Patent Application
20110280770
Not applicable.
Not Applicable.
1. Field of the Invention
This invention relates to radiopharmaceuticals, and more particularly to an improved apparatus and method for producing carrier free, ionic 62Cu.
2. Description of the Related Art
Positron Emission Tomography (PET) is an important and powerful diagnostic imaging tool; however, the distribution and regulatory issues associated with PET radiopharmaceuticals, such as 18F-FDG imply the need for generator-based imaging tracers. Proportional Technologies, Inc. (PTI) continues to refine the development of the 62Zn/62Cu generator that provides carrier free, ionic 62Cu (t1/2=9.7 min) and achieves instant high labeling yield with cGMP produced ligands in convenient lyophilized kit form. This system produces 62Cu with unexcelled levels of purity and specific activity because of the intrinsic characteristics of its anion exchange medium having a strong affinity exclusively for Zn. The current generation of the 62Zn/62Cu generator (the “microgenerator”) and the lyophilized kits can be nationally distributed, satisfies regulatory requirements for human use, and the feasibility for both animal and human studies has been demonstrated. The 62Cu half-life readily enables serial imaging studies, and the easy versatile coordination chemistry of copper provides wide ranging capabilities.
A rapidly emerging area for the diagnostic imaging is the development of new tumor-specific peptide-based radiopharmaceuticals. Since many peptide receptors are overexpressed on tumors, they make an attractive target for diagnostic imaging and radiotherapy with radiolabeled peptides. Producing these new radiopharmaceuticals successfully in the clinical setting requires in many instances very high specific activity (SPA) labeled peptide without any further HPLC purification.
Positron Emission Tomography (PET) is a unique imaging technique among the many diagnostic technologies available to clinicians. Unlike the majority of imaging techniques such as X-ray, SPECT, CT, ultrasound, MRI, or mammography that provide imaging of solid anatomical features, PET can measure functional characteristics of the tissue being imaged. As of 2004, approximately 900,000 PET scans were performed annually in the United States, and this number is predicted to grow to more than 2,000,000 by 2010. At a base cost of $3000-$6000 per PET scan, this represents a nearly $3 billion per year sector of the health care industry. PET is routinely used in combination with CT for monitoring treatment progress, and hybrid scanners capable of performing dual PET/CT scans during a single scanning operation are in common use. The vast majority of clinical studies currently employ the single metabolic tracer 18F-FDG.
Radioactive Copper Isotopes for PET Application: Copper offers simple coordination chemistry with a variety of ligands and has a group of promising positron-emitting radioisotopes, such as 60/61/62/64Cu. The very short half-life of 60Cu limits its use to a research setting with a dedicated cyclotron, although researchers at Washington University have successfully utilized this isotope in a clinical setting. The longer half-life of 61Cu potentially opens the door to distribution from regional radiopharmacies, but the lower abundance of the positron decay mode (only 60%) in 61Cu is problematic and the long half life of 3.41 hrs limits injectable dose and makes it impossible or at least very difficult to perform serial studies within a single clinic day. The much longer half-life of 64Cu circumvents the processing and shipment problem, but low positron decay abundance together with severe injected dose constraints imposed by dosimetry make it very difficult to obtain PET images with reliable imaging statistics within reasonable image acquisition times. All of the cyclotron produced copper isotopes, relying upon enriched isotopic plating for each production are incredibly expensive for animal/preclinical studies. They are probably impractical for human studies because of the need for cyclotron operated under cGMP conditions at all clinical facilities. 62Cu is the exception, which can be provided by an automated 62Zn→62Cu generator developed by Proportional Technologies, Inc. The 9.3 hour half-life of the 62Zn parent is amenable to GMP assembly of the generator at a central production facility followed by express courier delivery to clinical sites. The wide distribution of this generator is feasible from a small number of production centers strategically located all over the country. The 9.3 hr half life of the 62Zn parent is 5 times longer than the 110 minute 18F half life. That means the 62Zn/62Cu generator can be delivered over a radius that is 5 times greater. Thus a given center can serve 25 times the geographical area. Hence nearly 100 production facilities are required for 18F-FDG; only 4-8 centers are required for 62Zn/62Cu.
Success of the 62Zn/62Cu Microgenerator: PTI has developed an automated 62Zn/62Cu microgenerator that can work with three interchangeable lyophilized kits for instant synthesis of 62Cu bis(thiosemicarbazone), or 62Cu-BTS, radiopharmaceuticals.5-25 This system is being nationally distributed and readily satisfies FDA requirements. The 62Cu half-life of 9.7 min enables serial imaging studies and by allowing larger injected dose reduces patient imaging time. The 62Zn/62Cu microgenerator and associated BTS ligand kits have been used extensively by subcontracted clinical sites, interested third party academic and clinical researchers, and a major pharmaceutical company. PTI has successfully manufactured and delivered the microgenerator by overnight shipment with a private courier over 70 times. Table 1 summarizes the recipients of the microgenerator and the nature of the research conducted.
