Abstract:
One pot process of preparing multifunctional liposome drug is provided. In this one pot process, liposome reacted with radionuclide labeled solution, chemotherapy drug, and targeted ligand at appropriate temperature. The product in this invention for preparation multifunctional liposome drugs in for imaging, delivery and targeting in cancer diagnosis and therapy has proved to be more simple, convenient, effective and easier than the prior art is.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention relates to, one pot process of preparing multifunctional liposome drug for imaging, delivery and targeting in cancer diagnosis and therapy. 
       BACKGROUND OF THE INVENTION 
       [0002]    Liposomes, which are biodegradable and essentially non-toxic vehicles, can encapsulate both hydrophilic and hydrophobic drugs. In addition, liposomes can be used to carry radioactive compound as payloads. Liposomes can provide several advantages for bimodality radiochemotherapy for the following reasons:
   (1) Biocompatibility: Lipid and cholesterol used for liposome manufacture are common constitutes of cell membranes and therefore are easily metabolized.   (2) Enhanced permeability and retention (EPR) effect: Due to the unregulated tumor growth and location of endothelial lining in angiogenetic vasculature, the blood vessels in tumors have a tendency to leak, which induces the spontaneous accumulation of liposomes from blood circulation into the tumor. This phenomenon of concentration and localization of drugs in tumor tissues is called the enhanced permeability and retention effect. In addition, angiogenesis is the major mechanism of ascites fluid production.   (3) Varying uniform sizes: Liposome with variable homogeneous particle size ranges can readily be produced by using the extrusion techniques.   
 
         [0006]    Two diagnostic and therapeutic radionuclides,  188 Re and  186 Re, which have excellent physical properties. Bao et al. have developed a direct labeling method using  99m Tc-BMEDA complex to label the commercially available pegylated liposome doxorubicin. (J. Pharmacol Exp Ther, 308: 419-425, 2004). One pot process of preparing multifunctional liposome drug for imaging, delivery and targeting in cancer diagnosis and therapy has not found yet. 
       SUMMARY OF THE INVENTION 
       [0007]    The main object of the present invention is to provide one pot process of preparing multifunctional liposome drugs. In this one pot process, liposome reacted with radionulcude labeled solution, chemotherapy drug, and targeted ligand at appropriate temperature. 
         [0008]    The product in this invention for preparation multifunctional liposome drugs in for imaging, delivery and targeting in cancer diagnosis and therapy has proved to be more simple, convenient, effective and easier than the prior art is. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1  is the results of the cold competition receptor binding assay. 
           [0010]      FIG. 2  is the results of cytotoxic activity assay. 
           [0011]      FIG. 3  is the images revealed a high uptake. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0012]    The following abbreviations are employed: 
         [0000]    BMEDA: N,N-bis(2-mercaptoethyl)-N′,N′-diethylethylenediamine
 
