Abstract:
A method and an apparatus for the manufacture of 3′-deoxy-3′-[ 18 F]Fluorothymidine are disclosed. A fluorothymidine precursor, a reaction solvent for the fluorothymidine precursor, a source of [ 18 F] ions, and dilution water are combined to form a reaction mixture including a radiolabeled thymidine product, [ 18 F] ions, and residual reaction impurities. The reaction mixture is passed through a solid phase extraction cartridge. The solid phase extraction cartridge includes a solid sorbent to adsorb the radiolabeled thymidine product on the sorbent, and a portion of the residual reaction impurities pass through solid phase extraction cartridge without adsorbing on the sorbent. Any remaining impurities on the sorbent are washed off with water, and the radiolabeled thymidine product is eluted off the sorbent using an alkanol solvent. The eluted radiolabeled thynidine product is dried and reconstituted in water before any further extraction steps. The method and apparatus do not require the use of high-pressure liquid chromatography.

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS  
       [0001]     This application claims priority from U.S. Provisional Patent Application No. 60/501,922 filed Sep. 11, 2003.  
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH  
       [0002]     Not Applicable. 
     
    
     BACKGROUND OF THE INVENTION  
       [0003]     1. Field of the Invention  
         [0004]     The field of the invention is the manufacture of radiopharmaceuticals and particularly, the manufacture of 3′-deoxy-3′-[ 18 F]Fluorothymidine.  
         [0005]     2. Description of the Related Art  
         [0006]     Radiolabeled compounds are used as diagnostic imaging agents in positron emission tomography (PET). For example, 2-[ 18 F]fluoro-2-deoxy-D-glucose (FDG) is one tracer used in nuclear medical imaging. This molecule, labeled with the radionuclide  18 F, behaves in a way similar to glucose in the first step of its destabilization in the human body and allows a user to map and quantify this fundamental mechanism. It is indicated for diagnosis of numerous diseases. FDG and its preparation are described in U.S. Pat. Nos. 4,617,386, 4,794,178, 5,169,942, 5,264,570, 5,436,325, 5,759,513, 5,808,020, 5,932,178, and 6,172,207 (which are incorporated herein by reference along with all other publications cited herein).  
         [0007]     Another radiolabeled compound, radiolabeled thymidine, is currently being explored as a diagnostic imaging agent for positron emission tomography in the area of oncology. There is much excitement being generated about its possibilities. However, radiolabeled thymidine is difficult to produce so its use has been limited. The current method used to purify 3′-deoxy-3′-[ 18 F]Fluorothymidine employs high-pressure liquid chromatography (HPLC). This technique requires a column, high pressure pumps, and an ultraviolet detector which is a relatively complex system. Such systems can range in price from a few thousand dollars up to $30,000 and they require a 30 minute set up procedure and about 10 minutes for each synthesis use. Because of their complexity, prior systems are also less reliable, particularly in a clinical setting.  
         [0008]     Thus, the current method used to purify 3′-deoxy-3′-[ 18 F]Fluorothymidine presents many problems. For instance, the long purification time for 3′-deoxy-3′-[ 18 F]Fluorothymidine is undesirable given the 110 minute half life for the  18 F fluoride ion; and the automated 3′-deoxy-3′-[ 18 F]Fluorothymidine purification equipment has a high cost.  
         [0009]     Therefore, there is a need for a method and an apparatus for preparing purified 3′-deoxy-3′-[ 18 F]Fluorothymidine at lower cost with decreased purification times.  
       SUMMARY OF THE INVENTION  
       [0010]     The present invention provides a method and apparatus for preparing radiolabeled thymidine which include a solid-phase extraction purification method which can purify radiolabeled thymidine quickly and conveniently. The radiolabeled thymidine is trapped on a cartridge and then released in the appropriate eluent. This method replaces more complicated, costly, and time-consuming purification methods like high-pressure liquid chromatography purification. This method has also been incorporated into an automated synthesis unit to purify radiolabeled thymidine without human intervention after production has started.  
         [0011]     The solid-phase trapping method of the present invention reduces purification time from approximately 10 minutes to 2 minutes or less. The cost of the previous equipment needed for current purification is several thousand dollars depending on the exact set-up. The cost for this invention is only a few dollars per synthesis. The simple nature of this invention (i.e., lack of electronic parts, detectors, complex valves) improves reliability, which is very important in the clinical setting. This invention allows 3′-deoxy-3′-[ 18 F]Fluorothymidine to be made on a standard 2-[ 18 F]Fluoro-2-deoxy-D-glucose synthesis unit such as one of the TRACERlab Synthesizers available from GE Healthcare which include an HPLC unit and are widely available at many clinical sites.  
