Comparison of the [18F]-FDG and [18F]-FLT PET Tracers in the Evaluation of the Preclinical Proton Therapy Response in Hepatocellular Carcinoma.

PURPOSE: The main objective of the present study was to compare the 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) and 3'-[18F]fluoro-3'-deoxythymidine ([18F]-FLT) PET imaging biomarkers for the longitudinal follow-up of small animal proton therapy studies in the context of hepatocellular carcinoma (HCC). PROCEDURES: SK-HEP-1 cells were injected into NMRI nude mice to mimic human HCC. The behavior of [18F]-FDG and [18F]-FLT tumor uptake was evaluated after proton therapy procedures. The proton single-fraction doses were 5, 10, and 20 Gy, with a dose rate of 10 Gy/min. The experimental protocol consisted of 8 groups of 10 mice, each group experiencing a particular dose/radiotracer condition. A reference PET exam was performed on each mouse the day before the irradiation procedure, followed by PET exams every 3 days up to 16 days after irradiation. RESULTS: [18F]-FDG uptake showed a linear dose-dependent increase in the first days after treatment (37%, p < 0.05), while [18F]-FLT uptake decreased in a dose-dependent manner (e.g., 21% for 5 Gy compared to 10 Gy, p = 1.1e-2). At the later time point, [18F]-FDG normalized activity showed an 85% decrease (p < 0.01) for both 10 and 20 Gy doses and no variation for 5 Gy. Conversely, a significant 61% (p = 0.002) increase was observed for [18F]-FLT normalized activity at 5 Gy and no variation for higher doses. CONCLUSION: We showed that the use of the [18F]-FDG and [18F]-FLT radiolabeled molecules can provide useful and complementary information for longitudinal follow-up of small animal proton therapy studies in the context of HCC. [18F]-FDG PET imaging enables a treatment monitoring several days/weeks postirradiation. On the other hand, [18F]-FLT could represent a good candidate to monitor the treatment few days postirradiation, in the context of hypo-fractioned and close irradiation planning. This opens new perspectives in terms of treatment efficacy verification depending on the irradiation scheme.

The Role of Alcohol Dehydrogenase in Drug Metabolism: Beyond Ethanol Oxidation.

Alcohol dehydrogenases (ADHs) are most known for their roles in oxidation and elimination of ethanol. Although less known, ADHs also play a critical role in the metabolism of a number of drugs and metabolites that contain alcohol functional groups, such as abacavir (HIV/AIDS), hydroxyzine (antihistamine), and ethambutol (antituberculosis). ADHs consist of 7 gene family numbers and several genetic polymorphic forms. ADHs are cytosolic enzymes that are most abundantly found in the liver, although also present in other tissues including gastrointestinal tract and adipose. Marked species differences exist for ADHs including genes, proteins, enzymatic activity, and tissue distribution. The active site of ADHs is relatively small and cylindrical in shape. This results in somewhat narrow substrate specificity. Secondary alcohols are generally poor substrates for ADHs. In vitro-in vivo correlations for ADHs have not been established, partly due to insufficient clinical data. Fomepizole (4-methylpyrazole) is a nonspecific ADH inhibitor currently being used as an antidote for the treatment of methanol and ethylene glycol poisoning. Fomepizole also has the potential to treat intoxication of other substances of abuse by inhibiting ADHs to prevent formation of toxic metabolites. ADHs are inducible through farnesoid X receptor (FXR) and other transcription factors. Drug-drug interactions have been observed in the clinic for ADHs between ethanol and therapeutic drugs, and between fomepizole and ADH substrates. Future research in this area will provide additional insights about this class of complex, yet fascinating enzymes.

Clinical value of PET/CT with carbon-11 4DST in the evaluation of malignant and benign lung tumors.

