter citric acid fentanyl (for example with the form administration of sugared ingot; The people such as Shaiova, Support Care Cancer 12 (4): 268-273,2004); The chlorine nitrazepam is (for example with tablet form; The people such as Gremeau-Richard, Pain 108 (102): 51-57,2004); Capsicum element is (for example with the form of sugared ingot; The people such as Okuno, J.Cancer Integr.Med. 2 (3): 179-183,2004); Chloramines ketone is (for example with the form of oral cavity lotion; The people such as Slatkin, Pain Med.4 (3): 298-303,2003); And grain property leucocyte-macrophage bacterium colony-stimulating factor (GM-CSF)/grain property leucocyte bacterium colony-stimulating factor (G-CSF), laser therapy and the glutamine additive (people such as Duncan, Aliment.Pharmacol.Ther.18 (9): 853-874,2003).

The present invention's part is based on following test result.

Embodiment 1

1. introduce

1.1 ultimate principle

Two kinds of C3H mouses (C3H/HeJ and C3H/HeOuJ) difference is to exist or do not exist LPS receptor TLR4 (being present in the C3H/HeOuJ kind).C3H/HeJ mouse is responsive more for the lethal effect of total body radiation, yet does not develop into the oral mucositis with C3H/HeOuJ mice same degree behind local acute radiation snout.The ultimate principle of these differences is not also understood.

1.2 acute snout radiation model

Acute mice snout radiation model in the mice has been used for the radiation protection performance of confirmed test chemical compound.The process of oral mucositis is determined and is being carried out producing in radiation 10-12 days the peak value mucositis very much in this model.Acute mucositis has general toxicity seldom, seldom produces radiogenic animal dead.In current research, we use the dosage of 30Gy to bring out oral mucositis.

2. goal in research and summary

2.1 goal in research

Goal in research as described below will be estimated local acute radiation to two kinds of mice oral mucositis orders of severity and the influence of persistent period.Wild type C3H/HeOuJ mice and endotoxin resistant strain C3H/HeJ compare.Mucositis uses the dosage that points to the mice snout to bring out as the acute radiation of 30Gy.Several time points after radiation, one group of four mice of each experimental group are killed.When death, remove tongue and be divided into 3.The front portion 1/3rd of each tongue is fixed in the formalin to carry out histologic analysis subsequently.Extract to provide mRNA to come analysis of cells factor expression level at the middle part 1/3rd of each tongue.The rear portion of each tongue quick freezing in liquid nitrogen is used for following analysis.When death, from each animal blood sampling and preparation serum to prepare to carry out cytokine analysis subsequently.This research concentrates on proinflammatory cytokine TNF-α and IL-6.

2.2 research summary

Use 64 mices altogether.56 mices (being respectively 28 C3H/HeOuJ and 28 C3H/HeJ) directly gave the radiation that single dose is 30Gy to snout at the 0th day.In addition, 8 mices (4 C3H/HeOuJ and 4 C3H/HeJ) are as there not being radiating control animals to use.Animal is killed and obtain blood and tissue according to step described in the table 1.

The histology of table 1.C3H/HeJ and C3H/He0uJ mice oral mucosa effects of ionizing radiation and cytokine are relatively

3. research design

Use 64 mices (32 C3H/HeOuJ and 32 C3H/HeJ).As shown in table 2, mice is divided into four groups at random: the group of 28 animals (group 1 and group 2) is as the radiation group, or the group of 4 animals (group 3 and group 4) is as radiation matched group not.

Group	The mice kind	Quantity	Radiation 30Gy
Group	The mice kind	Quantity	Radiation 30Gy		1	C3H/HeOuJ WT	28	Be
2	The C3H/HeJ mutant	28	Be		1	C3H/HeOuJ WT	28	Be
2	The C3H/HeJ mutant	28	Be	3	C3H/HeOuJ WT	4	Not
4	The C3H/HeJ mutant	4	Not	3	C3H/HeOuJ WT	4	Not

Table 2. experimental group animal distributes

Weigh to animal the every day (0-14 days) in research cycle.At the 0th day, the single dose that the animals received in the

group

1 and 2 concentrates on snout was the radiation of 30Gy.Remaining animal of lead shield barrier protection.After radiation 2 hours, 6 hours, 24 hours (1 day), 3 days, 6 days, 10 days and 14 days, kill 4 animals and as described below collection blood and the tissue of group in 1 and 2.Kill the animal in group 3 and the group 4, downcut tongue, and collected blood at first day.The tongue of each animal resolves into 3 (front portion, middle part and rear portions) and each tongue is fixed in the formalin.Hematoxylin and eosin (H﹠amp by histologic analysis formalin fixed tongue; E) coloured part carries out the mucositis test.Carry out the mucositis evaluation according to the checking scale in the mode of covering.Use the cytokine TNF-α and the IL-6 of standard ELISA test test sera sample.

4. material and method

4.1 animal

Using body weight is the 5-6 C3H/HeOuJ in age in week and the C3H/HeJ mouse (JacksonLaboratories) of 22.3 grams.Animal uses earhole to number respectively and with the stable breeding of about 5 animals/cage groups.Before the research beginning, animal is conformed.In at least 2 days time, observe animal every day to eliminate the animal of performance state difference.

4.2 stable breeding

Study in 70+/-5 the temperature and the relative humidity of 50%+/-20% providing between the Animal House of filtered air.Set between Animal House to keep the minimum per hour ventilation volume of 12-15.The room connects automatic timer, in the bright/dark cycle that keeps connection in 12 hours and closed in 12 hours, does not have low-light.Use Bed-O-Cobs

Laying and jede Woche change once at least.Cage, top, bottle etc. use commercial detergent wash and air-dry.Before using, with these article packing and HIGH PRESSURE TREATMENT.The commercial sterilisation agent is used for disinfecting surface and material is introduced ventilating kitchen.Bottom every day cleaning and at least twice drag with mop and to wash weekly.Wall and cage support were used the moistening of dilution liquid lime chloride sponge in every month once at least.Cage card or label with appropriate information of Study of recognition, dosage, number of animals and experimental group place whole cages.Record temperature and relative humidity during the research, and reservation record.

4.3 diet

Animal supply Labdiet

5001 foods also arbitrarily provide water.

4.4 animal randomization and distribution

Before radiation with mice at random and expection be divided into four experimental grouies.Each animal is by numbering corresponding earhole identification with each.The cage card is used to discern each cage, and perhaps label uses research numbering, test group # and number of animals labelling.