The 62Zn/62Cu microgenerator is compatible with high throughput use in a clinical setting. This system has high potential to become a robust source of PET 62Cu agents with greater distribution capacity and lower cost than exists for currently available agents. As part of the generator development, PTI has also pioneered a facile, interchangeable kit-based synthesis of multiple 62Cu-BTS radiopharmaceuticals. The BTS agents utilized by PTI display a range of human clinical profiles, and all are on firm regulatory footing with existing INDs or NDAs in place. Most recently, two large clinical studies have been funded for clinical application of agent 62Cu-ETS at Indiana University/Purdue University at Indianapolis (IUPUI). These studies will focus on measurement of blood flow in head and neck and renal cancers and could solve the problem of providing a robust blood flow agent as a clinically practical alternative to 15O Water.
Since 1995, PTI has been actively engaged in the research and development of 62Cu-BTS PET imaging agents. The first of these compounds, 62Cu-Pyruvaldehyde bis(N4-methylthiosemicarbazone), or 62Cu-PTSM, a perfusion agent, has had an active IND application filed with the FDA since 1995 and was followed with a NDA for the myocardial perfusion rest/stress imaging agent, trade name MyoPET, using the first generation 62Zn/62Cu generator system in 2001. The NDA was granted approvable status from the FDA in a letter in 2002. This NDA is eligible to be continued addressing concerns in the approvable letter.
Commercial IND's have been filed for the perfusion agent, 62Cu-Ethylglyoxal bis(N4-methylthiosemicarbazone), also known as 62Cu-ETS, in 2006 and for the hypoxia imaging agent, 62Cu-Diacetyl bis(N4-methylthiosemicarbazone), also known as 62Cu-ATSM in 2007. The second generation generator system, or the “microgenerator,” used to create instant kit synthesis of the aforementioned compounds was included with the 62Cu-ETS IND; however, it works interchangeably with all three compounds.
In collaboration with the University of Wisconsin-Madison (UW), PTI has completed the Phase I and Phase II clinical study for 62Cu-ETS. An additional Phase II study protocol has also been approved at IUPUI by the FDA, which involves the evaluation of tumor perfusion utilizing 62Cu-ETS in head and neck cancer patients, employing 15O-water and 18F-FDG as comparators. A third Phase II study will soon begin at IUPUI after FDA approval is obtained to begin in renal cancer patients to measure tumor blood flow with 62Cu-ETS.
A Phase I clinical study for 62Cu-ATSM has also been complete by UW and the final report was submitted to the FDA in 2008. An animal feasibility study was completed by Duke University researchers comparing tumor hypoxia maps obtained from PET imaging using 62Cu-ATSM and 62Cu-PTSM autoradiographs and hypoxia maps generated through the use of an immunohistochemical marker in tumor-bearing rats. Table 2 summarizes the IND/NDA status for the various 62Cu-BTS agents and indicates the number of human subjects who have received these agents.
62Cu-PTSM
62Cu-ETS
62Cu-ATSM
Peptide application in PET: Over the last two decades, small peptide-based radiopharmaceuticals targeting specific receptors for PET tumor imaging and targeted radiotherapy has been recognized and has attracted intensive investigation and development. This is attributable to the unique peptide receptors prolifically expressed in many tumor cells at higher levels than in normal tissues. The best examples are somatostatin (SST) analogs27,33-77 (targeting SST receptor overexpressed in neuroendocrine tumors(NETs)) TOC27,38-45 and NOC46-51 peptide, RGD52-70 peptide analogs and multimers (targeting αvβ3-integrin receptor highly expressed on neoangiogenic vessels in various cancers) and α-melanocyte-stimulating hormone (α-MSH) analog Re(Arg11)CCMSH71-82 (targeting melanocortin-1 receptor in melanoma). These small peptides offer multiple advantages in imaging and therapy over proteins or antibodies. They can be easily synthesized and structurally manipulated to optimize their in vivo stability and improve their intrinsic high affinity to the corresponding peptide target receptor. They can be radiolabeled with isotopes such as 99mTc, 188Re, 177Lu, 212Pb, 111In, 86Y, 68Ga, 64Cu 71,73,77 after coupling to bifunctional chelators (BFCs).83 Because of their small size, they have much stronger ability to penetrate into tumors than large sized proteins or antibodies, they can reach peptide receptors on tumor cells efficiently, their localization and uptake into target cells and their clearance from the blood and non-target tissues is also very favorable. For example, the peak uptake time of 64Cu-radiolabeled RGD dimer in female nude mice bearing U87MG tumor cells is only about 15 min and competing tissues are well cleared by this time.67 Re(Arg11)CCMSH reaches the highest uptake at 30 min in TXM-13JQ human melanoma-bearing Scid mice84 and competing tissues are also well cleared. The promising TOC agent has been investigated in pilot patient studies in neuroendocrine tumours. Although the optimal imaging time was reported as 50 min in this study, the kinetic table provided showed almost no benefit to imaging after 20 minutes either in tumor uptake or background tissue clearance. With these fast tumor uptake and contrast times, short-lived 62Cu, labeled peptides are physically and chemically feasible in the tumor imaging applications.