OBJECTIVES: The aim of this study was to assess the clinical value of [11C]4DST uptake in patients with lung nodules, including benign and malignant tumors, and to assess the correlation between [11C]4DST uptake and proliferative activity of tumors in comparison with [18F]FDG uptake. METHODS: Twenty-six patients (22 males and 4 females, mean age of 65.5-year-old) were analyzed in this prospective study. Patients underwent [11C]4DST and [18F]FDG PET/CT imaging on the same day. Diagnosis of each lung nodule was confirmed by histopathological examination of tissue specimens at surgery, or during clinical follow-up after the PET/CT studies. To assess the utility of the semi-quantitative evaluation method, the SUVmax was calculated of [11C]4DST and [18F]FDG uptake by the lesion. Proliferative activities of each tumor as indicated by the immunohistochemical Ki-67 index was also estimated using surgical specimens of patients. Then the relationship between the SUVmax of both PET/CT and the Ki-67 index was examined. Furthermore, the relationship between the uptake of [11C]4DST or [18F]FDG and the histopathological findings, the clinical stage, and the clinical outcome of patients were also assessed. RESULTS: There was a positive linear relationship between the SUVmax of [11C]4DST images and the Ki-67 index (Correlation coefficients = 0.68). The SUVmax of [11C]4DST in the 26 lung nodules were 1.65 ± 0.40 for benign lesions, 3.09 ± 0.83 for adenocarcinomas (P < 0.001 between benign and adenocarcinoma), and 2.92 ± 0.58 for SqCCs (P < 0.001 between benign and SqCC). Whereas, the SUVmax of [18F]FDG were 2.38 ± 2.27 for benign lesions, 6.63 ± 4.24 for adenocarcinomas (n.s.), and 7.52 ± 2.84 for SqCCs (n.s.). The relationship between TNM tumor stage and the SUVmax of [11C]4DST were 2.54 ± 0.37 for T1, 3.48 ± 0.57 for T2, and 4.17 ± 0.72 for T3 (P < 0.005 between T1 and T2, and P < 0.001 between T1 and T3). In comparison with the TNM pathological stage, SUVmax of [11C]4DST were 2.63 ± 0.49 for stage I, 3.36 ± 0.23 for stage II, 3.40 ± 1.12 for stage III, and 4.65 for stage IV (P < 0.05 between stages I and II). In comparison of the clinical outcome, the SUVmax of [11C]4DST were 2.72 ± 0.56 for the no recurrence (No Rec.) group, 3.10 ± 0.33 for the recurrence-free with adjuvant chemotherapy after the surgery (the No Rec. Adjv. CTx. group) and 4.66 ± 0.02 for the recurrence group (Rec. group) (P < 0.001 between the No Rec and Rec. groups, and P < 0.005 between the No Rec. Adjv. CTx. and Rec. groups). CONCLUSIONS: PET/CT with [11C]4DST is as feasible for imaging of lung tumors as [18F]FDG PET/CT. For diagnosing lung tumors, [11C]4DST PET is useful in distinguishing benign nodules from malignancies. [11C]4DST uptake in lung carcinomas is correlated with the proliferative activity of tumors, indicating a promising noninvasive PET imaging of DNA synthesis in malignant lung tumors.

Nucleotide Analogues as Inhibitors of SARS-CoV-2 Polymerase, a Key Drug Target for COVID-19.

SARS-CoV-2 is responsible for the current COVID-19 pandemic. On the basis of our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously demonstrated that three nucleotide analogues (the triphosphates of Sofosbuvir, Alovudine, and AZT) inhibit the SARS-CoV RNA-dependent RNA polymerase (RdRp). We also demonstrated that a library of additional nucleotide analogues terminate RNA synthesis catalyzed by the SARS-CoV-2 RdRp, a well-established drug target for COVID-19. Here, we used polymerase extension experiments to demonstrate that the active triphosphate form of Sofosbuvir (an FDA-approved hepatitis C drug) is incorporated by SARS-CoV-2 RdRp and blocks further incorporation. Using the molecular insight gained from the previous studies, we selected the active triphosphate forms of six other antiviral agents, Alovudine, Tenofovir alafenamide, AZT, Abacavir, Lamivudine, and Emtricitabine, for evaluation as inhibitors of the SARS-CoV-2 RdRp and demonstrated the ability of these viral polymerase inhibitors to be incorporated by SARS-CoV-2 RdRp, where they terminate further polymerase extension with varying efficiency. These results provide a molecular basis for inhibition of the SARS-CoV-2 RdRp by these nucleotide analogues. If sufficient efficacy of some of these FDA-approved drugs in inhibiting viral replication in cell culture is established, they may be explored as potential COVID-19 therapeutics.