4.5 radiation

The verification in research beginning fortnight of machine scale.At the 0th day to organizing the radiation (30Gy/ dosage) that all animals in 1 and 2 gives single dose.Radiation uses 160 kilovolts of (15-ma) sources to produce at 50 centimetres of focal lengths, uses 0.35 millimeter copper filtration system sclerosis.Radiation is carried out with 121.5cGy/ minute speed.Animal was anaesthetized before radiation, and placed under the lead screen so that only snout exposes.

4.6 tissue and blood collecting and analysis

4.6.1 animal kills and tissue collecting

Animal in group 3 and the group 4 is not radiating control animal.The whole radiation sample that are measured as in the research of these animals provide baseline control.Each 4 animal in group 3 and the group 4 killed at the 1st day.

The several time points of animal during studying in group 1 and the group 2 kill.Kill four animals in every group at each time point.Time point is after the radiation 2 hours, 6 hours, 24 hours, 3 days, 6 days, 10 days and 14 days.

When killing, remove tongue and resolve into 3.The front 1/3rd of each tongue is fixed in the formalin to carry out histologic analysis subsequently.Extract to be provided for the mRNA of analysis of cells factor expression level the centre 1/3rd of each tongue.The aft section of each tongue quick freezing in liquid nitrogen also stores to carry out following analysis.

When killing, get about 1 milliliter of blood and prepare serum to prepare cytokine analysis subsequently from each animal.This research concentrates on proinflammatory cytokine TNF-α and IL-6.

4.6.2 cytokine ELISA

Use is carried out enzyme-linked immunosorbent assay (ELISAs) available from the test kit pair cell factor TNF-α and the IL-6 of R and D system.These test kits instruct according to producer and use.All test on the blood serum sample that is being stored in-80 ℃ and repeat.If do not collect enough samples to carry out IL-6 and TNF-α, diluted sample to 1: 2 or 1: 4, and whole tests are carried out in duplicate.All test uses 50 microlitre sample/holes to carry out.

4.6.3 histology

In histology's sample stuck-at-0% formalin normal saline and use standard technique to carry out the paraffin hydrocarbon histology.Coverslip uses hematoxylin and eosin (H﹠amp; E) dye, and check by the pathologist (board certified pathologist) that committee is assert.

4.7 evaluation of result

Significant difference between the test group uses unidirectional ANOVA to determine.Estimate the difference of body weight between experimental group.

5. result and discussion

5.1 survival rate

6 death took place altogether at the 10th day.This distributes in C3H/HeOuJ and C3H/HeJ group equally (in every group dead 3), and the result was at the 14th day animal dead only in every group.Other animal of radiation is subsequently replenished animal to provide, and the data of the 14th day time point are provided.

5.2 body weight (Fig. 1 and 2)

Every group average weight percent changes as shown in Figure 1.Body weight changes data and shows, two treated animals reduce it every day after radiation and begin the about 5% of body weight, and increases up to the 6th day body weight.From the 6th day up to the 13rd day, the C3H/HeJ mouse body weight remains on and does not increase and with respect between its initial weight increase 5%.The C3H/HeOuJ mice lost the about 10% of its body weight between the 7th day to the 9th day, and did not have weight increase before the 13rd day.In order to estimate the difference between two groups, calculate each animal area under a curve (AUC) and use unidirectional ANOVA assay difference.Average A UC data as shown in Figure 2.Unidirectional ANOVA the analysis showed that, has the significant difference of statistics (P=0.008) between two groups.

5.3 serum cytokines level

The cytokine of cytokine IL-6 and TNF-α is estimated by ELISA.

5.3.1 blood serum IL-6 concentration

In not radiating C3H/HeOuJ mice, the average serum concentration of IL-6 is 1.0pg/ml.Dropping in the 3rd day after the radiation before the 12.0pg/ml, these data were increased to 88.8pg/ml in 6 hours after radiation, and were increased to peak level 122.7pg/ml at the 6th day.The 10th day and the 14th day show the decline gradually from the 6th day observed peak level.In not radiating C3H/HeJ mouse, the average serum concentration of IL-6 is 13.8pg/ml.Except the 10th day time point, all other reading be in 25 and 42pg/ml between, yet blood serum IL-6 concentration is increased to 69.4pg/ml.These data as shown in Figure 3.

5.3.2 serum TNF-α concentration

In not radiating C3H/HeOuJ mice, average serum TNF-α concentration is 48.0pg/ml.In serum TNF-α concentration of these mices, have two peak values, one come across 2 hours after (168.7pg/ml) and one at the 10th day (410.2pg/ml).Time point between these two peak values, serum TNF-α concentration approach the level (31.6pg/ml to 87.2pg/ml) in radiation C3H/HeOuJ mice not.At the 10th day and the 14th day, this concentration was lower than not radiating matched group (being respectively 5.7pg/ml and 6.6pg/ml).In C3H/HeJ mouse, not radiation control mice has the average serum TNF-α concentration of 109.3pg/ml.Reading is usually less than this value after the radiation subsequently, 6 hours 10 days 133.8pg/ml of 14.2pg/ml to the after the radiation.These data as shown in Figure 4.

5.4 tongue histology

The part of each tongue is carried out conventional hematoxylin and eosin (H﹠amp; E) histological processing.The pathologist that these coverslipes are assert by committee then checks and estimates the number (comprising the differential cell type analysis) of inflammatory cell in epithelium and connective tissue pathology, epithelium mitosis, ulcer percentage ratio, Skeletal muscle injury, per 10 high power fields with the yardstick of 0-3, and little, in and the number of trunk.

5.4.1 Histological evaluation

The epithelium of each sample and connective tissue zone provide independent evaluation respectively.The evaluation of epithelium as shown in Figure 5.The average epithelial tissue that does not carry out radiating C3H/HeOuJ mice is evaluated as 0, and the time point situation also is like this after the whole radiation except that the 1st day (average ratings is 0.25), the 6th and 10 day (average ratings is 2).In C3H/HeJ mouse, average epithelial tissue evaluation is 0 at the All Time point except that the 6th day (being evaluated as 0.75).The data that average connective tissue is estimated as shown in Figure 6.There is not the average connective tissue of radiating C3H/HeOuJ mice to be evaluated as 2.Dropped to 0 and be increased to before 1.5 at the 3rd day, estimate after radiation 2 hours and drop to 0, after radiation, be increased to 1 on the 1st day at the 10th and 14 day.In C3H/HeJ mouse, average epithelial tissue evaluation is 1.25 in not having radiating mice, drops to 0.25 in 2 hours before being increased to 1.25 gradually on the 10th day after radiation.For the connective tissue in C3H/HeOuJ and the C3H/HeJ mouse, any time evaluation is the same not high or be higher than after the radiation any time evaluation after the Histological evaluation of radiation murine and the radiation.The current the unknown of this reason.

