Patent Application
20140097078
The present invention is relates to radiochemistry. More specifically, the present invention is directed to a device for and method of performing distillation during radiosynthesis.
The prevalence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has increased over the last three decades, leading to an increased need for a suitable PET imaging agent. Somatostatin receptors, mainly sub-type 2, have been shown to be over-expressed on the surface of GEP-NETs leading to the development of octreotide, a somatostatin analogue. Octreotide has been labelled with many isotopes, but the radioligand routinely used in the clinic remains to be [111In]-Pentetreotide (Octreoscan™, sold by Covidien, manufactured by Mallinckrodt, Inc., Maryland Heights, Mo., USA).
Alternatively, a fluorine-18 labelled octreotate analogue which can be used for positron emission tomography (PET) imaging has also been developed. Octreotate was chosen over octreotide, since improvement in receptor affinity has been shown replacing the threoninol to threonine (see, Reubi, J. C.; Schar, J. C.; Waser, B.; Wenger, S., Eur. J. Nucl. Med. Mol. Imaging 2000, 27, 273). A novel class of fluorine-18 labelled Octreotate analogues have been developed through incorporation of various linker moieties at the N-terminus of the octapeptide. The labelling was achieved via the copper catalysed azide-alkyne cycloaddition reaction (CuAAC), which has proved to be an efficient and selective radiolabelling technique.
[18F]FET-βAG-TOCA has been identified as a tracer for the imaging of somatostatin positive neuroendocrine tumours (see, Iddon, L.; Leyton, J.; Indrevoll, B.; Glaser, M.; Robins, E. G.; George, A. J. T.; Cuthbertson, A.; Luthra, S. K.; Aboagye, E. O., Bioorg. Med. Chem. Lett. 21, (10), 3122). FET-βAG-TOCA, can be efficiently labelled during a click reaction in five minutes at room temperature. The use of click chemistry as a method to introduce radioisotopes into PET tracers has become more frequent in recent years since it was first applied by Marik and Sutcliffe, see Tetrahedron Lett. 2006, 47, 6681. Click chemistry has the advantage of being selective, and therefore reactive functional groups are well tolerated. It often favours aqueous conditions which allows for more polar molecules such as peptides to be labelled. The reaction shows selectivity, giving only the 1,4-substituted triazole and is generally an efficient reaction done at ambient temperatures.
[18F]fluoroethyl azide (“[18F]FEA”), is an intermediate of [18F]FET-βAG-TOCA. [18F]FEA may be purified by distillation along with the reaction solvent, acetonitrile. However, the apparatus used to carry out distillation in a manual laboratory setting is the thermospray device developed by the assignee of the instant invention, as described in United States Patent Publication No. 20090312654. The thermospray device is a unit containing a heated, coiled tube of a suitable material (peek tubing, stainless steel) in which the product can be collected through the end of the tubing in an appropriate vial. As this is a manual operation in which high quantities of fluorine-18 (>20 mCi) can lead to high extremity doses, there is a lack in the art for performing the distillation method to purify [18F]FEA as part of a click chemistry reaction method on an automated platform. There is additionally a need in the art for performing both distillation and click chemistry reactions on an automated platform to be able to isolate levels of activity for a clinical dose (˜10 mCi/mL).
The present invention provides a distillation and click chemistry method which can be applied to an automated process. The present invention is able to isolate material which could be suitable for routine clinical imaging of neuroendocrine tumors. The present invention provides a cassette for automated radiosynthesis that incorporates two reaction vessels.
The present invention also provides a disposable synthesis cassette and a method for performing purification and click chemistry on an automated synthesizer. The cassette includes two reaction chambers. The cassette desirably allows for additional purification to take place off-cassette while further performing final formulation prior to dispensing.
The present invention further provides a kit for performing synthesis of a radiopharmaceutical. The kit includes components adapted to be used with an automated synthesizer for performing purification and click chemistry. The kit provides two reaction chambers.
The cassette and kit of the present invention are configurable to be particularly suitable for synthesizing fluorine-18 labelled octreotate analogue synthesized via click chemistry.