A library of nucleotide analogues terminate RNA synthesis catalyzed by polymerases of coronaviruses that cause SARS and COVID-19.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH2-dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19.

In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

Synthesis and Characterization of Specific Reverse Transcriptase Inhibitors for Mammalian LINE-1 Retrotransposons.

Retrotransposons are a type of transposable element (TE) that have amplified to astonishing numbers in mammalian genomes, comprising more than a third of the human and mouse genomes. Long interspersed element class 1 (LINE-1 or L1) retrotransposons are abundant and currently active retroelements in the human and mouse genomes. Similarly, long terminal repeat (LTR)-containing retrotransposons are abundant in both genomes, although only active in mice. LTR- and LINE-1-retroelements use different mechanisms for retrotransposition, although both involve the reverse transcription of an intermediate retroelement-derived RNA. Retrotransposon activity continues to effect the germline and somatic genomes, generating interindividual variability over evolution and potentially influencing cancer and brain physiology, respectively. However, relatively little is known about the functional consequences of retrotransposition. In this study, we have synthesized and characterized reverse transcriptase inhibitors specific for mammalian LINE-1 retrotransposons, which might help deciphering the functional impact of retrotransposition in vivo.

Synthesis and anti-HIV activity of L-2',3'-Dideoxy-4'-selenonucleosides (L-4'-Se-ddNs).

Based on the potent anti-HIV activity of L-2',3'-dideoxycytidine (L-ddC), L-2',3'-dideoxy-4'-selenonucleosides (L-4'-Se-ddNs) have been synthesized from natural chiral template, L-glutamic acid, using Pummerer-type condensation as a key step. All synthesized compounds were assayed for anti-HIV-1 activity, but none of them did show any significant antiviral activity up to 100 µM, probably due to conformational differences between L-ddC and L-4'-Se-ddC, induced by the bulky selenium atom, which might play an important role in phosphorylation by cellular kinase.

Structures and Relative Glycosidic Bond Stabilities of Protonated 2'-Fluoro-Substituted Purine Nucleosides.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H]+, and 2'-fluoro-2'-deoxyguanosine, [Guofl+H]+, are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H]+, stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H]+. Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H]+ adopts C2'-endo sugar puckering, whereas [Guofl+H]+ adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

High-efficiency electrochemical degradation of antiviral drug abacavir using a penetration flux porous Ti/SnO2-Sb anode.

Electrochemical degradation of antiviral drug abacavir was investigated by using a penetration flux porous Ti/SnO2-Sb anode prepared by sol-gel method. The effects of applied current density, initial pH, and inorganic anions on the degradation kinetics were systematically studied. Degradation efficiency more than 97% was performed in only 10 min at a current density of 0.2 mA cm-2. The corresponding degradation rate constant and the lowest electrical energy per order were calculated to be 0.36 min-1 and 6.5 mWh L-1, respectively. Extending the reaction duration to 5 h, 53.3% of TOC removal was observed. The results indicated that effective degradation of abacavir appeared in the penetration flux porous Ti/SnO2-Sb anode with a very low energy consumption. Furthermore, the electrochemical intermediate products and the reaction site during abacavir degradation were detected and recognized. The quantitative structure-activity relationship model revealed that the potential risks of abacavir to the aquatic organism, such as fish, greatly decreased after flowing through the penetration flux porous Ti/SnO2-Sb anode.

Synthesis of 1',2'-methano-2',3'-dideoxynucleosides as potential antivirals.

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1',2'- methano-2',3'-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.

How Long of a Dynamic 3'-Deoxy-3'-[18F]fluorothymidine ([18F]FLT) PET Acquisition Is Needed for Robust Kinetic Analysis in Breast Cancer?