DSPC: Distearoyl phosphatidylcholine
 
PEG: Polyethylene glycol
 
DSPE: Distearyl phosphatidylethanolamine
 
       BBN: Bombesin 
     DXR: Doxorubicin 
     DMF: N,N-dimethylformamide 
       [0013]    NHS: N-hydroxyl succinimidyl ester 
       Oct: Octreotide 
     Example 1 
     The Preparation of DSPE-PEG-BBN 
       [0014]    10 mg of bombesin was dissolved by adding 2 mL of DMF. After the bombesin completely dissolved, 18.94, of TEA was added to the solution and stirred for 1 hr under nitrogen gas. On the other hand, 21.5 mg of DSPE-PEG-NHS was dissolved in 2 ml of DMF to completely dissolve and then dropped into the above solution to stir for 24 hr under nitrogen gas. The solvent was removed after the reaction finished. An excess of chloroform was added to the resultant solid product and the solution kept standing to carry out precipitation and was then filtered through a filter paper No. 42. 
         [0015]    The precipitation was collected and dissolved in 2 mL water. The product was separated through a column of Sephadex G-25 with water as an eluent. The product was confirmed its location and purity by a BCA protein assay and then collected by removing the solvent through a lyophilized. The product also was analyzed by HPLC-ELSD through a column of XTerra MSC18(5 μm) with 90% water and 10% methanol as an eluent and 10 minutes as analytic time. The retention time was 4.5 minutes. The product was analyzed average molecular weight by MALDI-TOF/TOF as [M+H] + =3751 Da. 
       Example 2 
     One Pot Processes of Preparing  188 Re-DXR-liposome-BBN 
       [0016]    5 mg of BMEDA and 0.5 mL of 0.17 mol/L glucohepatonate and 120 μL (10 μg/μL) of stannous chloride were pipetted into a fresh vial, then flushing nitrogen gas for 2 minute to avoid the oxygenation of stannous chloride. 1 mL of highly specific activity of  188 Re-sodium perrhenate were added, then sealed vial. The sealed vial was heated in an 80° C. water-bath for 1 h. The vial was cool down at room temperature, adjust pH to neutrality (pH 6˜7) with 120˜150 μL of 5N NaOH by slowly pipetting. The labeling efficiency of the  188 Re-BMEDA complexes was checked by paper chromatography with normal saline as the eluent. The labeling efficiency of  188 Re-BMEDA complexes was 90˜100% (Rf: 1, free  188 Re; Rf: 0,  188 Re-BMEDA). 
         [0017]    10 μL of DSPE-PEG 2000 -BBN (40 mg/mL) and 188.5 μL DXR (10 mg/mL) and 1 mL of liposomes encapsulating (NH 4 ) 2 SO 4  were mixed with 0.5 mL of  188 Re-BMEDA solution, and then incubated in a 60° C. water-bath for 30 mins. Sephagrose CL-6B column (GE Healthcare Bio-Sciences AB, Sweden) chromatography with normal saline was used to separate  188 Re-DXR-liposome-BBN from free  188 Re-BMEDA and free DXR. Eluted  188 Re-DXR-liposome-BBN solution was collected in 0.5 ml into each tube for total 30 tubes. The yield of  188 Re-DXR-liposome-BBN was calculated according to the following standard formula: Labeling efficiency (%)=[100×(Radioactivity of fractions with  188 Re-DXR-liposome-BBN/(Total fraction radioactivity+column residue)]. The yield of  188 Re-DXR-liposome-BBN was 75˜85% ( FIG. 2 ). 
       Quality Control of  188 Re-DXR-liposome-BBN 
       [0018]    1 mL acidic isopropanol (81 mM in isopropanol) was mixed with 0.2 mL diluted DXR-loaded liposomes, the amount of doxorubicin trapped inside the liposome was determine with a spectrofluorometer (FP6200, JASCO) at an excitation wavelength of 475 nm and an emission wavelength of 580 nm. The concentration of liposomes was estimated by the phosphate assay (Bartlett, 1959). In this preparation(n=3), DXR-loaded liposomes contained 120˜160 μg/μmole phospholipid. Particle size of Liposome were measured by dynamic laser scattering with a particles analyzer (Nano ZS90, Malvern, UK). Particle sizes ranged from 90˜110 nm in diameter (Table 1). 
       DXR Encapsulating Efficiency Analysis of  188 Re-DXR-Liposome-BBN 
       [0019]    Condition MicroSpin column with 2504, normal saline (G50, GE Healthcare Bio-Sciences AB, Sweden), then add 254, liposome sample into the center of MicroSpin column. The column was centrifuged with 3000 rpm for 2 mins and the eluted solution was collected in a fresh tube. Then eluted the column with another 254, normal saline and collected the eluting solution in the same tube. Measured the amount of doxorubicin trapped inside the liposome. The DXR encapsulating efficiency of  188 Re-DXR-liposome-BBN was calculated according to the following standard formula: encapsulating efficiency (%)=100×{(the total volume of  188 Re-DXR-liposome-BBN after purification)×(the concentration of  188 Re-DXR-liposome-BBN after purification)/25×(the concentration of  188 Re-DXR-liposome-BBN before purification)}. The DXR encapsulating efficiency of  188 Re-DXR-liposome-BBN was larger than 85%. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The quality control of  188 Re-DXR-liposome-BBN for three batch. 
               
             
          
           
               
                 Batch No. 
                 980806 
                 980811 
                 980813 
               
               
                   
               
               
                 Re-188 encapsulating 
                  84.2% 
                 75.91% 
                 79.62% 
               
               
                 efficiency 
               
               
                 DXR encapsulating 
                 97.14% 
                 91.97% 
                 84.96% 
               
               
                 efficiency 
               
               
                 Drug/phospholipid 
                 135.01 
                 157.08 
                 123.29 
               
               
                 ratio (μg/μmole) 
               
               
                 Phospholipid conc. 
                 14.81 
                 12.73 
                 16.22 
               
               
                 (concentrate DXR 
                 mmole/mL 
                 mmole/mL 
                 mmole/mL 
               
               
                 conc. to 2 mg/mL) 
               
               
                 Particle size (nm) 
                 94.23 ± 25.12 
                 96.04 ± 30.27 
                 95.33 ± 28.61 
               
               
                   
               
             
          
         
       
     