         [0012]     In one aspect, the invention provides a method for preparing a radiolabeled thymidine product. In the method, a fluorothymidine precursor, a reaction solvent for the fluorothymidine precursor, a source of [ 18 F] ions, and dilution water are combined to thereby form a reaction mixture including a radiolabeled thymidine product, [ 18 F] ions, and residual reaction impurities. (The term “reaction mixture” does not require that all components of the reaction mixture participate in a chemical reaction.) The reaction mixture is passed through a solid phase extraction cartridge as a first extraction step. The solid phase extraction cartridge includes a solid sorbent to adsorb the radiolabeled thymidine product on the sorbent, and at least a portion of the residual reaction impurities pass through solid phase extraction cartridge without adsorbing on the sorbent. The solid phase extraction cartridge sorbent is preferably a reversed phase C-18 sorbent. Any remaining impurities on the sorbent are washed off with wash water, and the radiolabeled thymidine product is eluted off the sorbent using an alkanol solvent. The eluted radiolabeled thymidine product is dried and reconstituted in water before any further extraction steps.  
         [0013]     The solid phase extraction cartridge may be the only reversed phase solid phase extraction cartridge used in the method, and preferably, high pressure liquid chromatography is not used in the method. Preferably, the alkanol solvent is ethanol, and the reaction solvent is an aprotic solvent such as dimethyl sulfoxide. The dilution water that is combined with the fluorothymidine precursor, the reaction solvent for the fluorothymidine precursor, and the source of [ 18 F] ions is in an amount in excess of the reaction solvent, and the wash water used to wash off any remaining impurities on the sorbent is preferably not delivered by way of the reaction vessel.  
         [0014]     The reaction mixture may be passed through a filter before passing the reaction mixture through the solid phase extraction cartridge. The filter removes precipitates from the reaction mixture. Also, bypassing the filter after passing the reaction mixture through the filter for a time period is possible. Preferably, the steps of passing the reaction mixture through the solid phase extraction cartridge, washing off the remaining impurities on the sorbent, and eluting the radiolabeled thymidine product off the sorbent take two minutes or less.  
         [0015]     The reconstituted radiolabeled thymidine product may be passed through a solid phase extraction cartridge to further remove impurities and thereafter filtered to further remove impurities.  
         [0016]     In another aspect, the invention provides an apparatus for preparing a radiolabeled thymidine product. The apparatus includes a reaction vessel for containing a fluorothymidine precursor, a reaction solvent for the fluorothymidine precursor, a source of [ 18 F] ions, and dilution water to thereby form a reaction mixture including a radiolabeled thymidine product, [ 18 F] ions, and residual reaction impurities. A solid phase extraction cartridge is in fluid communication with the reaction vessel. The solid phase extraction cartridge includes a solid sorbent to adsorb the radiolabeled thymidine product on the sorbent. At least a portion of the residual reaction impurities pass through solid phase extraction cartridge without adsorbing on the sorbent. The apparatus includes a source of wash water in fluid communication with the solid phase extraction cartridge for providing wash water to wash off any remaining impurities on the sorbent with water, and a source of an alkanol solvent in fluid communication with the solid phase extraction cartridge for providing the alkanol solvent to elute the radiolabeled thymidine product off the sorbent using the alkanol solvent. The apparatus does not require the use of a high pressure liquid chromatography unit. Preferably, the wash water is not delivered by way of the reaction vessel.  
         [0017]     The apparatus may include a filter in fluid communication with the reaction vessel and the solid phase extraction cartridge such that the reaction mixture passes through the filter before the solid phase extraction cartridge. The filter removes precipitates from the reaction mixture. There may be provided a bypass conduit in fluid communication with the reaction vessel and the solid phase extraction cartridge for allowing the reaction mixture to bypass the filter.  
         [0018]     The apparatus may further include a controller and a controllable valve in electrical communication with the controller. The controllable valve is located between the reaction vessel and the bypass conduit. There is provided a pressure sensor in electrical communication with the controller. The pressure sensor provides a signal to the controller when the filter is clogged such that the controller adjusts the controllable valve to allow the reaction mixture to bypass the filter.  