The cassette and kit of the present invention additionally allows for preconditioning of an SPE cartridge which may be performed under a hood to maintain sterility of the cassette. The cassette and kit of the present invention also allows for provision of reagents in the second reaction chamber which may be performed under a hood to maintain sterility of the cassette.
The present invention may be used to purify the labelled intermediate, eg, [18F]FEA, of a synthesized compound in an automated process. The purification may include distillation of the intermediate prior to performing a click chemistry reaction. For example, the present invention is able to provide for the synthesis of FET-βAG-TOCA on an automated cassette-based platform by first distilling [18F]FEA and providing the distilled output to a click chemistry reaction.
The present invention provides a cassette, and additionally a kit of components, for performing a radiosynthesis method including a purification step via distillation and a cassette which allows this method to be performed in a substantially automated manner. The present invention incorporates two reaction vessels onto a cassette manifold in which to purify an intermediate of the radio synthesis product and to perform a click chemistry reaction. The second vessel added to the cassette allows for a reaction to occur at room temperature.
Synthesis device 100 may be a FASTlab® synthesizer sold by GE Healthcare, Liege, BE, which incorporates the software for operating cassette 110 in accordance with the method of the present invention. The software of the present invention is provided as a non-transitory computer readable storage medium with an executable program for performing the method of the present invention when cassette 110 is mounted to synthesis device 100. Synthesizer 100 is thus able to operate cassette 110 to conduct the steps of performing a chemical reaction in a first vessel at a first elevated temperature, heating the first vessel to a second elevated temperature to cause distillation, delivering a distilled reaction product from the first vessel to a second vessel; and performing a click chemistry reaction with the distilled reaction product in the second vessel. Desirably, the second elevated temperature is higher than the first elevated temperature. Additionally, the first and second vessels are desirably connected to a common manifold through which the reaction product and certain reagents may be conducted during performance of the process. For example, the delivering step desirably includes the step of directing the distilled reaction product from the first vessel though a portion of the manifold to the second vessel. Additionally, the method of the present invention may further include the steps of purifying the click chemistry product and formulating a final product from the purified click chemistry product, wherein the purifying step is performed in a purifying device connected to the manifold. The purifying device is thus desirably operated in coordination with said synthesizer device. The second vessel is desirably preloaded with reagents, although the present invention may include the step of placing click chemistry reagents in the second vessel
Cassette 110 is thus removably attachable to synthesis device 100 which cooperatively engages the cassette so as to be able to actuate each of the stopcocks and syringes to drive a source fluid with a radioisotope through the cassette for performance of a chemical synthesis process. Additionally, synthesis device 100 includes a heating cavity into which receives the first reaction vessel of cassette 110 therein so as provide the heat required for chemical reactions occurring therein. No heating is required for the second vessel. Synthesizer 100 is programmed to operate pumps, syringes, valves, heating element, and controls the provision of nitrogen and application of vacuum to the cassette so as to direct the source fluid into mixing with the reagents, performing the chemical reactions, through the appropriate purification cartridges, and selectively pumping the output tracer and waste fluids into appropriate vial receptacles outside the cassette. While the fluid collected in the output vial is typically input into another system for either purification and/or dispensement, synthesizer 100 and cassette 110 can also be connected to a separate purification system which returns a purified compound back to cassette 110 for further processing.
After product dispensement, the internal components of cassette 110 are typically flushed to remove latent radioactivity from the cassette, although some activity will remain. Cassette 110 thus can be operated to perform a two-step radiosynthesis process. By incorporating a second reaction vessel on the manifold, cassette 110 of the present invention is further able to provide simple purification so as enable click chemistry processes.
