Hayes Dougan

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents

Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.

15-(Para-[123I] iodophenyl) pentadecanoic acid obtained using mercuration and subsequent [123I] radioiodination

In an ongoing effort to obtain quantitative, rapid kit type labelling of [123I] radiopharmaceuticals, we have examined organomercury precursors of [123I] 15-(para-iodophenyl)-pentadecanoic acid (IPPA). Chloromercuri derivatives of phenyl pentadecanoic acid (PPA) and the PPA ethyl ester were obtained by mercuration utilizing mercuric trifluoroacetate in trifluoroacetic acid followed by treatment with acetic acid and hydrochloric acid. The most simple compound, chloromercuri PPA, proved insoluble at room temperature in the common solvents useful for radioiodination and purification. The study was extended in a systematic way to chloromercuri PPA ethyl ester and the acetoxy mercuri PPA ethyl ester. As expected, these two compounds posessed successively more useful ranges of solvent compatibility. Iodination and [123I] radioiodination were carried out with the three compounds of PPA. Chloromercuri PPA was dissolved with difficulty in acetic acid at 70°C and 71% radiochemical yield of [123I] IPPA was obtained during the course of a 5 minute reaction utilizing chloramine T. The chloromercuri PPA ethyl ester was dissolved in ethyl acetate/acetic acid (2/1 v/v) at room temperature and 87% radiochemical yield of [123I] IPPA was obtained following 10 minutes reaction. With the acetoxy mercuri PPA ethyl ester it was possible to conduct the radioiodination in ethanol again using chloramine T. A modest radiochemical yield (r. y.) (51%) of [123I] IPPA ethyl ester was obtained after 60 min. It was possible to enhance the radiochemical yield in the presence of lithium acetate (84% r. y.). The isomeric purity of the [123I] IPPA ethyl ester was unexpectedly high (99.9% para) when the radioiodination was conducted at room temperature.

Efficient production of 15-(para-[123I]iodophenyl) pentadecanoic acid

A one-step labelling procedure for 15-(para-[123I]iodophenyl) pentadecanoic acid (IPPA) has been developed. While previous work involved two or three stages each with its own solvent, the present work employs a mild emulsifier solution as a single solvent for both 123I labelling and injection. Incorporation of 123I was brought to a near quantitative level (98.5%) by exchange labelling in the presence of Cu(I). The reaction was shown to be para specific and there is no potential for toxic metal contamination (esp. thallium). Combined with simple conditions and reasonable time (100°C and 90 min), the features are advantageous for kit development. Organizational advantages are examined.

Meta iodophenylpentadecanoic acid (meta IPPA): Occurrence as a contaminant of para IPPA

Abstract While developing organomercury precursor routes for para [123I]IPPA, we discovered abundant meta IPPA ethyl ester following mercuration in trifluoroacetic acid (TFA) (20°C) and subsequent iodination. 1H-NMR and HPLC were employed for analysis and identification. The isomeric levels found were para(76.1%) >ortho(15.2%) >meta(8.7%) following 3.5 min mercuration, and meta(43.3%) >ortho(32.5%) >para(24.2%) following 3.5 days mercuration. The meta isomer is significant, ranging from 8.7 to 43% in our measurements, or 0.11 to 1.79 when calculated as the meta/para ratio. The meta isomer was resolved from the para and ortho isomers by means of HPLC using convenient new reverse phase and normal phase systems. Assays of meta [123I]IPPA agreed with measurements of the unlabeled isomer.

Differential solubility labelling and purification of ortho-iodo-hippuric acid with 123I

Abstract A simple method is described for the production of high quality clinical 123 I hippuran at TRIUMF. The first feature is that solid ortho -iodo-hippuric acid (IHA) is heated with aqueous [ 123 I]iodide creating a single phase where labelling occurs. When subsequently cooled to room temperature, IHA crystallizes. The second feature is employed at this stage. A density gradient is created with NaCl to remove the supernatant fluid from the IHA crystals. The IHA is then neutralized for pharmaceutical use. The product purity is consistantly >99% by TLC and by HPLC analysis that is described. The initial tag is typically 96% and the useful recovery is 85% by this process. Iodine-123 has been used up to 100 mCi per batch from the 500 MeV 133 Cs (p, 2p, 9n) 123 Xe reaction and from the 124 Xe (p, 2n) 123 Xe reaction.