Application Of ¹¹C And ¹⁸F Labeled PI3K Inhibitor GDC-0941 As Novel Molecular Probes In Breast Cancer

Purpose GDC-0941(Pictilisib)is an inhibitor of the phosphatidylinositol 3-kinase(PI3-K),which is under clinical trial phase 2.Approximately 70% of breast cancer has PI3K/Akt abnormal activation of this pathway.Non-invasive measurements of [11C]-GDC-0941 uptake in organs and tumors may provide additional information on pharmacokinetics of pictilisib.The purpose of the study was to radiolabel GDC-0941 with carbon-11 to evaluate its pharmacokinetics noninvasively and potency as a PET tracer to screen pictilisib-sensitive tumors.Methods Pictilisib radiolabeled with [11C]-methyl iodide using GE TRACERlab FXCPro.Normal KM mice performed micro-positron emission tomography(PET)scan with [11C]-GDC-0941 to evaluate its pharmacokinetics characteristic noninvasively.Two human breast cancer cell lines MCF-7(PIK3CA mutation,pictilisib-sensitive)and MDA-MB-231(PIK3CA wild-type,pictilisib-resistant)were chosen in this study.In vitro cell uptake assay and in vivo micro-PET and biodistribution of tissues was performed with [11C]-GDC-0941 in nude mice bearing xenografts of MCF-7 and MDAMB-231 cells to evaluate the potency of [11C]-GDC-0941 as a novel molecular probe.Results The in vitro cell assay showed that the internalization rate of both cells was 3.10±0.29% vs.1.42±0.06%(P =0.0001)?3.23±0.15% vs.1.58±0.09%(P<0.0001)?4.32±0.64% vs.2.14±0.10%(P =0.002)at 10,30 and 60 for MCF-7 and MDA-MB-231 cells,respectively.These results suggested good binding potency for [11C]-GDC-0941 in vitro.Dynamic micro-PET scans and biodistribution of tissues showed that hepatobiliary excretion and intestinal reuptake was the main metabolic pattern for [11C]-GDC-0941 in vivo.Static micro-PET scans on MCF-7 xenograft can be visualized while MDA-MB-231 tumors could not.Biodistribution of [11C]-GDC-0941 in two xenograft models showed the significant higher radioactivity accumulation in the MCF-7 xenografts at 60 min after tracer injection with 2.71±0.22%ID/g than that of MDA-MB-231 xenografts with 1.31±0.36%ID/g(P=0.025).Meanwhile,the tumor-muscle ratio of MCF-7 and MDA-MB-231 at 60 min was(3.22±0.54)and(1.28±0.30)at 60 min,which showed statistic significant differently(P=0.045).Micro-PET imaging showed no tumor was seen in the blocking of MCF-7 bearing mouse.Conclusion In this study,we first successfully prepared [11C]-GDC-0941.In vivo studies revealed that the liver and small intestine was the predominant excretory pathway of pictilisib.We show that [11C]-GDC-0941 identifies GDC-0941-sensitive tumors for the first time.These results pave the road for studies examining the benefit of GDC-0941 in patients with breast cancer or other tumors overactive PI3K/Akt/m TOR signal pathway.However,the high activity in liver and intestinal should be noticed and improved.In addition,because of the short half-life of 11C(approximately 20 minutes),long half-life radionuclide should be used,which spurred the development of 18 F labeled GDC-0941.Purpose This study aimed to add triethylene glycol into GDC-0941 and label it with a longer half-life nuclide 18 F to establish 18 F labeled PEG3 modified GDC-0941 as a novel PET molecular probe.In vitro/in vivo experiments of cancer cells were performed to evaluate the pharmacokinetic characterization of GDC-0941 in order to reduce hepatic uptake,improve the bioavailability of molecular probes and improve imaging quality.Methods 1.Preparation and Purification and Identification of Precursor GDC-0941-PEG-OTs: HO-PEG-OTs were prepared and then HO-PEG3-OTs and GDC-0941 were mixed in K2CO3 solution to obtain GDC-PEG3-OH compound was separated on a silica gel column to obtain the precursor GDC-PEG3-OTs.The product was identified by carbon and hydrogen spectra.2.Preparation of 18F-PEG3-GDC-0941: GE's automated synthesis equipment TRACERLAB FX-XN was used to synthesis,the product 18FPEG3-GDC-0941 was separated and purified by HPLC.3.Uptake of 18F-PEG3-GDC-0941 by different breast cancer subtypes: The expression of MCF-7 and PI3Kp110? in two breast cancer cell lines with different PI3Kp110? expression level,and the expression of the two cells was identified by Western blot.The uptake of 18F-PEG3-GDC-0941 cells at different time points(10,30,60 and 120min)was measured by ? detector.The dynamic imaging and biodistribution of Micro-PET at 120 min in the normal Kunming mice and the above-mentioned