5.4.2 inflammation

Calculate the average of inflammatory cell in per 10 high power fields of each time point for every kind of mice, and the results are shown in Fig. 7.The cell number of seeing in the Radiata connective tissue is not higher than two kinds of desired values in the mice, and in the C3H/HeOuJ mice after whole radiation time point be lower than desired value.In the C3H/HeOuJ mice, except the 10th day (cell number is higher than about 2 times of radiation matched group not (and being higher than after the radiation 2 hours and about 10 times of 6 hours time points)) and the 14th day (cell number of observation be higher than radiation matched group not about 50%), the number of observed inflammatory cell also is lower than not observed number in the radiation matched group at most of time points.For connective tissue Histological evaluation, observed reason the unknown of high number unexpectedly in Radiata not.In most of animals, under the whole circumstances, the main body of penetrating fluid is made up of lymphocyte, and mononuclear cell and macrophage constitute almost all non-lymphocytes.A large amount of polymorphonuclear cell (polymorphonucleocytes) (PMNs or neutrophil) is only seen (1OuJ and 2HeJ) from the 10th day time point three animals.

5.4.3 epithelial cell mitosis

The number of the mitosis figure of seeing in the counting epithelium layer, and for the mitosis average of every group of mice in per 10 high power fields of each time point as shown in Figure 8.The number of the mitosis figure of counting in the epithelium layer of C3H/HeOuJ mice tongue is very low usually, and is average 04 in radiation murine not, and number (observing 2.75 meansigma methods) the All Time point in addition that is lower than the 6th day.In C3H/HeJ mouse, the mitotic number subaverage of observing in radiation murine not is 0.1 C3H/HeOuJ mice.Yet this value was increased to 0.4 on the 10th day after radiation.

5.4.4 blood vessel

For each sample is counted the quantity of per 10 high power field medium vesselses, and calculate the average of every kind of mice at each time point.These data as shown in Figure 9.The quantity of per 10 high power field medium vesselses is not 26.6 in the radiation C3H/HeOuJ mice, and is 27.2 in the not radiating C3H/HeJ mouse.In the C3H/HeOuJ mice, dropped to before 8.5 at the 3rd day, number of blood vessel obviously dropped to 4.5 in 2 hours after radiation, rose to 20.6 at the 1st day, and raise at the 6th day and the 10th day before reaching peak value 33.7 on the 14th day.With respect to not this expression raising 27% of radiation matched group, and with respect to time point raising 648% in 2 hours.Interesting is to it may be noted that the not radiation matched group that killed at the 1st day has and the 1st day similar level of time point.In C3H/HeJ mouse, blood vessel quantity approaches not radiating matched group usually, reaches 18.3 minimum in 2 hours after radiation, and reaches 33.1 maximum after radiation on the 6th day.Change in order to estimate the blood vessel quality, estimated the number of trunk in per 10 high power fields and the gained number is represented with the percentage ratio of the blood vessel sum seen in identical 10 high power fields.The result of this analysis as shown in figure 10, and show the trunk number in the C3H/HeOuJ mice, seen by the meansigma methods 9.2% of not radiation control mice bring up to the 1st day peak value 26.5% of (after the radiation 24 hours), and reduce at all the other time durations of research.C3H/HeJ mouse does not have the matched group of being higher than level 13.2% in the radiation murine, and it was increased to peak value 18.7% on the 6th day after radiation, and in the 10th day and the level that dropped to below the matched group in 14 days after the radiation.

6. conclusion

1. during this research, the C3H/HeOuJ mice shows the body weight loss bigger than C3H/HeJ mouse, and observed result significantly (P=0.008) statistically when using unidirectional ANOVA test evaluation.

2. the plasma cell factor level the analysis showed that, not radiating C3H/HeJ control mice has the level higher than its C3H/HeOuJ homologue, unless the C3H/HeOuJ mice shows the increase bigger than C3H/HeJ in carrying out the radiating plasma cell factor, after radiation, observed the peak level of IL-6 and TNF-α on the 6th day.

3. in C3H/HeJ mouse, demonstrate variation very little on the histology.The C3H/HeOuJ mice demonstrated tangible epithelium disorder in the 6th day and 10 days after radiation.The Histological evaluation of connective tissue is higher in the contrast of radiation not C3H/HeOuJ mice, and descends in 2 hours to 6 days after radiation, is returned in the 10th day and 14 days after radiation and approaches control level.

4. the number of inflammatory cell demonstrates very little change in C3H/HeJ mouse, yet is increased to peak value on the 10th day in the C3H/HeOuJ mice after radiation, with these animals organize peak value histiocyte factor level consistent.Penetrant is mainly lymphocyte in nature.

5. demonstrating in C3H/HeJ mouse slightly at the observed mitotic number of epithelium layer increases, and the 10th day peak value display, yet notices the obvious peak value of mitogen activation at the 6th day in the C3H/HeOuJ mice.

6. in the analysis of observed number of blood vessel and size, in C3H/HeJ mouse, notice seldom and change, yet the C3H/HeOuJ mice after the radiation immediately (after the

radiation

2 and 6 hours) show the minimizing of number of blood vessel, and the overall increase of time point subsequently (after the radiation the 10th day and 14 days).In the C3H/HeOuJ mice, after radiation, noticed the increase of trunk percentage ratio in 24 hours.

Example II

1. introduce

1.1 ultimate principle

As described in example I, two kinds of C3H mouses (C3H/HeJ and C3H/HeOuJ) difference is to exist or do not exist LPS receptor TLR4 (being present in the C3H/HeOuJ kind).In above-mentioned test, confirmed that the C3H/HeOuJ kind is easy to infect the oral mucositis that is brought out by the focused radiation snout, yet the C3H/HeJ group has resistance relatively to radiation induced mucositis.The evaluation of the proinflammatory cytokine in these animals shows that these cytokines can be played a role by inducing of LPS receptor TLR4 in the C3H/HeOuJ mice in the morbidity of oral mucositis.Research purpose as described below is that assessing compound blocks the stimulation of TLR4 (eritoran) in the oral mucositis mouse model.

1.2 acute snout radiation model

Acute mice snout radiation model in the mice has been used for the radiation protection performance of confirmed test chemical compound.In this model the process of oral mucositis determine very much and carrying out radiation after produced the peak value mucositis in 10-12 days.Acute mucositis has general toxicity seldom, seldom produces radiogenic animal dead.In current research, we use the dosage of 30Gy to bring out oral mucositis.