         [0019]     In yet another aspect, the invention provides a method for preparing a radiolabeled thymidine product on a radiolabeled product synthesizer that has a reaction vessel and a high pressure liquid chromatography unit in a product flow path. In this method, the high pressure liquid chromatography unit is removed from the product flow path, and a fluorothymidine precursor, a reaction solvent for the fluorothymidine precursor, a source of [ 18 F] ions, and dilution water are combined in the reaction vessel to thereby form a reaction mixture including a radiolabeled thymidine product, [ 18 F] ions, and residual reaction impurities. The reaction mixture is passed through a solid phase extraction cartridge, which includes a solid sorbent to adsorb the radiolabeled thymidine product on the sorbent. In one form, the solid phase extraction cartridge sorbent is a C-18 sorbent. At least a portion of the residual reaction impurities pass through solid phase extraction cartridge without adsorbing on the sorbent. Any remaining impurities on the sorbent are washed off with wash water, and the radiolabeled thymidine product is eluted off the sorbent using an alkanol solvent such as ethanol.  
         [0020]     The dilution water that is combined with the fluorothymidine precursor, the reaction solvent for the fluorothymidine precursor, and the source of [ 18 F] ions is in an amount in excess of the reaction solvent (e.g., dimethyl sulfoxide). Preferably, the fluorothymidine precursor, the reaction solvent for the fluorothymidine precursor, the source of [ 18 F] ions, and water are combined in a reaction vessel, and the wash water used to wash off any remaining impurities on the sorbent is not delivered by way of the reaction vessel.  
         [0021]     The reaction mixture may be passed through a filter before passing the reaction mixture through the solid phase extraction cartridge so that the filter removes precipitates from the reaction mixture. The filter may be bypassed after passing the reaction mixture through the filter for a time period. Preferably, the steps of passing the reaction mixture through a solid phase extraction cartridge, washing off the remaining impurities on the sorbent, and eluting the radiolabeled thymidine product off the sorbent take two minutes or less.  
         [0022]     These and other features, aspects, and advantages of the present invention will become better understood upon consideration of the following detailed description, drawings, and appended claims. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0023]      FIG. 1  is a schematic diagram of a synthesizer for practicing the present invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0024]     Referring to  FIG. 1 , there is shown a synthesizer  10  according to the present invention for preparing 3′-deoxy-3′-[ 18 F]Fluorothymidine. A suitable general reaction scheme for preparing 3′-deoxy-3′-[ 18 F]Fluorothymidine is described by Machulla et al. at “Simplified labeling approach for synthesizing 3′-deoxy-3′-[ 18 F]fluorothymidine ([ 18 F]FLT)”,  Journal of Radioanalytical and Nuclear Chemistry,  24: 843-846, 2000. In synthesizer  10 , vessels are provided for containing various reactants used in preparing 3′-deoxy-3′-[ 18 F]Fluorothymidine. Specifically, vessel T is provided for containing O-18( 18 O) water including [ 18 F] fluoride ions (hereinafter the “target water”); vessel  1  is provided for containing potassium carbonate (K 2 CO 3 ) and Kryptofix™ 222 (k 222 ); vessel  2  is provided for containing a fluorothymidine (FLT) precursor and dimethylsulfoxide (DMSO) solvent; vessel  3  is provided for containing hydrochloric acid (HCl); vessel  4  is provided for containing an aqueous solution of sodium acetate; vessels  5  and  6  are provided for containing water; and vessel  7  is provided for containing ethanol. Controllable valves V 1  to V 14  are provided in various flow conduits for controlling the movement of fluid materials in the synthesizer  10 .  
         [0025]     In operation, the target water including O-18( 18 O) water and [ 18 F] fluoride ions (which may be synthesized using known techniques such as those described in U.S. Pat. Nos. 6,567,492 and 6,190,637) is delivered from vessel T through valve V 7  and passed through a [ 18 F] trap cartridge  12  such as an anion-exchange resin. The [ 18 F] trap cartridge  12  traps the [ 18 F] ions in the target water and the waste target water passes through valve V 7   b  into waste container  14 .  
         [0026]     An aqueous potassium carbonate solution and an acetonitrile (CH 3 CN) solution of Kryptofix™ 222 are then delivered from vessel  1 , and are passed through the [ 18 F] trap cartridge  12  to extract the [ 18 F] ions trapped by means of the [ 18 F] trap cartridge  12 . The Kryptofix™ 222 is an activating agent that activates the [ 18 F] by making it more reactive. The thus extracted [ 18 F] fluoride ion is sent via valve V 8  to a reaction vessel  16 . Subsequently, the reaction vessel  16  including the [ 18 F] fluoride ion, the potassium carbonate and the acetonitrile solution of Kryptofix™ 222 is heated under reduced pressure to eliminate moisture in the reaction vessel  16 .  
