Synthesis and composition of amino acid linking groups conjugated to compounds used for the targeted imaging of tumors

Abstract

The present disclosure relates to compounds that are useful as near-infrared fluorescence probes, wherein the compounds include i) a pteroyl ligand that binds to a target receptor protein, ii) a dye molecule, and iii) a linker molecule that comprises an amino acid or derivative thereof. The disclosure further describes methods and compositions for incorporating the compounds as used for the targeted imaging of non-small cell lung cancer (NSCLC) in human subjects. Conjugation of the amino acid linking groups increase specificity and detection of the compound. Methods and compositions for use thereof in diagnostic imaging are contemplated.

1 . A method of imaging a carcinoma that expresses a folate receptor in a subject in need thereof comprising
 a. administering to the subject an effective amount of a compound capable of binding to a cancerous cell having the formula:   
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt or isotope thereof, wherein: X is a single amino acid or a single amino acid derivative thereof, wherein the single amino acid or single amino acid derivative contains an —OH, —NH 2 , or —SH functional group, and Y is a dye that has a fluorescence excitation and emission spectra in the near infra-red range, wherein Y is represented by the formula: 
       
         
           
           
               
               
           
         
         wherein, R 1  is independently selected from the group consisting of O, S, N and C, and R 2  is independently selected from the group consisting of CH 2  and CH 2 CH 2 , and the compound enhances the fluorescence of the dye, Y; and
 b. fluorescent imaging of an area in the subject's body where the compound binds to a cancerous cell wherein the carcinoma is selected from the group consisting of cervical cancer, ovarian cancer, breast cancer, leukemia cancer, and lung cancer. 
 
       
     
     
         2 . The method of  claim 1  wherein the subject is a human. 
     
     
         3 . A method of in vivo identification of diseased tissue in a subject in need thereof, comprising
 (ii) administering a compound to the subject, wherein the compound has the formula:   
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt or isotope thereof, wherein: X is a single amino acid or a single amino acid derivative thereof, wherein the single amino acid or single amino acid derivative contains an —OH, —NH 2 , or —SH functional group, and Y is a dye that has a fluorescence excitation and emission spectra in the near infra-red range, wherein Y is represented by the formula: 
       
         
           
           
               
               
           
         
       
       wherein, R 1  is independently selected from the group consisting of O, S, N and C, and R 2  is independently selected from the group consisting of CH 2  and CH 2 CH 2 , and the compound enhances the fluorescence of the dye, Y;
 (ii) irradiating an area of subject's body containing diseased tissue with at least one excitation wavelength in the near infrared range; 
 (iii). fluorescent imaging the compound in the area of the subject's body wherein the compound has bound to or been taken up by the diseased tissue in the subject's body, in response to the at least one excitation wavelength, and 
 (iii) determining the location or surface area of the diseased tissue in the subject. 
 
     
     
         4 . The method of  claim 3  wherein the near infra red range in step (ii) is from about 600 nm to about 1000 nm. 
     
     
         5 . The method of  claim 3  wherein the near infra red range in step (ii) is from about 600 nm to about 850 nm. 
     
     
         6 . The method of  claim 3  wherein the excitation light is monochromatic or polychromatic. 
     
     
         7 . The method of  claim 3  wherein fluorescence image is visible and distinct from the auto-fluorescence of the surrounding tissue. 
     
     
         8 . The method of  claim 3  wherein the diseased tissue is located at an interior site in the subject. 
     
     
         9 . The method of  claim 3  wherein the diseased tissue is selected from the group consisting of cardiac, breast, ovarian, uterine, lung, endothelial, vascular, gastrointestinal, colorectal, prostatic tissue, endocrine tissue, and combinations thereof. 
     
     
         10 . The method of  claim 3  wherein the subject has a preoperative diagnosis of pulmonary adenocarcinoma. 
     
     
         11 . The method of  claim 10  wherein the preoperative diagnosis was determined by transthoracic needle or transbronchial needle aspiration. 
     
     
         12 . The method of  claim 10  further comprising the step of using an endoscopic device to deliver the excitation light to the site, to receive fluorescence emanating from the site within a body cavity, and to aid in formation of a direct image of the fluorescence from the diseased tissue. 
     
     
         13 . A method for utilizing a diagnostic procedure during surgery in a subject in need thereof, comprising:
 (i) administering to the subject a composition comprising a compound wherein the compound having the formula:   
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt or isotope thereof, wherein: X is a single amino acid or a single amino acid derivative thereof, wherein the single amino acid or single amino acid derivative contains an —OH, —NH 2 , or —SH functional group, and Y is a dye that has a fluorescence excitation and emission spectra in the near infra-red range, wherein Y is represented by the formula: 
       
         
           
           
               
               
           
         
       
       wherein, R 1  is independently selected from the group consisting of O, S, N and C, and R 2  is independently selected from the group consisting of CH 2  and CH 2 CH 2 , and the compound enhances the fluorescence of the dye, Y;
 (ii) irradiating an area of the subject's body containing diseased tissue with light having at least one excitation wavelength in the range from about 600 nm to about 850 nm; 
 (iii) fluorescence imaging the compound in the area of the subject's body wherein the compound has bound to or been taken up by the diseased tissue in the body part, wherein the targeting construct fluoresces in response to the at least one excitation wavelength; 
 (iv) determining the location or surface area of the diseased tissue in the subject; and 
 (v) removing at least a portion of the tumor tissue. 
 