2. goal in research and summary

2.1 goal in research

The target of research as described below is to check that the eritoran of subcutaneous administration is to the order of severity of radiation induced oral mucositis and the influence of persistent period.Mucositis uses the dosage that points to the mice snout to bring out as the acute radiation of 30Gy.Several time points after radiation, one group of four mice of each experimental group are killed.When killing, remove tongue and resolve into 3.The front portion 1/3rd of each tongue is fixed in the formalin to carry out histologic analysis subsequently.The middle part 1/3rd of each tongue is extracted providing mRNA to come the analysis of cells factor expression, and the rear portion of each tongue quick freezing and store and be used for following analysis in liquid nitrogen.When killing, from each animal blood sampling and prepare serum to prepare to carry out cytokine analysis subsequently.

2.2 research summary

In this research, use 54 mices altogether.48 C3H/HeOuJ mices are divided into 3 groups (group 1-3) 16 every group.As shown in table 2, other 6 animals are dropped into independent matched group (group 4).

3. research design

Use 54 male C3H/HeOuJ mices in about 22 grams of body weight and 6-7 age in week.There are 16 every group of three groups of experimental grouies, and 6 matched groups that do not carry out radiating animal.All animals have and were inserted into the jugular jugular vein intubate in left side at the 3rd day.Beginning in the 0th day, the animal via of group in 1 and 4 by intubate by the injection placebo that is administered twice every day.Before radiation in the 0th day 2 hours or be lower than beginning in 2 hours and lasted till that the animal of group in 2 was with the 2IV injection of 1 mg/kg administration eritoran every day the 10th day.Before radiation in the 0th day 2 hours or be lower than beginning in 2 hours and lasted till that the animal of group in 3 was with the 2IV injection of 10 mg/kg administration eritoran every day the 10th day.At the 0th day, the single dose that the animals received in the

group

1,2 and 3 is pointed to snout was the radiation of 30Gy.6 animals in the group 4 are as radiationless control animals (referring to table 2).8 animals of group each in 1,2 and 3 are killed and according to described collection step blood of table 2 and tissue.

Table 2: experimental group distributes

Group	Size of animal	Kind	Handle	Radiation	Kill time point
Group	Size of animal	Kind	Handle	Radiation	Kill time point	1	16 is male	C3H/HeOuJ	Placebo IV bid	30Gy is to snout	The 6th day 8, the 10th day 8
2	16 is male	C3H/HeOuJ	eritoran 1mg/kg IV bid	30Gy is to snout	The 6th day 8, the 10th day 8	1	16 is male	C3H/HeOuJ	Placebo IV bid	30Gy is to snout	The 6th day 8, the 10th day 8
2	16 is male	C3H/HeOuJ	eritoran 1mg/kg IV bid	30Gy is to snout	The 6th day 8, the 10th day 8	3	16 is male	C3H/HeOuJ	eritoran 10mg/kg IV bid	30Gy is to snout	The 6th day 8, the 10th day 8
4	6 is male	C3H/HeOuJ	Placebo IV bid	Do not have	The 10th day 6	3	16 is male	C3H/HeOuJ	eritoran 10mg/kg IV bid	30Gy is to snout	The 6th day 8, the 10th day 8

4. material and method

4.1 animal

Using body weight is the 5-6 week C3H/HeOuJ mice in age (JacksonLaboratories) of 21.3 grams.Animal uses earhole to number respectively and stable breeding separately.Before the research beginning, make animal conform.In at least 2 days time, observe animal every day to eliminate the animal that shows as state difference.

4.2 stable breeding

Described in example I part 4.2, study between Animal House.

4.3 diet

To animal supply Labdiet

5061 aseptic radiation rodent foods also arbitrarily provide water.

4.4 animal randomization and distribution

Before radiation with mice at random and expection be divided into three (3) individual experimental grouies.Each animal is by numbering corresponding earhole identification with each.The cage card is used to discern each cage, and perhaps label uses research numbering, test group # and number of animals labelling.

4.5 radiation

4.6 tissue collecting and analysis

4.6.1 histology

In histology's sample stuck-at-0% formaldehyde normal saline solution and use standard technique to carry out the paraffin hydrocarbon microscopic anatomy.Coverslip uses hematoxylin and eosin (H﹠amp; E) dyeing.

4.7 evaluation of result

5. result and discussion

5.1 body weight (Figure 11 and 12)

The average weight percent increase of every group of every day as shown in figure 11 in the research.The radiation matched group does not increase by 8.1% at the research period average, and average loss 0.8% in the placebo group.In accepting the group of eritoran, accepting to observe in the group of 1 mg/kg 4.0% average weight increases, yet is 0.2% body weight loss in the group of accepting 10 mg/kg.The analysis result of three winding rayings as shown in figure 12.In these three groups, there is not marked difference (P=0.261).When with radiation matched group not relatively the time, radiation matched group not and accept placebo (P＜0.001) and the radiation group of eritoran 10 mg/kg (P＜0.001) between have marked difference.

5.4 tongue histology

Each tongue carries out conventional hematoxylin and the eosin histology handles.Because all technical reasons are estimated 44 samples altogether.In these 44 samples, 6 are in not radiation matched group, 12 are in placebo group (6 at the 6th days, 6 at the 10th day), 14 are in eritoran 1 mg/kg processed group (7 at the 6th days, 7 at the 10th day), and 12 be in eritoran10 mg/kg processed group (7 the 6th day, 5 at the 10th day).

Based on the prevailing complete observation of complete data set for normally or normal basically.This is used for the explanation of 14 samples, and wherein 4 are not radiating matched group.Normal 6 (the 6th day 2/7 samples, and the 10th day 4/7 sample) that also are used for describing 14 of 1 mg/kg eritoran processed group, and 3 (the 6th day 1/7 samples, and the 10th day 2/5 sample) of 12 in the 10 mg/kg eritoran processed group.Only in 12 samples is called as normal (the 6th day sample) in the placebo treatment group.Hyperkeratosis finds in 14 samples that also wherein neither one is in not radiation matched group.Hyperkeratosis is the most common to be occurred in eritoran 10 mg/kg processed group, and wherein it is found in 7 (the 6th day 3/7 samples, and the 10th day 4/5 sample) in 12 samples.Hyperkeratosis 5 discoveries in 14 samples in eritoran 1 mg/kg processed group (the 61/7th sample, and the 10th day 4/7 sample).Only 2 samples are found hyperkeratosis in placebo group, one of each time point.Only in 5 samples, find epithelial hyperplasia, be in placebo group (the 6th day) yet in these samples 4 are in eritoran 10 mg/kg experimental grouies (2 of each time points) and the 5th.As if these observed results show the improvement that whole eritoran experimental grouies are suitable with respect to placebo group, and wherein high dose experimental group (10 mg/kg) shows hypertrophy and Hyperkeratotic tendency.