With additional reference to
Valves 2-24 include three open ports opening to adjacent manifold valves and to their respective luer connectors, cannulas, and syringe barrels. Valves 1 and 25 include three open ports, one port opening towards valve 2 and 24, respectively, on port opening upwards, and one port opening in fluid communication with manifold endports 118 and 120, respectively. Each valve includes a rotatable stopcock which puts any two of the three associated ports in fluid communication with each other while fluidically isolating the third port. Manifold 112 further includes, at opposing ends thereof, first and second socket connectors 121 and 123, each defining rearwardly-opening (ie, towards the synthesizer 100 to which is mounted) gas ports 121a and 123a, respectively. Synthesizer 100 includes 25 rotatable arms, each for engaging one of the stopcocks of cassette 110 and to position each stopcock according to a synthesis program, thereby enabling controlled flow through appropriate portions of cassette 110. In
Cassette 110 desirably includes a polymeric housing (not shown) having a planar major front surface and defining a housing cavity in which manifold 112 is supported. Cassette 110 includes a first reaction vessel 114 and a second reaction vessel 116. First reaction vessel 114 includes a vessel body 122 defining a reaction chamber 124 and three vessel ports 126, 128, and 130. Vessel ports 126, 128, and 130 are connected in individual fluid communication with valves 7, 8, and 25, respectively. Second reaction vessel 116 includes a vessel body 132 defining a reaction chamber 134 and three vessel ports 136, 138, and 140. Vessel ports 136, 138, and 140 are connected in individual fluid communication valves 9, 10, and 20, respectively. Reaction vessel 114 is sized to be placed within a heating cavity on the synthesizer 100 so that heat may be applied to the reaction occurring in chamber 124. Reaction vessel 116 is able to remain outside of the heating cavity on the synthesizer so that the reactions occurring therein are conducted at room temperature. Additionally, cassette 110 is connectable to an HPLC purification system 105 (in
A first reverse separations cartridge 142 is positioned between manifold positions 4 and 5 while a second separations cartridge 144 is positioned between manifold positions 22 and 23. First separations cartridge 142 is used for primary purification. Second separations cartridge 144 is used for solvent exchange, or formulation. A length of Tygon tubing 146 is connected between manifold valve 21 and a product collection vial 148 in which is dispensed the formulated drug substance. Vial 148 desirably supports a vent needle so as to allow gas within vial 148 to escape therefrom while the vial fills with the product fluid dispensed from cassette 110. While some of the tubings of the cassette are, or will be, identified as being made from a specific material, the present invention contemplates that the tubings employed in cassette 110 may be formed from any suitable polymer and may be of any length as required.
With continued reference to
Cassette 110 desirably includes an elongate hollow support housing 170 having a first end supported at valve 15 and an opposed second end supporting an elongate hollow spike 172 extending therefrom. Spike 172 is designed to pierce the septum of a water container 174 which desirably provides a supply of water-for-injection for use in the synthesis process. Cassette 110 further includes a plurality of pumps engageable by the synthesis device in order to provide a motive force for fluids through the manifold. Valves 3, 11, and 24 each support a syringe pump 176, 178, and 180, respectively, in fluid communication with the upwardly-opening valve port and each including a slideable piston reciprocably movable by the synthesizer device. Syringe pump 176 is desirably a 1 ml syringe pump that includes an elongate piston rod 177 which is reciprocally moveable by the synthesis device to draw and pump fluid through manifold 112 and the attached components.
Valve 6 supports an elongate hollow housing 182 having a cylindrical wall 182a defining an open elongate cavity 184. The radioisotope, in this example [18F]fluoride, is provided in solution with H2[18O] target water and is introduced at manifold valve 6. Connection of the source of the radioisotope is made to housing 182 prior to the initiation of synthesis. Valve 1 supports a length of tubing 186 extending to a waste collection vial 187 which collects the waste-enriched water after the fluoride has been removed by the QMA cartridge 142. The fluoride will be eluted from cartridge 142, using the K222/KHCO3 from vial housing 150, and delivered to the first reaction vessel 114, as will be described further hereinbelow.
A length of tubing 188 will be connected to valve 19 and extend to an external purification system 105, while another length of tubing 190 will be connected to valve 18 to return the fluid from the external purification system. The external purification system is desirably an HPLC system (no shown), although other purification systems are contemplated as being suitable for the present invention. Valve 17 supports a luer cap 192 thereon in order to seal the upwardly-opening valve port thereof.
