Patent Application: US-201214369460-A

Abstract:
provided is a composition of detection agents for living cells , especially epithelial tumor cells ; the composition contains 0 - 5 % folic acid , 0 - 10 % folic acid complex , 0 . 01 - 5 % methylene blue , 0 . 1 - 10 % carbohydrate reducing agent , 2 - 6 % acetic acid , and 3 - 95 % water . also provided is a preparation method for the composition of detection agents and kits containing the composition of detection agents .

Description:
it is shown in clinical studies that folic acid receptors are excessively expressed in the surface of most tumor cells , whereas are only present in few amount in normal cells . therefore , folic acid and folic acid complexes ( folic acid derivatives ) can serve as tumor specific targeting molecule for diagnosis and treatment of tumors [ 4 - 5 ] . in the invention , the term “ folic acid complex ” is defined as a complex formed from the binding of the folic acid molecule to one or more other components , wherein the carboxylic group of the folic acid binds to the one or more other components by coupling or conjugation . the one or more other components may be , for example , drugs , radioactive nuclides , dyes , genes , developing agents , and the like . examples of the folic acid complex are folic acid - mitomycin complex and folic acid - dtpa complex , and the like . in the composition and method of the invention , folic acid and / or folic acid complex may be administered simultaneously , or may be used alone . the folic acid complex used in the detecting agent composition described in the invention include folic acid conjugates formed by coupling of various small molecule compounds with folic acid , which include , but are not limited to folic acid — γ - cysteine , ( r )- 2 -( 2 -( r )- 3 , 4 - dihydroxy - 5 - carbonyl - 2 , 5 - dihydrofuran )- 2 - hydroxyethyl - 4 -( 6 -( 2 - amino - 4 - carbonyl - 3 , 4 - dihydropteridine ) methylamino ) benzoate , and the like . the amount of folic acid in the composition of the invention is 0 - 5 %, preferably 0 - 4 . 5 %, 0 - 4 %, 0 - 3 . 5 %, 0 - 3 %, 0 - 2 . 5 %, 0 - 2 %, 0 - 1 . 5 %, even more preferably 0 - 1 % by weight percentage . the amount of the folic acid complex in the composition of the invention is 0 - 10 %, preferably 0 - 8 %, 0 - 7 %, 0 - 6 %, 0 - 5 %, 0 - 4 %, 0 - 3 %, 0 - 2 %, even more preferably 0 - 1 % by weight percentage . methylene blue is a clinically commonly used dye . its mechanism of staining and color change is primarily based on its different colors in oxidation state and reduction state . specifically , methylene blue in reduction state is colorless , whereas aqueous solution of methylene blue in oxidation state exhibits a blue color . moreover , oxidized methylene blue , while present in the composition of the invention , may also exhibit a bluish green , blackish green ( brownish green ), and purple black color . the biological dye methylene blue has high affinity to cancer cells and melanin [ 7 ] , whose oxidation and reduction property cause methylene blue to appear different color change spectra in the oxidation and reduction states of the tumor tissues . such color changes can be directly identified by rapid visual observation . the anti - oxidation capacity of tumor tissues is significantly enhanced as compared to normal tissues in general . however , significant oxidative stress still exists in such tumor issues [ 6 ] . oxidative stress indicates oxidative lesion of the tissue , which is a period of occult pathological progression of the tissue . although the reduction property of tumor cells is enhanced , their oxidation - reduction balance is still inclined to oxidation , unless the tumor cells are in an apoptotic or inhibited state . therefore , in the intracellular environment of such vital cells , methylnene blue in the composition of the invention is oxidized to exhibit the green , bluish green , blackish green and purpose black color of the oxidation state . in the composition of the invention , the folic acid complex and a few amount of folic acid can form a folic acid - methylene blue complex with methylene blue . because the binding effect between the folic acid molecule and the folic acid receptor excessively expressed on the surface of the tumor cells , the folic acid - methylene blue complex can more easily enter into the cells with the promotion by acetic acid , and release methylene blue in reduction state . furthermore , in tumor cells in oxidative stress , methylene blue is rapidly oxidized and thereby generates color change . with different malignant degree of the tumor , methylene blue turns into dark bluish green , blackish green , and purple black