Connective tissue damage or destruction are being seen in 11 samples altogether, wherein 8 are in the placebo treatment group (3/6 at the 6th days, and 5/6 at the 10th day), 2 is eritoran 1 mg/kg processed group (all at the 6th day), one is in eritoran 10 mg/kg experimental grouies (the 6th day).Losing or the record in 7 samples that breaks of epithelium, and go up atrophoderma record in other 5 samples.In 12 samples with epithelium loss, 5 are in the placebo treatment group (2 at the 6th days, 3 at the 10th day), 5 are in eritoran 1 mg/kg processed group (entirely at the 6th day), and 2 are in eritoran 10 mg/kg groups (entirely at the 6th day).The cellularity that improves is seen in 10 samples, 2 are in placebo group (1 at the 6th days, 1 at the 10th day), 5 are in eritoran 1 mg/kg processed group (1 at the 6th days, 4 at the 10th day), and 3 are in eritoran 10 mg/kg processed group (1 the 6th day, 2 at the 10th day).Observed two class penetrants, circle cell or lymphocyte penetrant record in 8 samples at each group and time point uniform distribution, and are seen among in 6 of radiation matched groups not one.The mastocyte penetrant is observed in 9 samples, and wherein 7 are in placebo group (5 the 6th day, 2 at the 10th day), and other 2 samples are in eritoran 10 mg/kg processed group, the 6th day time point.Consider other observation uniform distribution of vasodilation and raising vascularity, perhaps be difficult to see to show any significant difference between the experimental group.As reduce shown in radiation-induced connective tissue damage and the mastocyte infiltration, these observations show that the eritoran treatment produces the tongue histology who improves.

5.3.1 the thickness of the tongue back side and the outside of belly

Each sample is estimated the minimal amount and the maximum number of the tongue back side and outside of belly epithelium layer.Number calculates average minimum and the maximum ga(u)ge of each experimental group at each time point thus.For the back side of tongue, the average minimal amount of cellular layer is not 6 cellular layers in the radiation matched group.In the placebo experimental group, the average of cellular layer was 2.7 at the 6th day, was 2.0 at the 10th day.In eritoran treatment group, the minimum average B configuration number of cellular layer is 2.8 (the 6th days) and 3.8 (the 10th days) in 1 mg/kg group, is 3.8 (the 6th days) and 3.3 (the 10th days) in 10 mg/kg groups.These data as shown in figure 13.The maximum average of back side epithelium layer is 8 in radiation matched group not.In the placebo experimental group, the average of cellular layer was 4.8 at the 6th day, was 3.5 at the 10th day.In eritoran treatment group, the minimum average B configuration number of cellular layer is 5.3 (the 6th days) and 6.4 (the 10th days) in 1 mg/kg group, is 6.2 (the 6th days) and 6.1 (the 10th days) in 10 mg/kg groups.These data as shown in figure 14.At the outside of belly, the minimum average B configuration number of cellular layer is 4 cellular layers in radiation matched group not.In the placebo experimental group, the average of cellular layer was 1.7 at the 6th day, was 0.9 at the 10th day.In eritoran treatment group, the minimum average B configuration number of cellular layer is 2.0 (the 6th days) and 2.8 (the 10th days) in 1 mg/kg group, is 3.0 (the 6th days and the 10th day) in 10 mg/kg groups.These data as shown in figure 15.At the outside of belly, the maximum average of epithelium layer is 6 in radiation matched group not.In the placebo experimental group, the average of cellular layer was 4.2 at the 6th day, was 2.4 at the 10th day.In eritoran treatment group, the minimum average B configuration number of cellular layer is 3.7 (the 6th days) and 4.6 (the 10th days) in 1 mg/kg group, is 5.8 (the 6th days) and 5.9 (the 10th days) in 10 mg/kg groups.These data as shown in figure 16.These observations show that eritoran seems the protective epithelium cellular layer, and 10 mg/kg groups show than the epithelium layer in 1 mg/kg the group bigger slightly protection, the particularly outside of belly.

6. conclusion

1. during studying, observe tangible mortality rate, yet too high mortality rate is not relevant with any one test group.

2. in three radiation experiments groups, do not seeing the statistics marked difference aspect the weight increase.

3. be described as quantity, epithelial hyperplasia and the Hyperkeratotic increase of normal sample and the reduction of connective tissue damage and mastocyte penetrant by measurement, with respect to the matched group of placebo treatment, the group of two use eritoran processing all shows the improvement on the tongue histology.

4. though the group of two use eritoran processing all shows the improvement on the tongue histology, has the tangible difference of described histology between 1 mg/kg and 10 mg/kg, but does not know which dosage demonstrates bigger improvement.

EXAMPLE III

1. introduce

1.1 ultimate principle

As mentioned above, two kinds of C3H mouses (C3H/HeJ and C3H/HeOuJ) difference is to exist or do not exist LPS receptor TLR4 (being present in the C3H/HeOuJ kind), and the C3H/HeOuJ kind is easy to infect the oral mucositis that is brought out by the focused radiation snout, yet the C3H/HeJ group has resistance relatively to radiation induced mucositis.In addition as mentioned above, the evaluation of the proinflammatory cytokine in these animals shows that these cytokines can be played a role by inducing of LPS receptor (TLR4) in the C3H/HeOuJ mice in the morbidity of oral mucositis.The purpose of testing described in the example II has proved the effect of eritoran in the oral mucositis model.The best administration time table of eritoran of having determined as described below.

1.2 acute snout radiation model

Acute mice snout radiation model has been used for the radiation protection performance of confirmed test chemical compound.The process of oral mucositis is determined and is being carried out producing in radiation 10-12 days the peak value mucositis very much in this model.Acute mucositis has general toxicity seldom, seldom produces radiogenic animal dead.In current research, we use the dosage of 30Gy to bring out oral mucositis.

2. goal in research and summary

2.1 goal in research

The target of this research is to check the influence of the eritoran timetable of intravenously administrable, to the order of severity of radiation induced oral mucositis and the influence of persistent period.Mucositis uses the dosage that points to the mice snout to bring out as the acute radiation of 30Gy.After radiation the 10th day, one group of four mice of each experimental group were killed.When killing, remove tongue and be fixed in the formalin to carry out histologic analysis subsequently.When killing, from each animal blood sampling and prepare serum to prepare to carry out cytokine analysis subsequently.These samples are used to measure tumor necrosis factor (TNF-α), interleukin-6 (IL-6) and blood plasma amyloid A (SAA) level.