     
     
         14 . The method of  claim 13  wherein the subject is a human. 
     
     
         15 . The method of  claim 13  wherein the diseased tissue being identified includes non-small cell lung cancer. 
     
     
         16 . The method of  claim 15  wherein the non-small cell lung cancer is selected from the group consisting of lung adenocarcinoma, squamous cells carcinoma, large cell lung carcinoma, neuroendocrine carcinoma, carcinoid tumors, metastatic tumors, and lymph node. 
     
     
         17 . The method of  claim 13  wherein the diagnostic procedure is minimally invasive pulmonary resection. 
     
     
         18 . The method of  claim 13  wherein the tumor tissue is an FR expressing tumor. 
     
     
         19 . The method of  claim 13  wherein preoperative CT and PET were obtained in the subject. 
     
     
         20 . The method of  claim 19  wherein the tumor size was determined to be in the range of 1.5 to 4.3 cm. 
     
     
         21 . The method of  claim 13  wherein the compound of step (i) is administered in the range of about 1.79 to about 2.58 mg. 
     
     
         22 . The method of  claim 13  wherein the compound of step (i) is administered about 3 to about 6 hours prior to resection. 
     
     
         23 . The method of  claim 13  wherein the compound of step (i) is administered intravenously. 
     
     
         24 . The method of  claim 13  wherein a lobe of the subject is imaged in situ using a NIR thoracoscopic camera. 
     
     
         25 . The method of  claim 24  wherein the camera is included in a NIR imaging system including an 10 mm, 30° thoracoscope; an excitation light source of 785 nm; and
 emission filters selecting for light ranging from about 800 nm to about 835 nm. 
 
     
     
         26 . The method of  claim 25  wherein in situ fluorescence is appreciated through the pleural surface with a tumor-to-background ratio range of about 2.7 to about 4.2. 
     
     
         27 . The method of  claim 13  wherein the method reveals nodules. 
     
     
         28 . The method of  claim 27  wherein the nodule a synchronous nodule. 
     
     
         29 . The method of  claim 27  wherein the nodule a synchronous adenocarcinoma. 
     
     
         30 . The method of  claim 29  wherein the synchronous adenocarcinoma is a subcentimeter carcinoma. 
     
     
         31 . The method of  claim 30  wherein the subcentimeter carcinoma is about 0.6 cm in length or diameter. 
     
     
         32 . The method of  claim 13  wherein the compound accumulates in FRα expressing tumors. 
     
     
         33 . The method of  claim 30  wherein the identification of subcentimeter pulmonary adenocarcinomas is improved. 
     
     
         34 . The method of  claim 28  wherein the identification of synchronous nodules aids the method of treatment of diseased tissue in the subject. 
     
     
         35 . A method for in vivo diagnosis of tumor tissue in a subject in need thereof, comprising:
 (i) administering a diagnostically effective amount of at least one biologically compatible fluorescing targeting construct containing a compound that has been determined to bind to or be taken up by the sample tumor cells and a fluorophore responsive to at least one wavelength of light in the range from about 600 nm to about 850 nm, wherein the compound having the formula:   
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt or isotope thereof, wherein: X is a single amino acid or a single amino acid derivative thereof, wherein the single amino acid or single amino acid derivative contains an —OH, —NH 2 , or —SH functional group, and Y is a dye that has a fluorescence excitation and emission spectra in the near infra-red range, wherein Y is represented by the formula: 
       
         
           
           
               
               
           
         
       
       wherein, R 1  is independently selected from the group consisting of O, S, N and C, and R 2  is independently selected from the group consisting of CH 2  and CH 2 CH 2 , and the compound enhances the fluorescence of the dye, Y; and
 (ii) contacting samples of tumor cells obtained from the subject with a plurality of detectably labeled compounds, each of which binds to or is selectively taken up by a distinct tumor type; and 
 (iii) diagnosing the location or surface area of the tumor tissue in the subject's body part by directly viewing fluorescence emanating from the targeting construct bound or taken up in the tumor tissue upon irradiation thereof with light providing the at least one excitation wavelength for the fluorescent targeting construct. 
 
     
     
         36 . The method of  claim 34  wherein the subject is a human. 
     
     
         37 . The method of  claim 34  further comprising the step of identifying the compounds is bound to or taken up by the sample tumor cells.