color , whereas in the presence of inflammatory lesion , cauliflower excrescence ( hpv virus infection ) or cin i lesion , the color of methylene blue is green or light bluish green . the “ carbohydrate reducing agent ” in the invention refers to any reductive carbohydrate , derivatives thereof , or combinations thereof . the carbohydrate includes , but is not limited to a monosaccharide , a disaccharide , or a polysaccharide . specifically , the carbohydrate reductive agent can be glucose , fructose , galactose , hexose , lactose , maltose , and the like . the “ carbohydrate derivative ” described in the invention is defined as derivatives such as polysaccharide , glycoprotein , organic acid and the like formed by polymerization , esterification , oxidation and the like of carbohydrate substances . the amount of the carbohydrate reducing agent in the composition and method of the invention is 0 . 1 - 10 %, preferably 0 . 3 - 8 %, 0 . 1 - 3 %, and 0 . 05 - 1 %. the carbohydrate reducing agent in the composition and method of the invention reduces the methylene blue in oxidation state to colorless methylene blue in reduction state . folic acid and / or the folic acid complex bind to the folic acid receptor on the surface of the tumor cell . in an acidic environment of ph 5 . 0 - 5 . 5 , folic acid and / or folic acid complex mediates the internalization and release of methylene blue in reductive state into the cytosol . methylene blue in reductive state participates in the oxidative stress of the tumor tissues . the methylene blue in reductive state turns into the oxidative state . meanwhile , the osmotic pressure of the intracellular fluid increases , causing the methylene blue in oxidative state to escape from the cells and immediately exhibit different color change . such escaped methylene blue may adhere to the carrier for the administration of the invention , thereby immediately causing the composition on the carrier to exhibit color change . specifically , the composition turns from light yellowish brown into dark bluish green , blackish green , and purple black color . meanwhile , the tumor cells have enlarged nuclei and increased nuclear protein , which are precipitated and coagulated by acetic acid to produce a transient response , in which the abnormal epithelial tissue is shown as a acetowhite responsive region , which may provide location for the pathological sampling of the abnormal epithelial tissue . the term “ acetic acid ” used in the invention is ethanoic acid . the composition of the invention comprises 2 - 6 w % acetic acid . the acetic acid used in the composition of the invention and the preparation method thereof provides an acidic ph , preferably ph 5 . 0 - 5 . 5 . in addition , the use of acetic acid helps the composition of the invention to rapidly penetrate the cell , thereby interacting with the content of the cell so as to promote the occurrence of the color development . moreover , as mentioned above , after methylene blue , which component is used to exhibit color change , escapes from the cell , the acetowhite response generated by acetic acid on the abnormal epithelial tissue further provides basis for the location of the abnormal epithelial tissue . cells that can be stained by the composition of the invention are tumor cells , preferably epithelial tumor cells . said epithelial tissues include , but are not limited to cervical epithelium , vaginal epithelium , gastrointestinal epithelium , oral epithelium , and the like . such cells may come from tissue biopsy samples . the cells of the invention may be derived from mammalian subjects , said mammals include , but are not limited to human . the invention is further illustrated in examples and comparative examples below . in the following examples and comparative examples , the cervical epithelial tissue is used as the object for inspection , and the pathological inspection results of cervical tissues are used as the standard for reference , the sensitivity is used to illustrate the detection rate of the detecting agent : the higher the sensitivity , the stronger the ability to find the abnormal epithelial tissue ; and the specificity is used to illustrate the accuracy of the detecting agent : the higher the specificity , the higher the match between the detected abnormal epithelial tissue and the pathological inspection results . in the invention , sensitivity and specificity are defined and calculated as follows : under normal temperature and pressure , each of the various components as specified in table 1 below were individually dissolved in aqueous solvent , to which the biological detecting agent methylene blue were added , agitated and