PURPOSE: To quantitatively evaluate the minimally required scanning time of 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) dynamic acquisition for accurate kinetic assessment of the proliferation in breast cancer tumors. PROCEDURES: Within a therapeutic intervention trial, 26 breast tumors of 8 breast cancer patients were analyzed from 30-min dynamic [18F]FLT-PET acquisitions. PET/CT was acquired on a Gemini TF 64 system (Philips Healthcare) and reconstructed into 26 frames (8 × 15 s, 6 × 30 s, 5 × 1 min, 5 × 2 min, and 2 × 5 min). Maximum activity concentrations (Bq/ml) of volume of interests over tumors and plasma in descending aorta were obtained over time frames. Kinetic parameters were estimated using in-house developed software with the two-tissue three-compartment irreversible model (2TCM) (K1, k2, k3, and Ki; k4 = 0) and Patlak model (Ki) based on different acquisition durations (Td) (10, 12, 14, 16, 20, 25, and 30 min, separately). Different linear regression onset time (T0) points (1, 2, 3, 4, and 5 min) were applied in Patlak analysis. Ki of the 30-min data set was taken as the gold standard for comparison. Pearson product-moment correlation coefficient (R) of 0.9 was chosen as a limit for the correlation. RESULTS: The correlation of kinetic parameters between the gold standard and the abbreviated dynamic data series increased with longer Td from 10 to 30 min. k2 and k3 using 2TCM and Ki using Patlak model revealed poor correlations for dynamic PET with Td ≤ 14 min (k2: R = 0.84, 0.85, 0.86; k3: R = 0.67, 0.67, 0.67; Ki: R = 0.72, 0.78, 0.87 at Td = 10, 12, and 14 min, respectively). Excellent correlations were shown for all kinetic parameters when Td ≥ 16 min regardless of the kinetic model and T0 value (R > 0.9). CONCLUSIONS: This study indicates that a 16-min dynamic PET acquisition appears to be sufficient to provide accurate [18F]FLT kinetics to quantitatively assess the proliferation in breast cancer lesions.

Critical assessment of approaches for molecular docking to elucidate associations of HLA alleles with adverse drug reactions.

Adverse drug reactions have been linked with genetic polymorphisms in HLA genes in numerous different studies. HLA proteins have an essential role in the presentation of self and non-self peptides, as part of the adaptive immune response. Amongst the associated drugs-allele combinations, anti-HIV drug Abacavir has been shown to be associated with the HLA-B*57:01 allele, and anti-epilepsy drug Carbamazepine with B*15:02, in both cases likely following the altered peptide repertoire model of interaction. Under this model, the drug binds directly to the antigen presentation region, causing different self peptides to be presented, which trigger an unwanted immune response. There is growing interest in searching for evidence supporting this model for other ADRs using bioinformatics techniques. In this study, in silico docking was used to assess the utility and reliability of well-known docking programs when addressing these challenging HLA-drug situations. The overall aim was to address the uncertainty of docking programs giving different results by completing a detailed comparative study of docking software, grounded in the MHC-ligand experimental structural data - for Abacavir and to a lesser extent Carbamazepine - in order to assess their performance. Four docking programs: SwissDock, ROSIE, AutoDock Vina and AutoDockFR, were used to investigate if each software could accurately dock the Abacavir back into the crystal structure for the protein arising from the known risk allele, and if they were able to distinguish between the HLA-associated and non-HLA-associated (control) alleles. The impact of using homology models on the docking performance and how using different parameters, such as including receptor flexibility, affected the docking performance were also investigated to simulate the approach where a crystal structure for a given HLA allele may be unavailable. The programs that were best able to predict the binding position of Abacavir were then used to recreate the docking seen for Carbamazepine with B*15:02 and controls alleles. It was found that the programs investigated were sometimes able to correctly predict the binding mode of Abacavir with B*57:01 but not always. Each of the software packages that were assessed could predict the binding of Abacavir and Carbamazepine within the correct sub-pocket and, with the exception of ROSIE, was able to correctly distinguish between risk and control alleles. We found that docking to homology models could produce poorer quality predictions, especially when sequence differences impact the architecture of predicted binding pockets. Caution must therefore be used as inaccurate structures may lead to erroneous docking predictions. Incorporating receptor flexibility was found to negatively affect the docking performance for the examples investigated. Taken together, our findings help characterise the potential but also the limitations of computational prediction of drug-HLA interactions. These docking techniques should therefore always be used with care and alongside other methods of investigation, in order to be able to draw strong conclusions from the given results.

Synthesis and Antiviral Evaluation of TriPPPro-AbacavirTP, TriPPPro-CarbovirTP, and Their 1',2'-cis-Disubstituted Analogues.