Syringe pumps 178 and 180 are each desirably a 5 ml syringe pump that includes n elongate piston rod 179 and 181, respectively, which are reciprocally moveable by the synthesis device to draw and pump fluid through manifold 112 and the attached components. Movement of fluid through manifold 112 is additionally coordinated with the positioning of the stopcocks of valves 1-25, the provision of a motive gas at gas ports 121a and 123a as well as by a vacuum, such as that applied to port 120 (through vial 135). The present invention contemplates that the motive gas and the water-for-injection may be pumped through manifold 112 so as to assist in operating cassette 110.
Cassette 110 is mated to an automated synthesizer, desirably a FASTlab synthesizer, having rotatable arms which engage each of the stopcocks of valves 1-25 and can position each stopcock in a desired orientation so as to direct fluid flow throughout cassette operation. The synthesizer also includes a pair of spigots, one of each of which insert into ports 121a and 123a of connectors 121 and 123 in fluid-tight connection. The two spigots respectively provide a source of nitrogen and a vacuum to manifold 112 so as to assist in fluid transfer therethrough and to operate cassette 110 in accordance with the present invention. The free ends of the syringe plungers 177, 179, and 181 are engaged by cooperating members from the synthesizer, which can then apply the reciprocating motion thereto within the syringes 175, 178, and 180, respectively. A bottle containing water is fitted to the synthesizer then pressed onto spike 170 to provide access to a fluid for driving compounds under operation of the various-included syringes. Reaction vessel 114 will be placed within the reaction well of the synthesizer and the product collection vial 148 and waste vial 135 are connected. The synthesizer includes a radioisotope delivery conduit which extends from a source of the radioisotope, typically either vial or the output line from a cyclotron, to a delivery plunger. The delivery plunger is moveable by the synthesizer from a first raised position allowing the cassette to be attached to the synthesizer, to a second lowered position where the plunger is inserted into the housing 182 at manifold valve 6. The plunger provides sealed engagement with the housing 182 at manifold valve 6 so that the vacuum applied by the synthesizer to manifold 112 will draw the radioisotope through the radioisotope delivery conduit and into manifold 112 for processing. Additionally, prior to beginning the synthesis process, arms from the synthesizer will press the reagent vials onto their respective cannulas at their manifold valves. Lastly, a conduit 133 is connected to port 120 and spans to a waste vial 135 so that the cavity of vial 135 is in fluid communication with port 120. Waste vial 135 is also pierced by a vent needle 137 which allows gas to pass therethrough but not liquid. A conduit 139 extends from vent 137 to a vacuum port (not shown) on the synthesizer. The synthesis process may then commence.
The present invention further contemplates providing cassette 110 as part of a kit which may be assembled so as to perform a radiosynthesis method. The kit desirably provides cassette 110 with the required lengths of tubing as well as the reagents to be placed in the reagent housings. Additionally the kit of the present invention provides a source of reagents to be provided in second reaction vessel 116 for the click chemistry reaction. The sources of reagents may be provided in one or more vials where one vial contains CuSO4(aq) and another vial contains βAG-TOCA which may be added to second reaction vessel 116. The kit desirably further provide other reagent containers positioned within the reagent housings of the manifold at the first position so that their respective septums are spaced from the underlying spikes of their respective valves, although these other reagent containers may be insertable into their respective reagent housings. It is further contemplated that reaction chamber 134 may be accessed by disconnecting one or more of the conduit lines connected thereto so as to place the desired reagents therein. The disconnection and connection of those conduit lines, and the delivery of those reagents, is desirably performed under a flow hood providing a suitably clean environment. Likewise, second cartridge 144 may be pre-conditioned under a hood in a suitably clean environment and connected to manifold 112 there as well.