dissolved , followed by the addition of the reducing agent glucose and agitated for 30 minutes , then analytical pure acetic acid was added , agitated and mixed to make a detecting agent composition . the detecting agent composition was dipped with a large swab and smeared onto a cervical epithelial tissue . the swab was immediately taken out and immediate observed the color change of the swab . if the color of the swab was light yellowish brown , it indicated that there was no lesion of the epithelial tissue ; if the color of the swab was light bluish green , it indicated inflammatory lesion , cauliflower excrescence ( hpv virus infection ) or cin i lesion ; if the color of the swab was dark bluish green , blackish green , and purple black , it indicated abnormal lesion of cin ii or higher . meanwhile , colposcopy was recommended to take live tissues from multiple sites for histopathological examination , and then the sensitivity and the specificity of the detecting agent were tested using the histopathological testing results as the standard . the testing results are listed in table 1 . the method of example 1 was repeated using the various components whose amounts were specified in the following table 1 , except that the folic acid complex folic acid γ - cysteine was used in place of folic acid . the testing results are listed in table 1 . the method of example 1 was repeated using the various components whose amounts were specified in the following table 1 , except that folic acid and the folic acid complex ( r )- 2 -( 2 -( r )- 3 , 4 - dihydroxy - 5 - carbonyl - 2 , 5 - dihydrofuran )- 2 - hydroxyethyl - 4 -( 6 -( 2 - amino - 4 - carbonyl - 3 , 4 - dihydropteridine ) methylamino ) benzoate were co - dissolved in the aqueous solvent . the testing results are listed in table 1 . the method of example 1 was repeated using the various components whose amounts were specified in the following table 1 , except that folic acid and the folic acid complex ( r )- 2 -( 2 -( r )- 3 , 4 - dihydroxy - 5 - carbonyl - 2 , 5 - dihydrofuran )- 2 - hydroxyethyl - 4 -( 6 -( 2 - amino - 4 - carbonyl - 3 , 4 - dihydropteridine ) methylamino ) benzoate were co - dissolved in the aqueous solvent , and the reducing agent , a hexose derivative was used in place of the reducing agent glucose . the testing results are listed in table 1 . under normal temperature and pressure , 5 ml acetic acid was added into 95 ml distilled water for mixing to form a 5 % acetic acid solution . the solution was dipped with a large cotton swab and smeared onto a cervical epithelial tissue . after waiting for one minute , the presence or absence of acetowhite region on the epithelial tissue was observed close to the boundary of the squamous column of the cervical , and its boundary , thickness , color and response time were observed . if there was an acetowhite region with clear boundary , it indicated lesion of cin i or higher . colposcopy was conducted to take live tissues from multiple sites for histopathological examination , and then the sensitivity and the specificity of the 5 % acetic acid solution were tested using the histopathological testing results as the standard . the testing results are listed in table 1 . under normal temperature and pressure , 10 g potassium iodide was dissolved into 100 ml distilled water , to which 5 g iodine was added , agitated and dissolved to make a lugol &# 39 ; s iodine solution . the solution was dipped with a large cotton swab and smeared onto a cervical epithelial tissue to observe whether the epithelial tissue was stained by iodine . normal epithelium exhibited reddish brown or black color , whereas abnormal epithelium exhibited thick mustard yellow or yellowish brown color . colposcopy was conducted to take live tissues from multiple sites for histopathological examination , and then the sensitivity and the specificity of the lugol &# 39 ; s iodine solution were tested using the histopathological testing results as the standard . the testing results are listed in table 1 . from the aforesaid table 1 it can be seen that the examples using the detecting agent composition of the present invention all have significant advantages in sensitivity and specificity as compared to the detecting agents in the comparative examples , indicating that the composition of the invention can be sensitively and specifically used for detecting abnormal epithelial tissue . 1 . qiao youlin , zhang wenhua , zhao fanghui , pan qinjing , li ling , “ screen , early diagnosis , and early treatment solutions for cervical cancer ”, july , 2003 ; 3 . canto m i , setrakian s , willis j , et al , methylene blue - 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