       Example 3 
     The Cold Competition Receptor Binding Assay of Bombesin, DSPE-PEG-BBN and Liposome-BBN 
       [0020]    Cold competition receptor binding assay was studied using human bombesin 2 receptor expressed in HEK-293 cells as the source of GRP receptor (PerkinElmer, Boston, Mass., USA). Assays were performed using FC96 plates and the Multiscreen system (Millipore, Bedford, Mass.). Binding of  125 I-Tyr 4 -Bombesin (PerkinElmer, Boston, Mass., USA) to PC-3 cell membranes (0.16 g per well) was determined in the presence of increasing concentrations (0.001 nmole/L to 1000 nmole/L) of Bombesin-finer, DSPE-PEG-BBN and Liposome-BBN in a buffer solution (20 mmol/L HEPES, pH 7.4, 3 mmol/L MgCl 2 , 1 mmol/L EDTA and 0.3% BSA) with a total volume of 250 μL per well. After incubation for 120 min at RT, membranes were filtered and washed with ice-cold Tris-HCl buffer (50 mmol/L). The filters containing membrane-bound radioactivity were counted using a Cobra II gamma-counter (Packard, Meriden, Conn.). The inhibitory concentration of 50% (IC 50 ) was calculated using a 4-parameter curve-fitting routine using the EXCEL software. 
         [0021]    As shown in  FIG. 1 , for the receptor binding with GRPR, the IC 50  of Bombesin-iner, DSPE-PEG-BBN and Liposome-BBN was 0.186, 0.627 and 4.480 nM respectively. The Ki of Bombesin-iner, DSPE-PEG-BBN and Liposome-BBN was 0.146, 0.494 and 3.527 nM respectively. 
       Example 4 
     The Cytotoxic Activity Assay of  188 Re-DXR-Liposome-BBN 
       [0022]    The cytotoxic activity assay of  188 Re-DXR-Liposome-BBN on PC-3 human prostate cancer cell line was measured with an Countess™ cell counter (Invitrogen, Carlsbad, Calif., USA). Adherent PC-3 cells were seeding on 25T flasks. After growth overnight, PC-3 cells were treated with a medium containing  188 Re-BMEDA (30.5 μCi/ml),  188 Re-Liposome-BBN ( 188 Re-LB, 30.5 μCi/ml), DXR-Liposome-BBN (LDB, 32 μg/ml),  188 Re-DXR-Liposome-BBN ( 188 Re-LDB, 32 μg/30.5 μCi/ml) or control (normal saline) at 37° C. for 1 h. Additional normal groups were performed without any addition of drugs in medium. After washing with cold PBS, cells were additionally incubated at 37° C. for 2 days. Cells were stained with Trypan Blue and analyzed for cell viability using a Countess™ cell counter. The cell viability was calculated using the following formula: 
         [0000]    
       
         
           
             
               Cell 
                
               
                   
               
                
               viability 
             
             = 
             
               
                 
                   Cell 
                    
                   
                       
                   
                    
                   
                     no 
                     . 
                     
                         
                     
                      
                     of 
                   
                    
                   
                       
                   
                    
                   experimental 
                    
                   
                       
                   
                    
                   or 
                    
                   
                       
                   
                    
                   control 
                    
                   
                       
                   
                    
                   group 
                 
                 
                   Cell 
                    
                   
                       
                   
                    
                   
                     no 
                     . 
                     
                         
                     
                      
                     of 
                   
                    
                   
                       
                   
                    
                   normal 
                    
                   
                       
                   
                    
                   group 
                 
               
               × 
               100 
                
               % 
             
           
         
       
     
         [0023]    As shown in  FIG. 2 , the results of cytotoxic activity assay demonstrated that  188 Re-LDB have the superior cytotoxic activity on PC-3 human prostate cancer cell line. The cell viability of  188 Re-LDB in this study is 28.6±3.7%. 
       Example 5 
     MicroSPECT Imaging and Images Semi-Quantification Analysis of Targeted  188 Re-Liposome-BBN 
       [0024]    Imaging was acquired using low-energy, high-resolution collimators at 1, 24, 48 and 72 hr after intravenous injection of  188 Re-Liposome-BBN. When the imaging acquisition, the mice were anesthetized with 1˜2% isoflurane in 100% O 2 . The energy window was set at 155 KeV±10˜15%, the FOV (Field of View) was 12.5 cm. SPECT imaging was followed by CT image acquisition (X-ray source: 50 kV, 0.4 mA; 256 projections) with the animal in exactly the same position. Images were calibrated to standardized uptake values (SUV). 
         [0025]    For calculate Standardised tumor uptake value (StUV), known radio activity Re-188 was performed as reference. The SUV was determined from the regions of interest (ROI) on the tumor with uptake. The SUV was calculated according to the following standard formula: 
         [0000]      (measured activity concentration (μCi/g)/[Injected Dose (μCi)/body weight (g)]
 
         [0026]    As shown in  FIG. 3 , the images revealed a high uptake in tumors at 1 and 24 h after intravenous injection. The SUV of  188 Re-Liposome-BBN in tumor was 1.54 and 1.25 at 1 and 4 h after injection, respectively.