Background

[PubMed]

A potential approach to the treatment of diseases and genetic disorders in humans is gene therapy [PubMed]. This is a technique whereby the absent or malfunctioning gene is replaced by a working gene to produce an enzyme or a protein to correct the progression of disease. Noninvasive molecular imaging technologies play an important role in the fields of gene therapy by monitoring gene expression continuously in living animals (1). Magnetic resonance imaging (MRI), optical imaging, ultrasound imaging, and radionuclide imaging (positon emission tomography (PET)/single photon emission computed tomography (SPECT)) modalities have been applied for gene therapy of cancer, cardiovascular, neurological, musculoskeletal, hepatic, immunological, diabetic, and inherited diseases in small animals (2-10). These noninvasive imaging technologies allow quantitative assessments of the magnitude, location, and duration of the transgene expression.

Gene therapy can be achieved either by ex vivo or in vivo methods. The gene can be delivered using a viral or nonviral vector, such as liposome and naked DNA. Retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus have been used as gene transfer vectors. Imaging transgene expression can help to optimize gene therapy. There are two imaging strategies, direct and indirect. Direct imaging uses a target-specific probe directly to the target. Indirect imaging involves coupling the target gene to a reporter gene, the gene expression of which can be tracked by a specific reporter gene probe. Currently, there are two approaches to image transgene expression, either by reporter enzyme using substrate probe or reporter receptor using receptor ligand probe. The enzymes include cytosine deaminase, β-galactosidase, tyrosinase, and herpes simplex virus type 1 thymidine kinase (HSV1-tk) or its mutant, HSV1-sr36tk. The most extensively studied reporter enzyme gene is HSV1-tk (8, 11). The reporter receptors are dopamine D₂ receptor (D₂R) (12) and somatostatin receptor subtype 2 (hSSTR2) (13).

HSV1-tk is a commonly studied suicide gene for cancer therapy of glioma, prostate cancer, leukemia, and lymphoma (10, 14). Suicide gene therapy is based on the enzymatic conversion of a nontoxic prodrug into a lethal drug by a transgene. Anti-HSV nucleoside analogs, such as acyclovir, ganciclovir, and penciclovir, are converted to monophosphates by HSV thymidine kinase. The cellular enzymes converted the monophosphates to di- and triphosphates, which inhibit mammalian DNA polymerase. HSV1-tk has also been widely studied as a reporter gene. The HSV prodrugs have been explored for HSV1-tk reporter gene imaging. The selective phosphorylation of the prodrug probes by HSV1-tk leads to trapping within the transduced cells and not significantly in the other cells. Many radiolabeled acycloguanosine and thymidine derivatives have been studied for HSV1-tk reporter gene imaging in vitro, in small animals, and in humans (15).

[¹⁸F]FHBG is a side-chain ¹⁸F-labeled analog of penciclovir. [¹⁸F]FHBG has been evaluated in vitro and in small animals and is a potential imaging agent for gene expression using PET. Noninvasive molecular imaging of therapeutic HSV1-sr36tk suicide gene therapy in mice was demonstrated with [¹⁸F]FHBG and [¹⁸F]fluorodeoxyglucose PET imaging (14). Coexpression of the reporter gene HSV1-tk and a therapeutic gene is opening up further opportunities for gene therapy (15).

Synthesis

[PubMed]

9-(4-Hydroxy-3-hydroxymethylbutyl)-guanine (penciclovir) was converted to a methoxytrityl derivative followed by tosylation. The tosylate was reacted with either tetrabutylammonium [¹⁸F]fluoride or [¹⁸F]KF in the presence of kryptofix 2.2.2, followed by acidic hydrolysis to produce 9-(4-[¹⁸F]fluoro-3-hydroxymethylbutyl)guanine ([¹⁸F]FHBG). [¹⁸F]FHBG was isolated by high-performance liquid chromatography (HPLC) purification. The radiochemical yield was 8.0-22.3%, with an average of 12% (corrected for decay). Synthesis time was 90-100 min, including HPLC purification with radiochemical purity >99% and average specific activity of 11.8 GBq/mmol (320 mCi/mmol) (16). Similar syntheses were later reported by others (17, 18). One-pot and automated syntheses were achieved to provide 10-15% yield in about 60 min (17, 19).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

[¹⁸F]FHBG was shown to accumulate in HSV1-tk expressing cells in cultures. HT-29 human colon cancer cells were infected with HSV1-tk (20). In vitro uptakes of [¹⁸F]FHBG in the transduced cells were 31 and 71 times higher than the nontransduced cells at 1 and 5 h, respectively. HPLC analysis showed 10% intact [¹⁸F]FHBG, 6% monophosphate, 54% diphosphate and 30% triphosphate in the transduced cells at 5 h.

H9c2 rat embryonic cardiac cells were infected with Ad4tk, an adenovirus expression a mutant HSV1-sr39tk and vascular endothelial growth factor (VEGF₁₂₁) independently (21). Northern blot analysis showed increasing HSV1-sr39tk and VEGF RNA expression in the transduced cells. Accumulation of [¹⁸F]FHBG and [¹⁴C]FIAU increased with increasing HSV1-sr39tk and HSV1-tk expression, respectively. Secretion of VEGF also increased with the reporter gene expressions. There is also a significant correlation of reporter gene probe uptake with VEGF production in vitro. Therefore, it may be feasible in future for monitoring angiogenesis gene therapy in vivo.

Animal Studies

Human Studies

[PubMed]

Human dosimetry was estimated in eight healthy volunteers by intravenous injection of 69-228 MBq (1.86-6.16 mCi) of [¹⁸F]FHBG (25). The average effective dose is 0.0159 mSv/MBq (58.8 mrem/mCi). The organs receiving the highest absorbed doses were the urinary bladder (0.0942 mGy/MBq or 0.349 rad/mCi), kidneys (0.0511 mGy/MBq or 0.189 rad/mCi), gut (0.0457 mGy/MBq or 0.169 rad/mCi), and liver (0.0223 mGy/MBq or 0.0825 rad/mCi). In the urine, 83% of the total activity was intact [¹⁸F]FHBG.

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