Bifunctional chelators are required to link the peptide to radionuclides. In selecting an appropriate BFC for coupling with a receptor peptide, two requirements are 1) that the binding of the radionuclide by the BFC be very rapid for instant labeling in the clinical setting and 2) that the BFC-metal chelate be very stable in vivo so that organs, such as the liver, do not develop high background levels of radioactivity during imaging studies. Among various chelators reported in the literature, macrocyclic tetraaza/triaza compounds NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) satisfies these requirements for labeling and has been clearly demonstrated as highly superior in terms of instant labeling at room temperature to DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), the bifunctional chelators most frequently reported in the literature. The techniques for peptide bifunctionalization with such chelators are well known and widely practiced.
High specific activity formulations are required for PET imaging with peptides, especially if the peptide is able to induce pharmacologic effects, in even trace amounts. A perfect example is ReCCMSH(Arg11), a promising peptide agent that targets the melanocortin-1 receptor in melanoma imaging and therapy. All peptide agents have to go through preclinical screening studies using small animals like mice. The tumors in mice are very small in volume, typically just 1 gram or less, accordingly resulting in fewer available receptor sites. The melanocortin-1 receptor expresses only around 3000 active sites per cell. For a typical cell diameter of 20 microns, the number of tumor cells in a 1 mL tumor is approximately 2.4×108. The number of ReCCMSH(Arg11) peptide molecules is 3.0×1011 per nanogram, given a typical M.W. of 2000. Thus the amount of peptide that saturates the 1 mL tumor is at most 2.4 ng (2.4×108×3000/3.01×1011) or 1.2 pmol. In reality, the number of effective receptors may be far less than this number, because some of receptors on cells packed within a tumor are not accessible for binding. Therefore, to be assured of remaining below the saturation level the amount of peptide in the injectable dose ultimately should be significantly under 1.2 pmol or around 0.5 pmol corresponding to only 1 ng of 2000 MW peptide to be sure to avoid overwhelming the tumor receptors and degrading the tumor signal to background. With the injected peptide over this limit, only a fraction of the injected dose can go to the receptors in tumor and a huge amount may actually target other nonspecific organs/tissues that are actually weak receptors. Assuming that small animal PET imaging studies require ˜0.1 mCi 62Cu injectable dose, the SPA for the labeled peptide must be higher than 83 Ci/μmol (0.1 mCi/1.2 pmol).
In the literature, the radiocopper (64Cu) SPA for bifunctional ReCCMSH peptide derivatives was only <0.6 Ci/μmol77 and the highest SPA reported was 19˜20 Ci/μmol labeled with the generator derived 68Ga.71 Both of these SPA for the labeled peptides were obtained over lengthy reaction time at elevated temperature or with microwave-assisted procedure, together with very cumbersome HPLC purification post labeling. The requirement for HPLC purification of the radiolabeled material makes such studies very demanding, cumbersome, and expensive. with development of point of use automated synthesis and purification equipment. Such complex methodologies in turn create a massive headache for the regulatory process since in essence GMP must be practiced in preparing each dose at the point of use.
Through the application of BTS kits developed for human studies with the current microgenerator, high purity 62Cu elution has demonstrated superior capability to achieve instant high labeling results in numerous human trials. If the same techniques can be transferred to the peptide application, the labeling of bifunctional peptides will be as simple as one minute mixing of the generator elution and a rehydrated lyophilized kit, the same as the application of the current microgenerator. This dramatically simplified methodology for the preparation of clinical injectable dose can also provide a rapid proven path for FDA approval for clinical use of the peptide. Clearly, an ultra pure copper isotope source would substantially benefit the investigation of the selection and efficacy evaluation of bifunctional peptides and in addition can provide the platform for rapid clinical translation of the diagnostic techniques. Our goal is to develop a new generation of 62Cu generator that can provide ultra high pure 62Cu2+ with unexcelled high specific activity, at least 10 times that of the current microgenerator that is limited to about 170 Ci/umol. The ultimate goal is to approach the theoretical SPA limit for the isotope (20,000 Ci/umol).