2.2 research summary

60 (60) C3H/HeOuJ mices derive from Jackson Laboratories.When shipping, these animals have been inserted with venous cannulation.As shown in table 4, these animals are divided into 6 groups, every group of 10 animals.

3. research design

Use 60 (60) the only male C3H/HeOuJ mices in about 22 grams of body weight and 6-7 age in week.Exist five (5) individual every group be the experimental group of ten (10) individual animals, and the matched group of not accepting radiating ten (10) individual animals.Since the 0th day, before the radiation 2 hours or be lower than 2 hours, administration placebo or eritoran 10 mg/kg described in animal among the group 1-6 such as the table 4.Be administered twice continuous every day by the 9th day from the radiation same day (the 0th day).Animal in group 1 and the group 2 is accepted placebo in the whole administration stage.Animal in the group 3 is accepted the eritoran of 10 mg/kg in the whole administration stage.The animal of group in 4 accepted the eritoran of twice 10 mg/kg every day from the 0th day to the 3rd day, accept twice placebo every day up to the end in administration stage then.The animal of group in 5 from the 0th day to the 2nd day every day accept twice placebo, accept the eritoran of twice 10 mg/kg then every day from the 3rd day to the 6th day, accept twice placebo every day up to the end in administration stage then.The animal of group in 6 from the 0th day to the 5th day every day accept placebo twice, accept the eritoran of twice 10 mg/kg then every day up to the end in administration stage.All medicine and placebo administration are undertaken by intravenous injection via venous cannulation.

Table 4 experimental group distributes

Group	Size of animal	Handle	Eritoran	Placebo	Dosage
Group	Size of animal	Handle	Eritoran	Placebo	Dosage		1	10 is male	Radiationless placebo		0-9 days	0.1mL
2	10 is male	Placebo		0-9 days	0.1mL		1	10 is male	Radiationless placebo		0-9 days	0.1mL
2	10 is male	Placebo		0-9 days	0.1mL	3	10 is male	0-9 days 10mg/kg bid of eritoran	0-9 days		0.1mL
4	10 is male	0-3 days 10mg/kg bid of eritoran	0-3 days	4-9 days	0.1mL	3	10 is male	0-9 days 10mg/kg bid of eritoran	0-9 days		0.1mL
4	10 is male	0-3 days 10mg/kg bid of eritoran	0-3 days	4-9 days	0.1mL	5	10 is male	3-6 days 10mg/kg bid of eritoran	3-6 days	﹠ was 7-9 days in 0-2 days	0.1mL
6	10 is male	6-9 days 10mg/kg bid of eritoran	6-9 days	0-5 days	0.1mL	5	10 is male	3-6 days 10mg/kg bid of eritoran	3-6 days	﹠ was 7-9 days in 0-2 days	0.1mL

Every day during the research (0-10 days), each animal is weighed to the degree of accuracy of 0.1 gram.After radiation the 10th day, all animals is killed and get its tongue be used for histologic analysis.When killing, get blood and with blood plasma-80 ℃ of storages.

4. material and method

4.1 animal

Using body weight is the 5-6 week C3H/HeOuJ mice in age (JacksonLaboratories) of 23.2 grams.Animal has the jugular vein intubate that Jackson Laboratories installed before delivery, and uses earhole to number respectively and scheme separately and support.Before the research beginning, make animal conform.In at least 2 days time, observe animal every day and show as the animal that is not in good state to eliminate.

4.2 stable breeding

Described in example I part 4.2, study between Animal House.

4.3 diet

To animal supply Labdiet

5001 aseptic radiating rodent foods also arbitrarily provide water.

4.4 animal randomization and distribution

4.5 radiation

4.6 tissue collecting and analysis

4.6.1 histology

4.6.2 cytokine ELISA

Use is carried out enzyme-linked immunosorbent assay (ELISAs) available from the test kit pair cell factor TNF-α and the IL-6 of R and D system.The ELISA test kit that definite use of blood plasma amyloid A derives from BiosourceInternational carries out.These test kits instruct according to producer and use.All test on the blood serum sample that is being stored in-80 ℃ and repeat.In all three tests, sample is tested in duplicate, and if do not collect enough samples to carry out IL-6, SAA and TNF-α, diluted sample to 1: 4.All test uses 50 microlitre sample/holes to carry out.

4.7 evaluation of result

5. result and discussion

The animal of 108 dress intubate is used for this research altogether.Since the limited utilization rate of C3H/HeOuJ mice, these animals during 6 weeks in 3 groups of processing of branch.At the 10th day, 57 survivals in these mices.In 51 mices that do not survive in the 10th day, 21 the 0th day death or euthanasia, 11 because anesthesia and the death of radiation relevant issues, and 10 because the death of intubate problem (condense in initial injection back owing to think, intubate does not stretch, and perhaps intubate is moved the outside to and death).During studying in other 30 animals of death or euthanasia, 2 death in the 1st day, and 5 death in the 2nd day, 4 death in the 3rd day, and 7 death in the 4th day, the 5th and 6 day each dead 3, the the 7th and 8 day each dead 1, and the 9th and 10 day each dead 2.Each organizes dead distribution relative equilibrium.Respectively do not observe nine (9) individual death in radiation matched group and the excipient matched group at each.In the group of using 10 mg/kg eritoran to handle in 0-10 days or 0-3 days, respectively observe seven (7) individual death.Nine death were observed up to the group of using 10 mg/kg eritoran to handle on the 6th day from the 3rd day, and 10 death were observed up to the group of using 10 mg/kg eritoran to handle on the 9th day from the 6th day.

5.2 body weight (Figure 17,18,19 and 20)

The average weight percent increase of research each group every day as shown in figure 17.The radiation matched group does not increase by 3.2% at the research period average, and average loss 12.1% in the placebo group.Accepting with 10 mg/kg in the group of eritoran, in the group of handling in 0-10 days, find 7.8% average weight loss, in the group of handling in 0-3 days, find 2.2% body weight net loss, in the group of handling in 3-6 days, find 7.3% body weight net loss, and in the group of handling in 6-9 days, find 8.9% body weight net loss.In order to determine whether observed difference is remarkable in body weight change, area under the averaged curve (AUC) data are carried out unidirectional ANOVA.Analysis result as shown in figure 18.Accept radiating three groups and obviously be different from unirradiated nonirradiated matched group, placebo group (P＜0.001), the group (P=0.014) that use eritoran handled from the 0th day to the 10th day and the group (P=0.025) of using eritoran to handle from the 6th day to the 9th day.The group of using the group of eritoran processing from the 0th day to the 3rd day or using eritoran to handle from the 3rd day to the 6th day significantly is not different from unirradiated nonirradiated matched group.Yet the group of using eritoran to handle from the 0th day to the 3rd day has than the significantly lower loss in weight of placebo group (P=0.030).The data of removing all dead animals during weight data uses and studies and getting reanalyse.Analysis result is shown in Figure 19 and 20.Except the group of using eritoran to handle from the 0th day to the 9th day obviously was not different from not the radiation matched group, the result that unidirectional ANOVA analyzes also seldom changed in analysis.