breast cancer cell bearing mice were injected into the tail vein with 18F-PEG3-GDC-0941.Results The labeling yield of 18F-PEG3-GDC-0941 was 65.25±3.85% and the specific activity was(43.25 ± 1.75)Ci / mmol(n = 3)using analytical HPLC.The stability of 18F-PEG3-GDC-0941 in human serum was good,and its radiochemical purity was above 95% after 4 hours.The Log P value of 18F-PEG3-GDC-0941 after modification with PEG was 1.12 ± 0.01(n = 3).Cell uptake experiments showed that uptake of 18FPEG3-GDC-0941 by MCF-7 and MDA-MB-231 increased with time,and cellular uptake of the latter was significantly lower than the former.The uptake rates of 18FPEG3-GDC-0941 at 10,30,60 and 120 min in MCF-7 and MDA-MB-231 cells were 2.17±0.23% vs.1.92±0.07%(P=0.11),2.92±0.29% vs.2.58±0.27%(P=0.13),3.27%±0.07% vs.2.85±0.08%(P=0.000),3.35±0.08% vs.3.04±0.02%(P=0.003).There was a significant difference in uptake rates between the two cells at 60 min and 120 min.Micro-PET dynamic imaging studies of normal Kunming mice showed that 18FPEG3-GDC-0941 still excreted through the liver and intestine.However,liver uptake was significantly lower than that of [11C]-GDC-0941,and excretion rate was faster.The T/M of MCF-7 mice reached 5.76±1.23 at 60 min while the MDA-MB-231 mice was only 3.00±1.10,which was significant lower than MCF-7 mice(P=0.01).However,both tumor-bearing mice have high blood uptake.Conclusions 18F-PEG3-GDC-0941 was successfully synthesized in this study.The radioactivity yield was high and the stability was good.The water solubility of the probe was increased by connecting PEG3.Initial in vivo studies showed that 18F-PEG3-GDC-0941 was still excreted by the liver and intestine,and was excreted through the kidney with small part,but its liver excretion rate was significantly faster than that of [11C]-GDC-0941.Higher uptake of 18F-PEG3-GDC-0941 in bearing breast cancer tumors with high expression of PI3Kp110? was found.18F-PEG3-GDC-0941 is a promising molecular probe for predicting the efficacy of GDC-0941.Purpose Triple negative breast cancer was one subtype of breast cancer with the worst prognosis.The metastasis occurred early,and the mortality was very high.Despite great advances in treatment,the overall survival of patients is still low due to lack of effective treatment targets.The aim of this study was to investigate the tumor suppressive effect on the triple-negative breast cancer by PI3 K inhibitor GDC-0941,and to observe the effect of 18F-FDG with micro-PET.Methods Mouse breast cancer 4T1 cells were cultured and inoculated subcutaneously(n=20)in the right forelimb of Balb/c mice.Observe every 2 days,measure the diameter of the tumor with a vernier caliper and record the body weight of the mice.GDC-0941 was dissolved in 0.5% methylcellulose and 0.2% Tween 80.Each mouse is administered in a volume of 0.1 ml / 10 g.Tumor size is calculated as follows: Tumor volume(mm3)= long diameter × short diameter 2/2.The mice were randomly divided into 2 groups when the tumor size was up to 250 mm3: the treatment group(n=10)with GDC-0941 management(100 mg / kg)daily;the control group(n=10)was treated with 0.5% methylcellulose and 0.2% Tween80.The GDC-0941 group and the control group were given 21 consecutive days.Three mice in each group were screened for micro-PET for before scanning(0w)and after treatment(3w).The tumor tissues were stained with p-Akt(Ser473),and the expression of p-Akt(Ser473)in the treatment group and the control group was compared.Results The effect on the growth of 4T1 tumor of PI3 K inhibitor GDC-0941 was not very obvious.There was no significant difference between treatment group and vehicle group(P=0.117,n=7/group)in tumor size.But the effect of inhibiting tumor lung metastasis was significant.The number of lung metastases in GDC-0941 group and vehicle group was(9 ± 3)and(44 ± 8),respectively,which showed significantly difference in the GDC-0941 group and the vehicle group(P=0.002).In addition,the results of 18F-FDG imaging at 3 weeks after treatment showed that the tumor / muscle values in the GDC-0941 group were significantly lower than those in the vehicle group,which was 4.43 ± 0.27(GDC-0941 group)and 6.65 ± 0.25(vehicle group),respectively(P=0.001,n=3/group).Conclusions GDC-0941 can inhibit lung metastasis in 4T1 mice,and 18F-FDG can be used to monitor its efficacy.This study paved the way for the further application of 18 F labeled GDC-0941 to predict the efficacy of GDC-0941.