“Nanogenerator”: One superior feature for the generator derived 62Cu in general is its unexcelled level of purity stemming from the chemical nature of the anion exchange column, the heart of the generator system. Kraus and his colleges have conducted thorough studies investigating the behavior of more than 60 metallic elements under different chloride concentration and their separation performance on anion exchange resin. Those with nuclear number smaller than 41 are 4 orders of magnitude more abundant than the lower Z metals, and therefore by far the highest possibility for contamination in ultra high purity solution. Among them, the only two metals can form chloride complexes at 2M concentration are Zn and Fe (III). Fe(III) chloride binds so weakly with the AG1X8 resin that it is completely absent after three column separation/purification steps during the 62Zn purification process. These three steps use different sizes of anion exchange resin column to purify 62Zn from the target dissolution solution at different manufacturing stages. Therefore Zn, the chemical form of the mother isotope of the 62Cu generator, is the only metal that complexes strongly in 2M chloride eluant solution (1.8 M NaCl, 0.2 M HCl) utilized in the generator and is very strongly bound to the anion exchange resin column; and no other metal impurities (other than higher Z metals with natural abundance 4 orders of magnitude lower) can form anion complexes. Moreover, non-metallic materials are carefully selected for the construction of the entire generator fluid path, further limiting metal contamination. Inappropriate selection of generator materials can significant compromise the purity of the elution. One example is one type of commercial 68Ge/68Ga generator, based on a TiO2 solid column. Elutions of this system often suffers large metal contamination such as Fe(III), Zn(II) and Al(III), in the range of 2100±1300 μg/L, 5050±147 μg/L and 1080±125 μg/L, respectively.89 Fe and Al come from the metal column matrix whereas Zn is due to the accumulation of 68Ga decay which is an intrinsic problem for all types of 68Ge/68Ga generator. Complicated post treatment of the Ga generator elution has become a must for any high specific activity peptide labeling and will surely be a big hurdle for FDA approval for any clinical application. The 62Zn/62Cu generator is largely immune to the problems discussed above and its major contamination source arises from trace metal levels in the chemicals (NaCl, water and HCl) formulating the eluant solution and to a lesser extent from metals washed from the materials in the fluid path. This can easily be mediated by treatment of all the chemicals with Chelex resin, a highly selective chelator for transition metal cations, and vigorous wash of the generator fluid path before the usage.
The 62Cu SPA for the current microgenerator is typically 80-170 Ci per micromole of total copper. In comparison, the specific activity of 64Cu from a typical supplier is only 2.6-12.8 Ci/μmol at the end of the cyclotron production, and is reduced by a factor of 4 to 0.65-3.2 Ci/μmol at a clinical site due to the decay during overnight transportation period. Thus the SPA of the current microgenerator is already 25-120 times compared to commercially available 64Cu sources. However, typical small animal studies require ˜0.1 mCi of 62Cu injection to obtain acceptable quality of PET imaging and given 10 min time for the labeling and injection preparation, requiring 1.2-2.5 pmol (or 2.4-5 ng of peptide, given a typical M.W. of ˜2000) be injected. Though the SPA of the current microgenerator is superior to any other systems, it falls far short of the optimal value (0.5 pmol). Clearly it is fundamentally important to develop an even higher specific activity generator that can provide 62Cu with at least 10 times higher SPA for peptide investigation in the small animal and preclinical studies. We refer to this generator as a nano-generator because the ultimate goal is to produce doses with SPA high enough for the convenient, labeling of one nanogram (˜0.5 pmol) of peptide for preclinical investigation.