         [0027]     After the mixture in the reaction vessel  16  is sufficiently dried, the fluorothymidine (FLT) precursor and dimethylsulfoxide (DMSO) solvent are delivered from vessel  2  through valve V 2  into the reaction vessel  16 , which is then heated. The fluorothymidine (FLT) precursor used may be a dimethoxytriphenylmethyl protected anhydrothymidine known as 5′-O-(4,4′ dimethoxytriphenylmethyl)-2-3′-anhydrothymidine. Other protected anhydrothymidines may also be suitable. In one example embodiment, 10 milligrams of 5′-O-(4,4′ dimethoxytriphenylmethyl)-2-3′-anhydrothymidine dissolved in 1 milliliter of DMSO are delivered from vessel  2  through valve V 2  into the reaction vessel  16 .  
         [0028]     Hydrochloric acid (HCl) is then delivered through valve V 3  to the reaction vessel  16  to deprotect the 5′-O-(4,4′ dimethoxytriphenylmethyl)-2-3′-anhydrothymidine. An aqueous solution of sodium acetate is then delivered through valve V 4  to the reaction vessel  16  to adjust the pH of the reaction mixture towards neutral. Preferably, the aqueous solution of sodium acetate includes dilution water in an amount such that the volume ratio of dilution water to DMSO in the reaction vessel is at least 7 to 1 and preferably 9 to 1. After the reaction, [ 18 F] fluoride ions, Kryptofix™ 222, potassium carbonate, acetonitrile, HCl, sodium acetate, water and the radiolabeled 3′-deoxy-3′-[ 18 F]Fluorothymidine may be present in the reaction vessel  16 . Some of these (e.g., acetonitrile) may not remain in the reaction vessel  16 .  
         [0029]     The reaction mixture of any of the [ 18 F] fluoride ions, Kryptofix™ 222, potassium carbonate, acetonitrile, HCl, sodium acetate, dilution water and the radiolabeled 3′-deoxy-3′-[ 18 F]Fluorothymidine are then delivered from the reaction vessel  16  through valves V 9  and V 10  and valve V 11  side  11   a  and through frit  32  (pore size=2 microns) to a solid phase extraction cartridge  34 . Suitable solid phase extraction cartridges include those sold commercially as the Waters Sep-Pak® Plus C-18 solid phase extraction cartridge or the Waters Sep-Pak® C-18 Light. These types of cartridges are also described in U.S. Pat. Nos. 4,211,658 and 4,250,035, and their use is described in “Solid Phase Extraction Applications Guide and Bibliography” Sixth Edition 1995 published by Waters. The C-18 reversed-phase solid sorbent is a strongly hydrophobic silica-based bonded phase with a monofunctional bonding chemistry and a —Si(CH 3 ) 2 C 18 H 37  surface functionality. The nominal pore size is 125 Angstroms and the particle size range is typically 55-105 micrometers.  
         [0030]     The use of frit  32  serves to filter the reaction mixture prior to loading on the solid phase extraction cartridge  34 . It has been discovered that water may precipitate reaction mixture components, which would clog solid phase extraction cartridge  34 , rendering it ineffective. Various counter measures are taken to avoid precipitate clogging of the solid phase extraction cartridge  34 . For example, the frit (pore size=2 micron) blocks the appropriate amount of solid reaction mixture components to prevent the solid phase extraction cartridge from clogging.  
         [0031]     Also, a frit bypass line  44  is provided if there is a need to have the option to bypass the frit  32  because it gets partially blocked, slowing the filtration. The cartridge  34  can still work normally when the bypass line  44  is used, provided that most of the reaction mixture has been filtered. The limits of solid material deposition on the cartridge  34  have been explored and with the synthesizer  10  the process described above is reliable.  
         [0032]     With respect to solid phase extraction cartridge selection, the Waters Sep-Pak® C-18 solid phase extraction cartridge is suitable because C-18 tends to retain lipophilic compounds when they are loaded in hydrophilic mixtures. The DMSO is miscible with water and needs to be greatly diluted with water for the concept to work best. The concept was explored and dilution can be optimized to allow retention of  18 F-FLT (3′-deoxy-3′-[ 18 F]Fluorothymidine). The pressure must also be controlled, to keep the flow slow enough to allow the  18 F-FLT to interact with the solid-phase material in the solid phase extraction cartridge  34 .  
