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The ultimate radiochemical nightmare: upon radio-iodination of Botulinum neurotoxin A, the introduced iodine atom itself seems to be fatal for the bioactivity of this macromolecule.

van Uhm JI, Visser GW, van der Schans MJ, Geldof AA, Meuleman EJ, Nieuwenhuijzen JA - EJNMMI Res (2015)

Bottom Line: The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A.The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom.BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.

ABSTRACT

Background: Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 ((125)I) via IODOGEN-coated BoNT-A method.

Methods: Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 μg) was labelled with (125)I by coating with 2.5 μg IODOGEN, in a total reaction volume of 250 μL and a reaction time of 90 s. (125)I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of (125)I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay.

Results: Cetuximab (50 μg) labelling with (125)I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq (125)I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 μg) labelling with (125)I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of (125)I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom.

Conclusions: BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A. The bioactivity of a BoNT-A molecule is, however, lost upon the introduction of an iodine atom into the tyrosine moiety of this sensitive molecule.

No MeSH data available.


HPLC chromatograms of125I-cetuximab after PD10 column purification. Channel A shows the UV absorption of cetuximab at 280 nm at a retention time of 26 min (large peak at 42 min is from ascorbic acid). Channels B and C represent the radioactive signal of 125I-cetuximab from the 73.1 MBq and 150.0 MBq reaction demonstrating retained and impaired integrity, respectively.
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Fig3: HPLC chromatograms of125I-cetuximab after PD10 column purification. Channel A shows the UV absorption of cetuximab at 280 nm at a retention time of 26 min (large peak at 42 min is from ascorbic acid). Channels B and C represent the radioactive signal of 125I-cetuximab from the 73.1 MBq and 150.0 MBq reaction demonstrating retained and impaired integrity, respectively.

Mentions: Labelling of 50 μg cetuximab with 15 MBq 125I using IODOGEN-coated mAb method in 250 μL reaction volume and reaction time of 90 s resulted in a labelling yield of 70% to 80%. There was no difference in labelling yield by using 35 μg till 2.5 μg IODO-GEN. Also, no substantial increase in labelling yield was observed for a reaction time above 90 s. The same conditions and an increase of amount of radioactivity to 150 MBq resulted in a labelling yield of 71% to 81% (Table 2). ITLC showed that the radiochemical purity of the products was ≥99% after size exclusion chromatography (PD10). The phosphor imager analysis of the SDS-PAGE gel of the 73.1 MBq, 112.2 MBq and 150.0 MBq reactions is depicted in Figure 2. It revealed unaffected integrity with respect to molecular weight: only the presence of the major 150 kD BoNT-A band and a low amount (0.1% to 0.4%) of free iodine was observed. The HPLC analysis resulted in an unaffected integrity up to the 73.1 MBq reaction and a partly impaired integrity increasing for the 112.2 MBq and 150.0 MBq reactions (shown for 73.1 and 150.0 MBq reaction in Figure 3). Since the molecular weight was unaffected (Figure 2) and any formed S-Cl bonds are reduced by ascorbic acid, this remarkable finding indicates that a too heavy load of iodine atoms induces a conformational change of the mAb molecule. It was, therefore, decided that BoNT-A should not be labelled with an I/BoNT-A molar ratio higher than two, even though the immunoreactivity of 125I-cetuximab did not change by increasing the amount of radioactivity (Table 2). To determine the influence of HEPES and lactose on the labelling reaction, both were added to the reaction mixture. Lactose had no effect on the labelling yield, but addition of HEPES induced a decrease in labelling yield from 80.7% to 43.5% (Table 3). The addition of IODOGEN/acetonitrile reagent in two portions increased the labelling efficiency from 43.5% to 51.8%. The radiochemical purity was >98.5%. As before, the product with 125I/mAb molar ratio of 2.3 showed immunoreactivity of ≥95% and unaffected integrity of the product.Table 2


The ultimate radiochemical nightmare: upon radio-iodination of Botulinum neurotoxin A, the introduced iodine atom itself seems to be fatal for the bioactivity of this macromolecule.

van Uhm JI, Visser GW, van der Schans MJ, Geldof AA, Meuleman EJ, Nieuwenhuijzen JA - EJNMMI Res (2015)

HPLC chromatograms of125I-cetuximab after PD10 column purification. Channel A shows the UV absorption of cetuximab at 280 nm at a retention time of 26 min (large peak at 42 min is from ascorbic acid). Channels B and C represent the radioactive signal of 125I-cetuximab from the 73.1 MBq and 150.0 MBq reaction demonstrating retained and impaired integrity, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385017&req=5