The proposed generator has an extremely miniaturized column, 10× smaller than the current generator that is eluted with nearly 10× smaller volume Producing a specific activity that is assured to be ˜10 times higher. Further improvement will be achieved by rigorous cleaning of all solutions with Chelex and by eliminating all materials from the fluid path that have the potential to bind metals. The current generator employs a glass eluant reservoir, a glass column and glass wool packing in the column, all sources of potential metal binding sites. The generator contains only a single eluant reservoir and a small column with a volume of 5 μL that is eluted by a modest pressurization in the sealed eluant reservoir, as shown in
The background to the present invention and related art is best understood by reference to Applicant's own prior work. Applicant's issued patents and pending applications that may be relevant, including; (1) U.S. Pat. No. 5,573,747 entitled, “Method for Preparing a Physiological Isotonic Pet Radiopharmaceutical of 62CU; (2) U.S. Pat. No. 6,264,597 entitled, “Intravascular Radiotherapy Employing a Safe Liquid Suspended Short-Lived Source”; and (3) U.S. patent application Ser. No. 10/571,202, entitled, “Miniaturized 62Zn/62CU Generator for High Concentration and Clinical Deliveries of 62Cu Kit Formulation for the Facile Preparation of Radiolabeled Cu-bis(thiosemicarbazone) Compound.” Each of these listed patents are hereby incorporated by reference in their entirety for all purposes, including, but not limited to, supplying background and enabling those skilled in the art to understand, make and use in Applicant's present invention.
The present invention includes an improved 62Zn/62Cu generator for producing radiopharmaceuticals. The improvements comprising utilization of a significantly reduced generator column size and materials of construction that prevent contamination.
To realize an important improvement in delivered specific activity and to lower cost of delivery in clinical applications, we present the “nanogenerator” with 10 fold reduced elution volume and substantially optimized materials of construction providing at least 10 times, and potentially 100 times, higher SPA. The proposed generator will be eluted in a small volume of 30 μL and combined with various bifunctionalized peptides or ligands via synthesis kits to produce an injectable dose instantly by simply mixing at room temperature. With polished design and better selected construction materials for the nanogenerator and an improved synthesis cell, the ultimate goal is to push the specific activity of the derived 62Cu to levels approaching 19,400 Ci/μmol (the theoretical maximum), and that of the final radio-synthesized dose to 1000 Ci/μmol. Such a system will afford preparation of preclinical doses for mouse studies containing less than 1 ng of a typical peptide of MW 2000 (0.5 pmol). Reaching such levels is of profound importance because many peptide targets in small animals are expressed at only a few thousand receptors per cell or about 7×1011 active sites per gram of tumor, dictating tumor saturation and compromised uptake at a level of less than 2.4 ng of injected peptide. The proposed miniaturization will also strongly aid the commercialization by lowing cost and complexity of distribution.
Construction of Nanogenerator: The construction of a pilot nanogenerator is shown in
Five prototype nanogenerators have been assembled using the above miniaturized column. To assess the potential radiation effects on the smaller resin bed, activities ranging from 9 mCi to 230 mCi were explored. For each generator, elution samples of 30- and 60-second duration were collected frequently over a period of two days. Sample activity levels were assayed in a dose calibrator (Capintec CRC-15) at the setting of 499 and decay corrected to obtain elution profiles and yield estimates. Following decay of 62Cu (>4 hrs post elution), samples were counted in a germanium detector to measure 62Zn breakthrough.
In summary, the dramatic change in the new design is to abandon glass or metal materials, to minimize the metal contamination. The vast reduction in column size (see
62Cu Yield: The five nanogenerators described above were tested over 2-3 days of simulated clinical/animal use in the pilot studies. These tests included two units loaded with <⅕ of the planned activity to explore the feasibility of design, and two full size units to validate the performance and safety of the system and one full size unit deployed to a remote collaborator's facility (University of Missouri) for actual animal PET scan and biodistribution study. All generators were loaded the afternoon before the first day of preclinical/animal use testing. Table 3 lists the averaged yield and breakthrough data on the day of the simulated preclinical use for the five microgenerators, as compared to a typical current clinical 50 μL bed volume microgenerator. Extensive in-house testing has been performed for the two full size generators, showing that the average yield (62Cu eluted/62Zn on column) produced in the first 30 seconds of elution were greater than current microgenerator yields and consistently approached the theoretical maximum yield of 90% for 30 minute inter-elution equilibration time. Furthermore, high yields were maintained under conditions of frequent elution throughout the two day testing period.
62Zn breakthrough
<5.8 × 10−8 b
74% c
On the simulated clinic day we eluted the R&D Nanogenerator (Z240) a total of 15 times, in an elapsed time of more than 8 hours.
62Zn Breakthrough: The generator works under the assumption that the parent isotope is trapped on the selected column media and only the daughter isotope is flushed out in the elution. However, there is always certain level of parent isotope even in trace amounts leaching into the elution. The presence of the relatively long-lived isotope in the elution may cause contamination issue if its level is high. The breakthrough of 62Zn is defined by the 62Zn level measured in each elution over the amount loaded on the column at the time of measurement.
