The importance of high specific radioactivity in the performance of 68Ga-labeled peptide.

The use of (68)Ga-labeled peptides in diagnosis, dosimetry, therapy planning and follow-up of response to chemo- and radiotherapy requires accurate quantification of tracer binding characteristics in vivo, which may be influenced by the specific radioactivity (SRA) of the tracer. Systematic study of the complexation reaction of DOTA-D-Phe(1)-Tyr(3)-Octreotide (DOTATOC, where DOTA is the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) with (67)Ga, (68)Ga, (69,71)Ga and in the presence of competing metal cations [Al(III), Fe(III), In(III)] was performed using conventional and microwave heating techniques and assessed by mass spectrometry. Saturation binding of (68)Ga-DOTATOC to Rhesus monkey brain slices was performed using frozen section autoradiography. High SRA was necessary in order to characterize the saturation binding of (68)Ga-DOTATOC to somatostatin receptors in Rhesus monkey brain sections. The complexation of Ga(III) with DOTATOC suggested more favorable formation compared to Fe(III) and In(III). The microwave heating mode might influence the selectivity of the complexation reaction, especially when comparing the behavior of Ga(III) and In(III). Al(III) was less critical with contamination and could be tolerated up to a concentration equal to that of the peptide bioconjugate. The SRA of (67)Ga-DOTATOC and (67)Ga-NODAGA-TATE (NODAGA-Tyr(3)-Octreotate, where NODAGA is 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid) exceeded literature data by a factor of 7 and 5-15, respectively. High SRA was critical for providing sufficient contrast and accurate quantification of PET images. Microwave heating mode apart from the acceleration of the labeling reaction also improved the selectivity of the complexation reaction towards gallium. Fe(III) was shown to be the most critical competitor deteriorating the (68)Ga-labeling efficiency.

Autoradiography with positron emitting isotopes in positron emission tomography tracer discovery.

Rapid development of labeling chemistry and generation of new chemical entities for biologic interactions via combinatorial chemistry and high throughput screening gives great potential for the development of new positron emission tomography (PET) tracer candidates. It must, however, be realized that a large fraction of these candidates will fail to characterize the desired biochemistry in vivo due to undesirable properties that are not relevant to providing a specific signal in vitro. A full characterization of a PET tracer is a lengthy and expensive procedure, and it is necessary to establish confidence via a number of assays that a tracer will provide useful data in the target species (generally human). These assays should also serve as a background to choose or to reject a tracer at an early time point, conserving valuable resources and time. Autoradiography performed as an ex vivo binding technique or as ex vivo recording of in vivo tracer distribution are, in this respect, very important tools. Autoradiography binding methods allow a range of frozen tissues to be sectioned and incubated with the PET tracer, and with due caution and controls with selective blockade of binding, quantitative values can be obtained with respect to tracer binding and its regional distribution. The method is easy to learn and set up, and should be included in all PET research and development labs. Ex vivo autoradiography of selected organs or whole animals gives qualitative images of a tracer's distribution with high resolution and is especially valid for 18F-labeled tracers. When tracer administration is not limited by mass due to specific radioactivity, 11C-tracers can also be readily used. This method is attractive to use as a complement to small animal imaging due to its high resolution and anatomical correlate. Living slice autoradiography is a more cumbersome method, but has an advantage of utilizing live tissue that retains certain metabolic functions. The use of these different methods in the validation of tracers and for supplying complementary information is illustrated.

Effects of androgens on aromatase activity and 11C-vorozole binding in granulosa cells in vitro.

BACKGROUND: Locally produced androgens and estrogens are important in the hormonal regulation of follicular development. The present study aimed to further elucidate the mechanism through which androgens exert their ambivalent effects on aromatization. METHODS: Non-cultured human granulosa-luteal cells (GC) were treated with different concentrations of androstenedione (A4), testosterone (T), 5alpha-androstane-3,17-dione (5alpha-A) and dihydrotestosterone (DHT). The effects on aromatase activity were evaluated in a tritiated water assay (incubation time 2 h) and the availability of aromatase active sites was measured in a radiotracer-binding assay using the non-steroidal competitive aromatase inhibitor [11C]-vorozole (incubation time 15 min). RESULTS: A4, T and 5alpha-A caused dose-dependent inhibition of both aromatase activity and [11C]-vorozole binding; IC50-values for both inhibition processes were calculated for these three steroids, revealing A4 as the most potent inhibitor and T and 5alpha-A as moderate inhibitors. At low concentrations (0.01 and 0.1 micro M), DHT stimulated aromatase activity but did not affect [11C]-vorozole binding. At the higher concentrations tested (1 and 10 micro M) DHT suppressed both processes thus weakly binding the aromatase active site. CONCLUSION: Because the incubation time in the tritiated water assay was short, the stimulation by DHT at low concentrations might therefore most likely include mechanisms other than new synthesis of aromatase protein such as allosteric action of DHT upon aromatase or liganded androgen receptor-aromatase interaction.