Formulation development and manufacturing of a gastrin/CCK-2 receptor targeting peptide as an intermediate drug product for a clinical imaging study.

A DOTA-gastrin analogue (APH070) which, when labelled with (111)In, has high affinity for the gastrin/CCK-2 receptor (3nM) and low tumour to kidney ratio in animal models, has been formulated and manufactured for a clinical study. Oxidation of the peptide methionine residue greatly reduces receptor affinity, therefore development work focused on producing a stable intermediate drug product (iDP) whilst ensuring that the formulation, container, closure and manufacturing process did not inhibit the extremely sensitive radiolabelling reaction (itself a source of oxidation). Stress testing revealed that APH070 was stable at 2-8 degrees C at pH 6-9. Addition of an antioxidant (monothioglycerol) to the peptide formulation reduced stability when compared to buffer alone. Use of FluroTec (4023/50) stoppers (rather than FluroTec Plus (4110/40)) increased both the stability and radiolabelling efficiency of APH070. Long term stability (6 months) of the final formulation (1mg/ml APH070 in 0.01 M pH 7.2 phosphate buffer) stored at 5 degrees C in type I glass vials with FluroTec (4023/50) stoppers was 98.6+/-0.2% and 98.4+/-0.1% for upright and inverted samples, respectively. Clinical scale radiolabelling of the final formulation routinely achieves the specification of >85% (111)In-APH070 (unoxidised) stable for up to 2h after dilution with 0.9% w/v saline solution. Specific uptake of the radiopharmaceutical in CCK-2R-expressing AR42J tumours in nude mice has been demonstrated.

Imaging apoptosis in vivo using 124I-annexin V and PET.

Abnormal regulation of apoptosis is an important pathogenic mechanism in many diseases including cancer. Techniques to assess apoptosis in living organisms are limited and, in the case of solid organs, restricted to histological examination of biopsy samples. We investigated the use of (124)I-annexin V, which binds to phosphatidylserine (PS) on the surface of apoptotic cells, as a potential positron emission tomography (PET) radioligand for the noninvasive measurement of apoptosis in vivo. Annexin V and a similar-sized protein, ovalbumin, were directly labelled with (124)I. We report the validation of (124)I-annexin V in vitro and in an animal model of liver apoptosis that has not previously been used to test iodinated annexin V. Also, for the first time, we report metabolite analysis of (124)I-annexin V and the correlation of (124)I-annexin V uptake with apoptotic density (AD). Sixfold more (124)I-annexin V was associated with Jurkat cells after apoptosis induction, indicating that PS binding by annexin V was preserved after iodination. (124)I-ovalbumin did not demonstrate increased uptake in apoptotic cells. In normal BDF-1 mice, the radioligand was rapidly cleared, but some in vivo dehalogenation resulted in the accumulation of activity in the thyroid and stomach content. PET images demonstrated uptake of (124)I-annexin V but not (124)I-ovalbumin in apoptotic liver lesions. In vivo (124)I-annexin V uptake, derived from PET images, correlated with histologically derived AD (r=.86, P<.01). These results demonstrate that (124)I-annexin V is localised to anti-Fas-induced apoptosis, in contrast to (124)I-ovalbumin, which did not show preferential uptake in the apoptotic liver.