Cassette 110 may be configured for the production of [18F]FET-βAG-TOCA, although one of skill in the art will understand that variations in the reagents and operation of the cassette will allow for the production of other radiotracers utilizing either or both of distillation and click chemistry. The automated processes described hereinbelow were all performed using cassette 110 on a FASTlab synthesizer device. First reaction vessel 114 was positioned in the heating well of the FASTlab synthesizer.
The drying of fluorine-18 may be carried out in the first vessel 114 using a known operating sequence, such as that used by the FDG sequence file of synthesizer 100 when operating an FDG synthesis cassette of the prior art. Addition of 2-azidoethyl-p-toluenesulfonate in a solution of MeCN to Reaction vessel 114 is carried out using syringe pump 178 (5 mL syringe), opening valve 11 and adding to reaction vessel 114 through valve 7. The reaction is then heated to 80° C. for 15 min in reaction vessel 114. To distil the solution a low flow of nitrogen (˜100 mbar) is passed into reaction vessel 114 via valve 7, valve 8 is opened and the solution is distilled into Reaction vessel 116 through valve 10, with valves 17-25 being set in open communication so as to allow the exhaustion of the line with a low vacuum (−100 mBar) applied to vial connected to end port 120. The vacuum applied to the vial is provided by a second connection (not shown) between the vial 135 and the synthesizer 110.
Entry 1 in Table 1 above shows the most promising results to date. The [18F]FEA synthesised on the FASTlab has been used to carry out a click reaction with one of the alkynes designed for Octreotate (AH114667). The click chemistry proceeded as previously found using [18F]FEA from the thermospray distillation. There appears to be some loss of activity through to the waste bottle and it appears that some activity is trapped within the cassette manifold but this has not been measured to date.
Additionally experiments have been carried out that utilise the FASTlab platform for addition of the click reagents, sodium ascorbate, and bathophenanthroline disodium salt (BDPS). The CuSO4 and alkyne, AH114667 were added to the reaction vessel 116 prior to the synthesis commencing.
Additional reference is now made to
The fluorine-18 was drawn into the activity inlet reservoir (at valve 6) under vacuum and loaded onto the QMA cartridge 142. The K222/KHCO3 solution was then taken up into syringe 176 (at valve 3) and used to elute QMA cartridge 176 into reaction vessel 114 through valve 7 of the manifold. Reaction vessel 114 was then heated to remove the solvent. The precursor (A) was taken up into syringe 178 (at valve 11) and then added to A (200 μL) and heated to 80° C. for 15 minutes. The distillation was then performed at 120° C., nitrogen was applied to vessel 114 through valve 7, valve 8 was opened to the manifold and valve 10 of vessel 116 was opened to allow the [18F]fluoroethyl azide to enter, with a low vacuum applied to vessel 116 through valve 20. Following distillation, the Na-ascorbate solution was taken up into syringe 180 (at valve 24), and added to vessel 116 through valve 20. The BPDS was similarly directed through valve 14 to vessel 116. On addition of the reagents, N2 was applied to the reaction mixture to ensure mixing. After 5 minutes at room temperature the reaction was diluted with H2O (1.5 mL) and passed through valve 19 to the HPLC module for purification. The product was collected into a vial, diluted further with H2O (6 mL) and taken up through valve 18 into syringe 2. The diluted product was then subsequently applied to the tC18 cartridge 144 and eluted further with water. A flow of N2 was passed through the tC18 cartridge 144 to remove any solvents. The tC18 cartridge 144 was eluted with EtOH/PBS (1.5 mL) into the final product vial which contained a PBS solution (9 mL) for final formulation. The solution was then passed through a 0.22 μm sterile filter (PALL, Acrodisc HT Tuffryn Membrane, low protein binding).