Rapid Cartridge Chromatography for the Assay of Labeling Yield: A rapid radiochemical labeling assay using cartridge chromatography has been developed and validated against HPLC.21 This assay uses the Oasis HLB cartridge from Waters. A well-trained clinical technician can follow simple instructions to complete the test in approximately one minute. Briefly an Oasis® cartridge is conditioned with 1 mL ethanol, followed by 2 mL of normal saline. An 1-100 μL aliquot of the labeled ligand solution is mixed with 900 μL of water or saline, loaded onto the conditioned cartridge and the cartridge is rinsed with 2 mL of normal saline into a 3 mL syringe. The syringe and the cartridge are assayed in a Capintec CRC-15R (setting=499) dose calibrator. The reported radiochemical purity is calculated as the radioactivity on the cartridge divided by the sum of the total radioactivity used in the assay (saline wash+residual radioactivity on the cartridge).
Specific 62Cu Activity produced by the Nanogenerator: The 62Cu specific activity for the nanogenerator has been evaluated by titrating ⅓ (10-μL) of one entire elution with small additions of H2ATSM ligand and the labeling yield of the synthesized 62Cu-ATSM was determined with cartridge chromatography. According to the labeling yield of the previous titration, the addition amount of ligand was changed for the next titration whereas the ligand concentration maintained at 0.1 μg/mL via appropriate dilution. This ligand concentration for titration is the concentration identical for the regular 62Cu labeling using the current microgenerator and the 0.4 μg H2ATSM lyophilized kit; and has been proven adequate with extensive testing. A typical titration curve is displayed below in
The 62Cu specific activity of the current microgenerator has also been evaluated as 15 ng total copper per elution using the same titration approach. Given the typical clinic elution, dose as 20-40 mCi, the specific activity for the microgenerator is 1.3-2.7 Ci/μg. Thus the nanogenerator has increased the 62Cu specific activity by 8-11 fold. Due to nature of the generator column producing the 62Cu, the cold copper level generally doesn't correlate with the eluted activity dose, instead is related to the column size and the associated elution volume. With significant minimization of the generator column down to 5 μL from 50 μL, the generator elution volume decreased accordingly by a factor of 10 to 30 μL from 250 μL. Consistently, the cold copper level per each elution has been lowered approximately by a factor of 8 to 1.9 ng from 15 ng.
Pilot manufacture of NOTA functionalized peptide: To investigate the labeling performance of the nanogenerator, a pilot batch of NOTA-2-GGG-Arg11 peptide (provided by our collaborator, Dr. Thomas Quinn, University of Missouri) lyophilized kits (see
Labeling Yield and SPA for Peptide Labeling: In most of the pilot evaluation testing and animal study, the peptide labeling procedure was: A) one ligand vial was rehydrated with 100 μL water as a master solution; B) an aliquot (usually 10-μL) of this solution was mixed with the same volume of 2M NaOAc buffer in a low peptide binding centrifuge (CF) tube; C) 30-uL elution was taken from the nano-generator into the CF tube; D) the reaction solution was mixed vigorously for a few seconds and allowed to stand for one minute. The labeled dose required dilution with 450-μL water to reach isotonicity ready for animal/patient injection. Following labeling and dilution, the 62Cu concentration was in the range of 2.5-10 mCi/0.1 mL. Due to the limitation of the max injectable dose, the prepared isotonic solution usually needed a second dilution with normal saline to reach desired dose concentration.
#SPA = yield % × labeling activity (mCi)/peptide amount
Table 4 summarizes some typical results for the labeling using the three full size nanogenerators on the simulated clinic day. Instant high labeling yield was achieved within 2 min after the mixing of peptide and 62Cu elution, as long as a sufficient amount of peptide was added and vigorous thorough mixing was provided. It has been observed that the properly vigorous mixing of the initial reaction solution in a small volume (˜50 μL) is the key for good labeling results. Tests A-E are labeling using only partial eluted activity (10%). They were conducted to test reaction parameters while lowering the personal exposure to radiation. The lowest peptide amount with good labeling yield was 50 ng, equivalent to 500 ng for an entire elution. Tests F-M are labeling using all the eluted activity. During the pilot investigation, an entire nanogenerator elution could be consistently labeled with high yield using 500 ng peptide with >95% yield but become relatively less reliable with 250 ng peptide. In the table, the highest SPA for the 62Cu labeled peptide reached 237 Ci/umol (test J); however the labeling yield was relatively low (87%) with less peptide applied for the labeling. For those that achieved instant >95% high labeling yield, the highest SPA was 167 Ci/μmol (test E) using 10% of the total elution (4.3 mCi) and 131 Ci/mol (test G) using the entire elution (34 mCi). In comparison, the highest yield in literatures with 68Ga was only ˜20 Ci/μmol, accomplished by cumbersome HPLC post purification after the labeling.71 It should be emphasized that the peptide dose size in the full elutions ranged from 27 mCi to 36 mCi compatible with very high statistics patient studies.