Herein we describe the synthesis of lipophilic triphosphate prodrugs of abacavir, carbovir, and their 1',2'-cis-substituted carbocyclic analogues. The 1',2'-cis-carbocyclic nucleosides were prepared by starting from enantiomerically pure (1R,2S)-2-((benzyloxy)methyl)cyclopent-3-en-1-ol by a microwave-assisted Mitsunobu-type reaction with 2-amino-6-chloropurine. All four nucleoside analogues were prepared from their 2-amino-6-chloropurine precursors. The nucleosides were converted into their corresponding nucleoside triphosphate prodrugs (TriPPPro approach) by application of the H-phosphonate route. The TriPPPro compounds were hydrolyzed in different media, in which the formation of nucleoside triphosphates was proven. While the TriPPPro compounds of abacavir and carbovir showed increased antiviral activity over their parent nucleoside, the TriPPPro compounds of the 1',2'-cis-substituted analogues as well as their parent nucleosides proved to be inactive against HIV.

Microsolvated Model for the Kinetics and Thermodynamics of Glycosidic Bond Dissociative Cleavage of Nucleoside D4G.

Using the microsolvated model that involves explicit water molecules and implicit solvent in the optimization, two proposed dissociative hydrolysis mechanisms of 2',3'-didehydro-2',3'-dideoxyguanosine (d4G) have been first investigated by means of M06-2X(CPCM, water)/6-31++G(d,p) method. The glycosidic bond dissociation for the generation of the oxacarbenium ion intermediate is the rate-determining step (RDS). The subsequent nucleophilic water attack from different side of the oxacarbenium ion intermediate gives either the α-product [(2S,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-ol] or ß-product [(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-ol] and is thus referred to as α-path (inversion) and ß-path (retention). Two to five explicit water molecules (n = 2-5) are considered in the microsolvated model, and n = 3 or 4 is the smallest model capable of minimizing the activation energy for α-path and ß-path, respectively. Our theoretical results suggest that α-path (n = 3) is more kinetically favorable with lower free energy barrier (RDS) of 27.7 kcal mol-1, in contrast to that of 30.7 kcal mol-1 for the ß-path (n = 4). The kinetic preference of the α-path is rationalized by NBO analysis. Whereas thte ß-path is more thermodynamically favorable over the α-path, where the formation of ß-product and α-product are exergonic and endergonic, respectively, providing theoretical support for the experimental observation that the ß-cleavage product was the major one after sufficient reaction time. Comparisons of d4G with analogous cyclo-d4G and dG from kinetic free energy barriers and thermodynamic heterolytic dissociation energies were also carried out. Our kinetic and thermodynamic results manifest that the order of glycosidic bond stability should be d4G < cyclo-d4G < dG, which agrees well with the reported experimental stability order of d4G compounds and analogues and gives further understanding on the influence of 6-cyclopropylamino and unsaturated ribose to the glycosidic bond instability of d4G.

A chiral template-driven synthesis of a 3'-deoxy-3'-18F-fluorothymidine precursor.

An asymmetric synthesis of a 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) precursor has been developed wherein the deoxysugar moiety was synthesized using a novel Ga-mediated allylation of (R)-2,3-cyclohexylideneglyceraldehyde as the key step. The synthesis deviates significantly from the previous syntheses of the 18F-FLT precursors wherein the expensive starting material, thymidine was used.

Sex as a Biologic Variable in Preclinical Imaging Research: Initial Observations with 18F-FLT.

The study objective was to investigate whether sex influences 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) uptake and tissue distribution in mouse models of cancer. Methods:18F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18F-FDG biodistribution was measured for comparison. 18F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmacokinetic analysis demonstrated higher early renal 18F-FLT activity and greater accumulation of 18F-FLT in the urinary bladder in male than female mice. The differential pattern of 18F-FLT biodistribution between the sexes seen with 18F-FLT was not observed with 18F-FDG. Increased tumoral 18F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18F-FLT to assess tumor proliferation.

3'-Deoxy-3'-[18F]Fluorothymidine Uptake Is Related to Thymidine Phosphorylase Expression in Various Experimental Tumor Models.