The initial step of the synthesis is a nucleophilic displacement of the tosylate group of 2-azidoethyl-p-toluenesulfonate (A) by the [18F]fluoride anion to yield [18F]fluoroethyl azide (‘B’ from
The second step in the synthesis sequence was to incorporate the CuAAC reaction onto the FASTlab platform. During stability testing it could be seen that the βAG-TOCA was not stable for >20 minutes in the presence of either Na-ascorbate or BPDS, but was stable for >4 h in the presence of CuSO4. Thus, this approach was modified, in order to avoid degradation, by adding the Na-ascorbate and BPDS after the distillation of [18F]fluoroethyl azide was complete. The CuSO4(aq) and βAG-TOCA were added to reaction vessel 116 before the start of the synthesis. To add the desired quantities of Na-ascorbate (100 μL) and BPDS (100 μL) required careful manipulation of the pressure within the reaction vessels and manifold. The manifold was pressurised initially, followed by vessels 114 and 116. This ensured that no negative pressures were present that could move the solution quickly into the wrong compartment. The Na-ascorbate was then withdrawn from its reagent vial using syringe 180. In doing this process the manifold was filled with the Na-ascorbate solution. Valve 17, which had been sealed with luer fitting 192 on its upstanding port, was then oriented to prevent any solution from backtracking into its reagent vial. The contents of the syringe were then emptied through conduit 133 into the waste vial 135 and then nitrogen was passed via reaction vessel 114 to the syringe (position 24). The Na-ascorbate solution was then passed into reaction vessel 116 through valve 20 with the assistance of the N2 filled syringe, ie, the N2 was applied through port 121a, and a vacuum was pulled through the waste vial connected to end port 120. The same procedure was then repeated during addition of BPDS. Once both reagents had been added to the reaction mixture a gentle flow of nitrogen was passed into 116 to ensure that the solution was homogenous. Despite the total volume of the reaction increasing to 405 μL (vs the manual method total of about 205 μL), the reaction shows completion after 5 minutes at room temperature. This approach proved successful, during HPLC purification, as [18F]FET-βAG-TOCA was the major radiolabelled product (>90%). Once the product had been collected from the HPLC purification it was diluted with water and loaded onto a tC18 cartridge ready for formulation. It was found that EtOH/PBS (50:50) could be used to elute the product (1.2-1.3 mL). The isolated end of synthesis yield of [18F]FET-βAG-TOCA from fluorine-18 (10-100 mCi) was 12-23% (n=7), with a total synthesis time of 80 minutes.
The use of higher levels of fluorine-18 (0.5-1 Curie) was also investigated. Using 1 Curie of fluorine-18 resulted in significant radiolysis of the parent (only 62% parent at T=Om) which is believe due to HPLC purification and tC18 formulation. Although radiolysis occurred during isolation, once the product had been fully formulated (6% EtOH/PBS (10 mL)) it appeared to be stable up to 6 h. During this experiment, the EOS yield after aseptic filtration was 13%. The starting fluorine-18 was then reduced to see enough material could be isolated for a clinical dose, whilst at the same time reducing radiolysis. It was found that starting with 0.5 Curies allowed the isolation of sufficient activity (67-90 mCi (10.3 mL)) and analysis showed 91% of intact parent.
As a result, the present invention has been shown to provide an automatable cassette, and a kit therefor, which may be operated to isolate the final formulated product in EOS yields of 10-18% with radiochemical purity (97%) suitable for routine clinical imaging of neuroendocrine tumors. Those of skill in the art will recognized that the present invention may be modified to synthesize other compounds without departing from the teachings herein.
Additional experiments attempted to add ascorbic acid to the existing HPLC eluent (0.5% w/v), add a sodium ascorbate solution to the HPLC fraction collection vial (5 mg/mL (5 mL)) and elute the tC18 cartridge with a sodium ascorbate/EtOH solution (5 mg/mL/1% EtOH). Unfortunately, due to addition of ascorbic acid to the HPLC eluent, the purification became inefficient, showing very little elution of the stable reagents. Despite the preparative HPLC issues, the radiolysis was reduced and showed 97% parent at T=0 min (
While the particular embodiment of the present invention has been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the teachings of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation. The actual scope of the invention is intended to be defined in the following claims when viewed in their proper perspective based on the prior art.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/041204 | 6/7/2012 | WO | 00 | 12/9/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61531212 | Sep 2011 | US | |
| 61494934 | Jun 2011 | US |