Pilot Animal Studies using 62Cu labeled Peptide: Biodistribution and PET studies of radiolabeled 62Cu-NOTA-2-GGG-Arg11 were performed in B16/F1 flank tumor bearing C57 mice utilizing the newly designed nanogenerator with the 5 μg lyophilized peptide kits. Fourteen male and female, 6-8 week old mice were injected with B16/F1 cells (1,000,000/mouse) in the flanks or shoulders while anesthetized to induce solid tumors. B16/F1 solid tumors appeared in 14 days post inoculation. Radiolabeled peptides were injected through the tail vein at an average of 0.27 mCi per dose administration for biodistribution studies and an average of 0.7 mCi for imaging, achieving specific activities of 111.2-140.4 Ci/μmol (mean=122.1 Ci/μmol), and an average of 94.0% radiolabeling yield without a purification step after synthesis. Animals in the biodistribution studies were sacrificed by cervical dislocation followed by pneumothorax at 10, 20, and 40 min post administration, major organs removed, weighed, and radioactivity quantitated in a windowed gamma counter. A competitive blocking experiment was performed at 20 min post administration, in which 20 μg of non-labeled NDP-MSH peptide was co-injected to demonstrate receptor specificity.
Uptake was measured for the tumor, blood, heart, lung, liver, small intestine, intestine, kidneys, muscle, carcass and paper, and skin. The biodistribution data is presented in
A preliminary PET imaging study was also performed, in which two anesthetized animals were used for PET imaging studies starting at 10 min post administration of the radiolabeled peptide and statically imaged with a microPET-Focus scanner (Siemens Preclinical Solutions, Knoxville, Tenn.) for 20 min (see
The biodistribution of 62Cu-NOTA-2-GGG-Arg11 compared well to previously published studies using the melanoma targeting peptide conjugated with metal chelators DOTA or CBTE2A and radiolabeled with 64Cu.77,80 Tumor uptake of DOTA and CBTE2A conjugated 64Cu labeled peptides at 30 min post injection was 9.68±1.51% ID/g and 8.45±1.42% ID/g, respectively, compared to 9.43±2.69% ID/g for 62Cu-NOTA-2-GGG-Arg11 at 40 min post injection. The result is even more impressive since the 64Cu labeled peptides were HPLC purified prior to injection where as the 62Cu labeled peptide was injected directly from the kit preparation without additional purification. The biodistribution pattern of 62Cu-NOTA-2-GGG-Arg11 was similar to the 64Cu labeled peptides, however the overall disappearance of radioactivity from the normal organs and tissues was slightly slower. This slight difference in clearance kinetic was attributed to the presence of the GGG spacer. The use of a more hydrophilic spacer or removal of the spacer would likely enhance the clearance of the 62Cu NOTA conjugated peptide from the body, without affecting tumor uptake and retention.
Competitive binding studies were performed with NOTA-2-GGG-Arg11, NOTA-2-GSG-Arg11, and NOTA-2-Arg11 peptides to determine their IC50 values. Concentrations of the NOTA conjugated peptides ranging form 10-13 to 10-5 M were examined for their abilities to competitively displace the superpotent MSH analog 125I-NDP from binding the melanocortin-1 receptor present on cultured B16/F1 cells. The IC50 values for NOTA-2-GGG-Arg11, NOTA-2-GSG-Arg11, and NOTA-2-Arg11 peptides were 1.97×10−9, 5.99×10−9 and 1.25×10−9, respectively (
In vitro cell binding studies were performed with 62Cu-NOTA-2-GGG-Arg11 to demonstrate tumor cell targeting. Tissue culture wells containing 1 million cells each were incubated with 320,000 cpm of 62Cu-NOTA-2-GGG-Arg11 for various times between 5 min and 50 min then washed and counted. The melanoma cells demonstrated rapid uptake of the radiolabeled peptide that reached a maximum of 10% at 10 min post application and remained constant for 50 min (see
#Less injection volume leads to less injectable dose.
+Common max injectable volume for small animals like mice.
