Efficient Preparation of ^(18)F Labeled Protein Using a Novel Hydrazone-Formation Method

Abstract

^(18)F labeling of proteins or peptides is getting more and more important as the distribution of positron emission tomography (PET) increases. Although several methods for labeling proteins with ^(18)F have been reported, most of them showed low yields and complicated procedures. The author report a novel and efficient method which includes preparation of [^(18)F]fluorobenzaldehyde ([^(18)F]FBA) and successive conjugation with hydrazinonicotinic acid-human serum albumin conjugate (HYNIC-HSA) by formation of hydrazone. HYNIC-HSA, which also can be used for labeling ^(99m)Tc, was prepared by reacting various number of N-hydroxysuccinimide or tertafluorophenyl ester of HYNIC with HSA. The molar ratios of HYNIC to HSA were from 8:1 to 32:1. Unreacted HYNIC was removed using a PD10 size-exclusion column. The number of conjugated HYNIC was determined by measuring OD340 nm after conjugation with p-nitrobenzaldehyde. [^(18)F]FBA was prepared by nucleophilic substitution of [^(18)F]fluoride to 4-trimethylaminobenzaldehyde triflate in the presence of tetrabutylammonium bicarbonate. [^(18)F]FBA was purified by passing ion exchange cartridges (IC-H and QMA) and adsorbed to a C_(18) Sep-Pak cartridge. The adsorbed [^(18)F]FBA was eluted with 50% ethanol. HYNIC-HSA was added to the solution and conjugated with [^(18)F]FBA by the formation of hydrazone. The labeling efficiency was checked by ITLC-SG and saline. ^(18)F-HSA was purified using PD10 column. Biodistribution of ^(18)F-HSA, ^(99m)Tc-HSA and [^(18)F]FBA in mice were investigated at 10, 20 and 60 min after intravenous injection, respectively. The number of conjugated HYNIC molecules per HSA ranged from 5.2 to 23.2 according to the reaction conditions. The labeling efficiency of [^(18)F]FBA was 67±15.7%. The radiochemical purity was over 99% after purification. The labeling efficiency of HYNIC-HSA with [^(18)F]FBA was between 25% and 85%. The labeling efficiency increased as the number of conjugated HYNIC, concentration of HYNIC-HSA, or temperature increased. The labeling efficiency of control HSA was always less than 10%. These results showed that the conjugation of HYNIC to HSA significantly increased conjugation of [^(18)F]FBA. According to the biodistribution study, the results of ^(18)F-HSA were similar with ^(99m)Tc-HSA, while the blood uptake of [^(18)F]FBA was very low than that of ^(18)F-HSA and ^(99m)Tc-HSA. We concluded that ^(18)F-HSA was successfully labeled using a novel method which includes the formation of hydrazone between [^(18)F]FBA and HYNIC-HSA. This method can be applied for ^(18)F labeling of other proteins or peptides.;서론 : 양전자단층촬영기(PET)의 보급이 증가함에 따라 ^(18)F 표지 단백질 혹은 펩타이드의 중요성이 증가하고 있다. 단백질 혹은 펩타이드에 ^(18)F을 표지하는 방법은 여러 가지가 보고되고 있지만 대부분 표지효율이 낮고 표지방법이 복잡하다. 저자는 [^(18)F]fluorobenzaldehyde ([^(18)F]FBA)와 hydrazinonicotinic acid-human serum albumin conjugate (HYNIC-HSA) 사이에 hydrazone을 형성시켜 ^(18)F-HSA을 만드는 새로운 방법을 보고하고자 한다. 방법 : HSA에 다양한 몰수의 HYNIC N-hydroxysuccinimide ester 혹은 HYNIC tetrafluorophenyl ester를 반응시켜 HYNIC-HSA를 만들었다. HYNIC의 HSA에 대한 반응 몰비는 8:1에서 32:1까지 증가시켰다. 반응하지 않은 HYNIC은 PD10 column을 사용하여 제거하였다. HSA에 결합한 HYNIC의 수는 p-nitrobenzaldehyde와 반응시킨 후 340 nm에서 흡광도를 측정하여 계산하였다. Tetrabutylammonium bicarbonate 존재하에서 [^(18)F]fluoride를 4-trimethylammonium benzaldehyde triflate에 친핵치환반응을 이용하여 [^(18)F]FBA를 만들었다. 제조한 [^(18)F]FBA를 이온교환카트리지(IC-H, QMA)를 사용하여 정제하고 C_(18) Sep-Pak에 흡착시킨 후 50% ethanol로 유출하였다. 여기에 HYNIC-HSA를 부가하여 hydrazone을 형성시켜 ^(18)F-HSA를 제조하였다. 표지효율은 ITLC-SG/saline을 사용하여 측정하였고, 표지된 ^(18)F-HSA는 PD10 column을 사용하여 정제하였다. ^(18)F-HSA와 [^(18)F]FBA 의 마우스에서의 체내분포를 시간별(10, 20, 60분)로 확인하고 ^(99m)Tc-HSA의 결과와 비교하였다. 결과 : HSA에 결합한 HYNIC의 수는 반응조건에 따라 5.2에서 23.2 범위에 있었다. [^(18)F]FBA 의 표지효율은 67±15.7%였으며, 정제후 방사화학적순도는 99% 이상이었다. ^(18)F-HSA의 방사능 표지효율은 25% 에서 85%였다. 표지효율은 HSA에 결합한 HYNIC의 갯수가 많을수록, 반응한 HYNIC-HSA의 농도가 높을수록, 반응온도가 높을수록 증가하였다. HYNIC을 결합시키지 않은 HSA만을 사용하였을때 표지효율은 10% 이하였다. 이것은 HAS에 HYNIC을 도입하는 것이 HAS와 [^(18)F]FBA의 결합을 증가시킨다는 것을 나타낸다. ^(18)F-HSA의 마우스를 이용한 체내분포실험결과는 ^(99m)Tc-HSA와 유사하였다. 결론 : 우리는 [^(18)F]FBA 와 HYNIC-HSA 사이에 hydrazone을 형성시켜 ^(18)F-HSA를 성공적으로 표지하였다. 이 방법은 ^(18)F을 다른 단백질이나 펩타이드들에 표지하는데에도 사용할 수 있을 것이다.