5.3 tongue histology

Each tongue carries out conventional hematoxylin and the eosin histology handles, and coverslip is observed in the mode of covering.Estimate 57 samples altogether, and wherein 9 are in not radiation matched group, 9 are in placebo group, 11 are in the group of handling with 10 mg/kg eritoran from the 0th day to the 9th day, 11 are in the group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day, 9 are in the group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day, and 9 are in the group of handling with 10 mg/kg eritoran from the 6th day to the 9th day.Three parts of each sample are estimated following parameter: inflammatory cell among the mitosis in epithelium evaluation, connective tissue evaluation, inflammation evaluation, per 10 high power fields (hpf), ulcer percent, the every 10hpf (neutrophil, lymphocyte and monocyte/macrophage percent) number, every 10hpf be medium and small, in and the number of trunk, and the number of mastocyte among every 10hpf.

5.3.1 epithelium evaluation

As described in the 4.7.1 part, estimate epithelial tissue with 4 of 0-3 yardsticks.These are estimated as shown in figure 21.Not radiating animal all has estimates 0.Because this group is handled from the 0th day to the 9th day with 10 mg/kg eritoran, the placebo group has average ratings 1.1.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has average ratings 0.45.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has average ratings 0.89.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has average ratings 0.75.

5.3.2 connective tissue evaluation

As described in the 4.7.1 part, estimate connective tissue with 4 of 0-3 yardsticks.These are estimated as shown in figure 22.Not radiating animal all has estimates 0.The placebo group has average ratings 0.4, and the group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has average ratings 0.6.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has average ratings 0.4.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has average ratings 0.6.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has average ratings 0.8.

5.3.3 inflammatory evaluation

As described in the 4.7.1 part, estimate inflammatory with 4 of 0-3 yardsticks.These are estimated as shown in figure 23.Not radiating animal all has estimates 0.The placebo group has average ratings 0.4, and the group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has average ratings 0.5.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has average ratings 0.4.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has average ratings 0.6.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has average ratings 0.8.

5.3.4 mitosis number

Mitotic number is counted in 10 high power fields (hpf).These data as shown in figure 24.Not radiating animal has the meansigma methods of 1.2 mitosiss/10hpf.The placebo group has the meansigma methods of 3.9 mitosiss/10hpf.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the meansigma methods of 2.3 mitosiss/10hpf.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the meansigma methods of 1.5 mitosiss/10hpf.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the meansigma methods of 1.6 mitosiss/10hpf.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the meansigma methods of 1.5 mitosiss/10hpf.

5.3.5 ulcer percent

Estimate the ulcer percent of each sample.These data as shown in figure 25.Not radiating animal does not have ulcer.The placebo group has 13.3% average ulcer.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has 13.2% average ulcer.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has 2.7% average ulcer.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has 16.7% average ulcer.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has 10.0% average ulcer.

5.3.6 inflammatory cell infiltration

The inflammatory cell infiltration that exists in each sample is calculated by the sum of counting inflammatory cell among every 10hpf, and estimates cell type by estimating penetrant inner cell percent, and it is neutrophil, lymphocyte or monocyte/macrophage.The number of inflammatory cells destination data is listed in Figure 26, and neutrophil percent is listed in Figure 27, and lymphocyte percentage is listed in Figure 28, and monocyte/macrophage percent is listed in Figure 29.Not radiating animal has the meansigma methods of 9.3 cell/10hpf, has the average composition of 98.9% lymphocyte and 1.1% monocyte/macrophage, does not observe neutrophil.The placebo group has the meansigma methods of 44.9 cell/10hpf, has the average composition of 10.6% neutrophil, 86.7% lymphocyte and 2.8% monocyte/macrophage.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the meansigma methods of 43.6 cell/10hpf, has the average composition of 13.6% neutrophil, 82.7% lymphocyte and 4.5% monocyte/macrophage.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the meansigma methods of 33.3 cell/10hpf, has the average composition of 6.4% neutrophil, 93.2% lymphocyte and 0.5% monocyte/macrophage.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the meansigma methods of 31.5 cell/10hpf, has the average composition of 7.2% neutrophil, 91.1% lymphocyte and 1.1% monocyte/macrophage.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the meansigma methods of 52.1 cell/10hpf, has the average composition of 8% neutrophil, 91.0% lymphocyte and 1.0% monocyte/macrophage.

5.3.7 blood vessel

The number of blood vessel that exists in each sample is calculated by the sum of counting every 10hpf medium vessels, and by count this sample medium and small, in and the number of trunk estimate vessel size.Data are shown in Figure 30-32.Radiata does not have the meansigma methods of 5.6 blood vessel/10hpf, observes the average composition of 63.3% little blood vessel, 20.7% medium vessels and 16.0% trunk.The placebo group has the meansigma methods of 8.8 blood vessel/10hpf, has the average composition of 63.9% little blood vessel, 22.3% medium vessels and 13.9% trunk.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the meansigma methods of 9.4 blood vessel/10hpf, has the average composition of 74.6% little blood vessel, 16.1% medium vessels and 9.3% trunk.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the meansigma methods of 8.2 blood vessel/10hpf, has the average composition of 72.5% little blood vessel, 14.9% medium vessels and 12.6% trunk.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the meansigma methods of 7.0 blood vessel/10hpf, has the average composition of 67.9% little blood vessel, 20.5% medium vessels and 11.6% trunk.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the meansigma methods of 7.5 blood vessel/10hpf, has the average composition of 72.1% little blood vessel, 17.3% medium vessels and 10.6% trunk.

5.3.8 mastocyte

The number of mastocyte is determined by cell number among the every 10hpf of counting in each sample.Data as shown in figure 33.The every 10hpf of Radiata does not have 23.7 mastocytes.The every 10hpf of placebo group has 26 mastocytes.The every 10hpf of group that handled with 10 mg/kg eritoran from the 0th day to the 9th day has 18.4 mastocytes.The every 10hpf of group that handled with 10 mg/kg eritoran from the 0th day to the 3rd day has 24.4 mastocytes.The every 10hpf of group that handled with 10 mg/kg eritoran from the 3rd day to the 6th day has 24.2 mastocytes.The every 10hpf of group that handled with 10 mg/kg eritoran from the 6th day to the 9th day has 24.5 mastocytes.