         [0033]     After delivering the reaction mixture to the solid phase extraction cartridge  34 ,  18 F-FLT and a small amount of impurities are retained on the solid-phase material in the solid phase extraction cartridge  34 . The  18 [F] fluoride ions, Kryptofix™ 222, potassium carbonate, acetonitrile, HCl, sodium acetate, and water move through the cartridge  34  and are sent to waste vessel  47  by way of  25  valve V 13 .  
         [0034]     Water is then delivered through valves V 5 , V 10  and V 11  and through the solid phase extraction cartridge  34  to wash the remaining impurities off the solid-phase sorbent material in the solid phase extraction cartridge  34 . Washing the cartridge  34  with water works well to remove the hydrophilic  18 F fluoride ions, and one key is using an adequate supply of water to ensure complete fluoride removal. In one embodiment, the impurities are washed away from the cartridge  34  using about 15.5 milliliters of water. The remaining impurities and water are sent to waste vessel  47  by way of valve V 13 . Purified  18 F-FLT remains on the solid-phase material in the solid phase extraction cartridge  34 .  
         [0035]     Ethanol is then delivered through valves V 12 , V 10  and V 11  through the solid phase extraction cartridge  34  to elute the purified  18 F-FLT off the solid-phase material in the solid phase extraction cartridge  34 . The ethanol and purified  18 F-FLT mixture is then delivered to vessel  58  by way of valve V 13 . In one embodiment, the purified product is eluted from the cartridge  34  using 1 milliliter of ethanol. Ethanol is a suitable eluent, and it has a low toxicity in humans. It also evaporates at low temperatures (to avoid decomposition of the product), and was found to adequately elute the product from the cartridge  34 . The ethanol and purified  18 F-FLT mixture in the vessel  58  are heated to remove the ethanol. After evaporation of the ethanol, the purified  18 F-FLT is dissolved in water delivered to vessel  58  by way of valve V 6 . Water (not saline) is used because the addition of ions to the mixture would interfere with removal of residual  18 F fluoride ions at a later step.  
         [0036]     Water may not dissolve the  18 F-FLT product adequately. Therefore, the water and  18 F-FLT mixture in the vessel  58  may be delivered through valve V 14  after reconstitution to a second [ 18 F] fluoride trap  92  similar to the [ 18 F] trap cartridge  12  and a filter  67  (0.2 micron pore size) for filtering and delivery to vessel  73  prior to any further fluoride removal and sterilization. This prevents clogging of cartridges and sterilization filters. Usually, HPLC would be used to remove  18 F fluoride ions, but the second [ 18 F] fluoride trap  92  was used as an alternative and was found to effectively reduce fluoride in the product to make the product suitable for use.  
         [0037]     The entire synthesizer  10  may be operated by a controller using suitable software for control of, among other things, valves V 1  to V 14 . This provides many advantages including, for example, software accommodation of the frit bypass line  44 . A pause may be built-into the frit filtering step. The valves are positioned such that a pressure drop can be observed by a user to ensure that the pressure is dropping as expected while material is passing through the frit  32 . If pressure does not drop during observation, it indicates that the frit  32  is clogged, and frit bypass line  44  should be used.  
         [0038]     Various reaction mixtures are described in the literature for labeling fluorothymidine. The example mixture described above for the invention incorporates dimethylsulfoxide (DMSO) as a solvent. DMSO was desired as a solvent in this purification process because it dissolves the precursor molecule better than the alternative solvents. The need for optimal dissolution (for increased labeling efficiency) was important due to the fact that solid phase extraction methods can be inefficient at purification. The negative aspects of using DMSO were taken into account (high boiling point, tendency to leave residual) and determined to be tolerable with taking the appropriate counter measures such as (i) DMSO dilution with large excess of water (at least 7 to 1 water to DMSO) to prevent DMSO from eluting  18 F-FLT off the cartridge  34  while loading the diluted reaction mixture. (the large excess of water also starts to minimize effect of residual DMSO due to decreasing concentration), and (ii) water wash phase delivery of water through an independent path from the reaction vessel to prevent residual DMSO in the reaction vessel  16  from contaminating the lines and solid phase extraction cartridge  34 .  
         [0039]     Although the present invention has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.  
       INDUSTRIAL APPLICABILITY  
       [0040]     The invention relates to the manufacture of radiopharmaceuticals and particularly, the manufacture of 3′-deoxy-3′-[ 18 F]Fluorothymidine.