Fig3: HPLC chromatograms of125I-cetuximab after PD10 column purification. Channel A shows the UV absorption of cetuximab at 280 nm at a retention time of 26 min (large peak at 42 min is from ascorbic acid). Channels B and C represent the radioactive signal of 125I-cetuximab from the 73.1 MBq and 150.0 MBq reaction demonstrating retained and impaired integrity, respectively.
Mentions: Labelling of 50 μg cetuximab with 15 MBq 125I using IODOGEN-coated mAb method in 250 μL reaction volume and reaction time of 90 s resulted in a labelling yield of 70% to 80%. There was no difference in labelling yield by using 35 μg till 2.5 μg IODO-GEN. Also, no substantial increase in labelling yield was observed for a reaction time above 90 s. The same conditions and an increase of amount of radioactivity to 150 MBq resulted in a labelling yield of 71% to 81% (Table 2). ITLC showed that the radiochemical purity of the products was ≥99% after size exclusion chromatography (PD10). The phosphor imager analysis of the SDS-PAGE gel of the 73.1 MBq, 112.2 MBq and 150.0 MBq reactions is depicted in Figure 2. It revealed unaffected integrity with respect to molecular weight: only the presence of the major 150 kD BoNT-A band and a low amount (0.1% to 0.4%) of free iodine was observed. The HPLC analysis resulted in an unaffected integrity up to the 73.1 MBq reaction and a partly impaired integrity increasing for the 112.2 MBq and 150.0 MBq reactions (shown for 73.1 and 150.0 MBq reaction in Figure 3). Since the molecular weight was unaffected (Figure 2) and any formed S-Cl bonds are reduced by ascorbic acid, this remarkable finding indicates that a too heavy load of iodine atoms induces a conformational change of the mAb molecule. It was, therefore, decided that BoNT-A should not be labelled with an I/BoNT-A molar ratio higher than two, even though the immunoreactivity of 125I-cetuximab did not change by increasing the amount of radioactivity (Table 2). To determine the influence of HEPES and lactose on the labelling reaction, both were added to the reaction mixture. Lactose had no effect on the labelling yield, but addition of HEPES induced a decrease in labelling yield from 80.7% to 43.5% (Table 3). The addition of IODOGEN/acetonitrile reagent in two portions increased the labelling efficiency from 43.5% to 51.8%. The radiochemical purity was >98.5%. As before, the product with 125I/mAb molar ratio of 2.3 showed immunoreactivity of ≥95% and unaffected integrity of the product.Table 2

Bottom Line: The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A.The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom.BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.

ABSTRACT

Background: Botulinum neurotoxin A (BoNT-A) is a highly neurotoxic drug and frequently used in patients. Knowledge on the optimal way of administration of BoNT-A and its subsequent distribution is still rather limited. An accurate method for monitoring these processes might be the use of radiolabelled BoNT-A. In this paper, we report our feasibility study on labelling BoNT-A with high-dose iodine-125 ((125)I) via IODOGEN-coated BoNT-A method.

Methods: Using cetuximab as model substrate for BoNT-A, a miniaturization of the IODOGEN-coated mAb method was developed with special attention to the minimum required amount of the oxidant IODOGEN, while the amount of substrate, reaction volume and reaction time were downsized. Labelling efficiency and radiochemical purity were determined by TLC, integrity by SDS-PAGE and HPLC and immunoreactivity by cell-binding assay. BoNT-A (50 μg) was labelled with (125)I by coating with 2.5 μg IODOGEN, in a total reaction volume of 250 μL and a reaction time of 90 s. (125)I-BoNT-A was purified by size exclusion chromatography (PD10 column) using ascorbic acid solution (5 mg/ml, pH = 5) as eluent. Quality analysis of (125)I-BoNT-A was performed by an in vitro bladder strip model, an electrochemiluminescence assay and an Endopep assay.

Results: Cetuximab (50 μg) labelling with (125)I (15 to 150 MBq) resulted in a labelling efficiency of 70% to 80%, a radiochemical purity of >99%, an immunoreactivity of >95% and a retained integrity on SDS; HPLC analysis revealed partly affected integrity when 110 to 150 MBq (125)I was used, i.e. when the averaged I/mAb molar ratio exceeded 3. Addition of HEPES (20 mM) and lactose (1.25%) (lyophilized BoNT-A contains HEPES and lactose) decreased the labelling efficiency to 44% to 54%. BoNT-A (50 μg) labelling with (125)I (97.2 to 98.3 MBq) resulted in labelling efficiency of 51% to 52% with a radiochemical purity >98.5%, a specific activity of 150.5 to 152.9 MBq/nmol and an I/BoNT-A molar ratio of 1.86 to 1.90. The in vitro bladder strip model showed no bioactivity of (125)I-BoNT-A when compared to unlabelled BoNT-A. The electrochemiluminescence and Endopep assay demonstrated around 10% and 15% bioactivity of (125)I-BoNT-A compared to unlabelled BoNT-A, respectively. The remaining bioactivity correlates within the Poisson distribution with the amount of BoNT-A molecules that does not bear an iodine atom.

Conclusions: BoNT-A was successfully radio-iodinated with an activity high enough to enable in vivo measurement of nanograms of BoNT-A, which could be used in studying optimization of administration techniques of BoNT-A. The bioactivity of a BoNT-A molecule is, however, lost upon the introduction of an iodine atom into the tyrosine moiety of this sensitive molecule.

No MeSH data available.