PURPOSE: We recently reported that high thymidine phosphorylase (TP) expression is accompanied by low tumor thymidine concentration and high 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) uptake in four untreated lung cancer xenografts. Here, we investigated whether this relationship also holds true for a broader range of tumor models. PROCEDURES: Lysates from n = 15 different tumor models originating from n = 6 institutions were tested for TP and thymidylate synthase (TS) expression using western blots. Results were correlated to [18F]FLT accumulation in the tumors as determined by positron emission tomography (PET) measurements in the different institutions and to previously published thymidine concentrations. RESULTS: Expression of TP correlated positively with [18F]FLT SUVmax (ρ = 0.549, P < 0.05). Furthermore, tumors with high TP levels possessed lower levels of thymidine (ρ = - 0.939, P < 0.001). CONCLUSIONS: In a broad range of tumors, [18F]FLT uptake as measured by PET is substantially influenced by TP expression and tumor thymidine concentrations. These data strengthen the role of TP as factor confounding [18F]FLT uptake.

[18F]fluorothymidine PET Informs the Synergistic Efficacy of Capecitabine and Trifluridine/Tipiracil in Colon Cancer.

In cancer therapy, enhanced thymidine uptake by the salvage pathway can bypass dTMP depletion, thereby conferring resistance to thymidylate synthase inhibition. We investigated whether sequential combination therapy of capecitabine and trifluridine/tipiracil (TAS-102) could synergistically enhance antitumor efficacy in colon cancer xenograft models. We also examined 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) PET as a means to predict therapeutic response to a sequential combination of capecitabine and trifluridine/tipiracil. [3H]FLT uptake after 5-fluorouracil treatment in vitro and [18F]FLT uptake after capecitabine (360 mg/kg/day) in athymic nude mice (Balb/c-nu) with xenografts (n = 10-12 per group) were measured using eight human colon cancer cell lines. We determined the synergistic effects of sequential combinations of 5-fluorouracil and trifluridine in vitro as well as the sequential combination of oral capecitabine (30-360 mg/kg) and trifluridine/tipiracil (trifluridine 75 or 150 mg/kg with tipiracil) in six xenograft models (n = 6-10 per group). We observed significant increases in [3H]FLT uptake in all cell lines and [18F]FLT uptake in five xenograft models after 5-fluorouracil and capecitabine treatment, respectively. Increased [18F]FLT uptake after capecitabine followed by extinction of uptake correlated strongly with tumor growth inhibition (ρ = -0.81, P = 0.02). The effects of these combinations were synergistic in vitro A synergy for sequential capecitabine and trifluridine/tipiracil was found only in mouse xenograft models showing increased [18F]FLT uptake after capecitabine. Our results suggest that the sequential combination of capecitabine and trifluridine/tipiracil is synergistic in tumors with an activated salvage pathway after capecitabine treatment in mice, and [18F]FLT PET imaging may predict the response to capecitabine and the synergistic antitumor efficacy of a sequential combination of capecitabine and trifluridine/tipiracil. Cancer Res; 77(24); 7120-30. ©2017 AACR.

Whole-Body Distribution of Leukemia and Functional Total Marrow Irradiation Based on FLT-PET and Dual-Energy CT.

This report describes a multimodal whole-body 3'-deoxy-3'[(18)F]-fluorothymidine positron emission tomography (FLT-PET) and dual-energy computed tomography (DECT) method to identify leukemia distribution within the bone marrow environment (BME) and to develop disease- and/or BME-specific radiation strategies. A control participant and a newly diagnosed patient with acute myeloid leukemia prior to induction chemotherapy were scanned with FLT-PET and DECT. The red marrow (RM) and yellow marrow (YM) of the BME were segmented from DECT using a basis material decomposition method. Functional total marrow irradiation (fTMI) treatment planning simulations were performed combining FLT-PET and DECT imaging to differentially target irradiation to the leukemia niche and the rest of the skeleton. Leukemia colonized both RM and YM regions, adheres to the cortical bone in the spine, and has enhanced activity in the proximal/distal femur, suggesting a potential association of leukemia with the BME. The planning target volume was reduced significantly in fTMI compared with conventional TMI. The dose to active disease (standardized uptake value >4) was increased by 2-fold, while maintaining doses to critical organs similar to those in conventional TMI. In conclusion, a hybrid system of functional-anatomical-physiological imaging can identify the spatial distribution of leukemia and will be useful to both help understand the leukemia niche and develop targeted radiation strategies.