Summary of Nanogenerator performance: In summary, the results of pilot studies showed that a dramatically miniaturized column can actually perform significantly better than the current microgenerator with respect to important parameters, including yield and breakthrough, and that such performance can be maintained over the course of expected clinical use. Pilot study results also showed that pressure-driven elution was highly reproducible and that anticipated variability in delivered volume was within acceptable limits. The final pilot generator demonstrated that the system could be shipped overnight to a remote facility and produced clinically acceptable doses (>20 mCi) of 62Cu throughout the day of use flawlessly producing elutions for animal studies. These findings strongly support the feasibility of the proposed nanogenerator design.
The schematic of the 62Zn/62Cu nanogenerator manufactured for pilot studies is also shown in
The column was significantly reduced, from 50 uL to 5 uL (with respect to previous generation), which will result in less contamination since the same dose is eluted in 10× less volume. The surface area is reduced by a factor of 4, resulting in less exposure to possible impurities, if any, that may leech from the generator components. These features are incorporated into the design of a third generation 62Zn/62Cu generator which provides a source of ultra pure 62Cu2+ with high specific activity >2,000 Ci/umol approaching the theoretical SPA limit of 62Cu, 19,400 Ci/umol. We refer to this generator as a nano-generator because the ultimate goal is to produce doses with SPA high enough for the convenient, labeling of one nanogram (˜0.5 pmol) of compound for preclinical investigation.
62Zn loading line for generator column.
The overall production and utilization of the Nanogenerator is a simple and straightforward process. The anion exchange column is loaded with parent isotope 62Zn to a desired level relative to the time of its clinic use. After passing the standard quality control tests, the generator is packaged, labeled and released to the clinic site. At the clinical setting, the nanogenerator is received and ready to be used for multiple elutions. The 62Zn/62Cu nanogenerator has a exceptional labeling flexibility; we have previously shown that the it can work with three interchangeable lyophilized kits for instant synthesis of 62Cu bis(thiosemicarbazone), or 62Cu-BTS, radiopharmaceuticals1-20 as well as other peptides21. A rapid radiochemical labeling assay using cartridge chromatography has been developed and successfully validated against HPLC.17
In summary, results from our pilot studies have shown that a dramatically miniaturized column can actually perform significantly better than the previous generation of the 62Zn/62Cu generator with respect to important parameters, including yield and breakthrough, and that such performance can be maintained over the course of expected clinical use. Pilot study results also showed that pressure-driven elution was highly reproducible and that anticipated variability in delivered volume was within acceptable limits. The final pilot generator demonstrated that the system could be shipped overnight to a remote facility and produced clinically acceptable doses (>20 mCi) of 62Cu throughout the day of use flawlessly producing elutions for animal studies and potentially for human use.
This device seeks to undertake the final innovative steps in bringing 62Cu to clinical reality as a practical low cost PET tracer, coupling with methods of synthesis that can be accomplished in the clinicians' hands using instant kit technology afforded by metal chelation chemistry, which eliminates the cost of setting up and operating hundreds of GMP facilities that took over a decade to complete for 18F-FDG. This high cost, complex, and time intensive process is the reason of the stagnant PET field. Our approach is to utilize an almost trivial, simple inorganic system to deliver purified 62Zn and load this parent agent on generators, an incredibly straightforward process compared to organic synthesis processes required for 18F agents, and approximately 25 times fewer processing facilities are required. The miniaturization proposed here is very important to this distribution process since weight and size of the delivered generator will be substantially reduced and loading systems can be far more compact. The final innovation and the focal one is the development of a means of delivery of a radiopharmaceutical at an absolute world record level of specific activity allowing targeting cell signaling at levels not dreamed of with any other method. This technology will provide this capability not as a narrow research dream but rather can truly make a broad spectrum of PET agents available that will move rapidly through the regulatory process and finally facilitate getting the powerful PET technique to the patient! We have shown in previous studies and ongoing FDA process that very effective perfusion agents can be produced that could eventually carry the brunt of the diagnostic load in myocardial perfusion imaging. It is very important to realize that the innovation of this proposal is not in the details of making a column smaller, or using the right materials to avoid metal contamination, but it is about putting all of these things together and producing a technology that can serve as the base for shoring up a very valuable field of diagnostic imaging that is currently foundering.
8. Haynes, N. G., et al. Performance of a 62Zn/62Cu generator in clinical trials of PET perfusion agent 62Cu-PTSM. J Nucl Med 41, 309-314 (2000).
17. Thrash, T., Yue, Z., Stephens, A., Bui, H. & Lacy, J. L. QC evaluation of 62Cu bis(thiosemicarbazone) instant synthesis kits for PET imaging. Journal of Nuclear Medicine 49, 309P (2008).
Claims priority to provisional application 61/334,015.
| Number | Date | Country | |
|---|---|---|---|
| 61334015 | May 2010 | US |