5.4 plasma cell factor level

Use commercially available ELISA test kit to measure TNF-α, IL-6 and SAA serum levels.

5.4.1 serum TNF-alpha levels

Radiata does not have serum TNF-alpha levels of 43.0pg/ml.The placebo group has the average serum TNF-alpha levels of 63.8pg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the average serum TNF-alpha levels of 62.0pg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the average serum TNF-alpha levels of 20.3pg/ml.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the average serum TNF-alpha levels of 40.2pg/ml.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the average serum TNF-alpha levels of 119.1pg/ml.Data as shown in figure 34.

5.4.2 blood serum IL-6 level

Radiata does not have the blood serum IL-6 level of 48.7pg/ml.The placebo group has the average serum IL-6 level of 154.2pg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the average serum IL-6 level of 85.6pg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the average serum IL-6 level of 19.7pg/ml.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the average serum IL-6 level of 50.4pg/ml.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the average serum IL-6 level of 119.3pg/ml.Data as shown in figure 35.

5.4.3 Serum SA A level

Radiata does not have the Serum SA A level of 597 mcg/ml.The placebo group has the average serum SAA level of 427 mcg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 9th day has the average serum SAA level of 344 mcg/ml.The group of handling with 10 mg/kg eritoran from the 0th day to the 3rd day has the average serum SAA level of 279 mcg/ml.The group of handling with 10 mg/kg eritoran from the 3rd day to the 6th day has the average serum SAA level of 475 mcg/ml.The group of handling with 10 mg/kg eritoran from the 6th day to the 9th day has the average serum SAA level of 652 mcg/ml.Data as shown in figure 36.

6. conclusion

1. in this research, there is not evidence to show the toxicity of eritoran in mortality rate or loss in weight data.As in the early stage research, mortality rate is higher, however each the group in uniform distribution.

2. with respect to the group of using placebo treatment, the mice of using eritora to handle at 0-3 days is showing tangible the improvement aspect losing weight.

3. the level of the oral mucositis of observing in placebo group is lower than expection, makes to be difficult to estimate the influence of eritoran to the oral mucositis level.

4. compare with the early stage observation of this therapeutic scheme effect, may be because influence be seldom observed in the mucositis of observed reduced levels in the placebo group the group of handling with 10 mg/kg eritoran from the 0th day to the 9th day.

5. in accepting radiating group, the group of using eritoran to handle at 0-3 days has minimum epithelium evaluation, connective tissue evaluation and ulcer percentage ratio, shows that it suffers than other group damage still less.This group also has minimum inflammatory evaluation and next to the lowest inflammatory cell and mitosis number.

6. in accepting radiating group, the group of using eritoran to handle at 0-3 days has minimum TNF-α, IL-6 and SAA blood plasma level, shows the effectiveness of these systems in reducing inflammatory responses.

Claims

1. reduce the method for the mouth or the gastrointestinal mucositis order of severity in the patient, described method comprises the step that contains blocking-up Toll sample receptor 4 active compound compositions to patient's administration.

2. according to the process of claim 1 wherein that chemical compound is a lipid A analogue.

3. the method for claim 2, wherein lipid A analogue is in the following formula scope:

R wherein ¹Be selected from down group:

R ²Be straight or branched C5-C15 alkyl;

R ³Be selected from straight or branched C5-C18 alkyl

R ⁴Be selected from straight or branched C4-C20 alkyl and

R ⁶Be selected from hydroxyl, halogen, C1-C5 alkoxyl and C1-C5 acyloxy;

A ¹And A ²Be independently selected from:

OH，

o-z-Co ₂H

Wherein Z is a straight or branched C1-C10 alkyl; Or its pharmaceutically acceptable salt or its phosphate ester.

4. the method for claim 3, wherein lipid A analogue has following structure

Or be its pharmaceutically acceptable salt or its phosphate ester.

5. the method for claim 4, wherein lipid A analogue has following structure

Or be its pharmaceutically acceptable salt or its phosphate ester.

6. the process of claim 1 wherein that mucositis is a stomatitides.

7. the process of claim 1 wherein that mucositis is a gastrointestinal mucositis.

8. the process of claim 1 wherein that described patient suffers from mucositis.

9. the process of claim 1 wherein that the patient does not suffer from mucositis and but is in the catarrhal risk of development.

10. the method for claim 9, wherein the administration of catarrhal development by compositions suppresses among the patient.

11. the method for claim 10, wherein the administration of catarrhal development by compositions prevents among the patient.

12. the process of claim 1 wherein that the patient is the cancer patient.

13. the process of claim 1 wherein that the patient carries out recently, will carry out at once, perhaps currently carrying out head or cervical region radiation therapy, or stem cell or bone marrow transplantation.

14. the process of claim 1 wherein described dosing step make the patient be in the treatment of risk of mucositis development before, simultaneously or afterwards or its combination carry out.

15. the method for claim 14, wherein said dosing step is carried out the patient before being in the treatment of mucositis developing risk.

16. the method for claim 14, wherein said dosing step is carried out simultaneously with the treatment that makes the patient be in the mucositis developing risk.

17. the method for claim 14, wherein said dosing step is carried out the patient after being in the treatment of mucositis developing risk.

18. the method for claim 14, wherein said dosing step and the treatment that makes the patient be in the mucositis developing risk are carried out simultaneously, are included in addition to make the patient be in 0-3 days administration said compositions step at least once after the treatment of mucositis developing risk.

19. the method for claim 14, the treatment that wherein makes the patient be in the mucositis developing risk comprises radiation therapy.

20. the method for claim 14, the treatment that wherein makes the patient be in the mucositis developing risk comprises chemotherapy.

21. the process of claim 1 wherein that the said composition topical is to the patient.

22. the process of claim 1 wherein that compositions is administered into the patient by intravenous infusion.

23. the method for claim 1 comprises the step that applies antimicrobial therapy to the patient in addition.

24. the method for claim 23, wherein antimicrobial therapy is an antibiotherapy.

25. blocking-up Toll sample receptor 4 active chemical compounds preparation be used for the patient reduce mouthful or the medicine of the gastrointestinal mucositis order of severity in application.

26. the application in the claim 25, wherein said chemical compound are the lipid A analogue of following formula structure,

Or its pharmaceutically acceptable salt or its phosphate ester.