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Purpose [18F]-labeled positron emission tomography (PET) radioligands permit in vivo assessment of Alzheimer’s disease biomarkers, including aggregated neurofibrillary tau (NFT) with [18F]flortaucipir. Due to structural similarities of flortaucipir with some monoamine oxidase A (MAO-A) inhibitors, this study aimed to evaluate flortaucipir binding to MAO-A and MAO-B and any potential impact on PET interpretation. Methods [18F]Flortaucipir autoradiography was performed on frozen human brain tissue slices, and PET imaging was conducted in rats. Dissociation constants were determined by saturation binding, association and dissociation rates were measured by kinetic binding experiments, and IC50 values were determined by competition binding. Results Under stringent wash conditions, specific [18F]flortaucipir binding was observed on tau NFT-rich Alzheimer’s disease tissue and not control tissue. In vivo PET experiments in rats revealed no evidence of [18F]flortaucipir binding to MAO-A; pre-treatment with MAO inhibitor pargyline did not impact uptake or wash-out of [18F]flortaucipir. [18F]Flortaucipir bound with low nanomolar affinity to human MAO-A in a microsomal preparation in vitro but with a fast dissociation rate relative to MAO-A ligand fluoroethyl-harmol, consistent with no observed in vivo binding in rats of [18F]flortaucipir to MAO-A. Direct binding of flortaucipir to human MAO-B was not detected in a microsomal preparation. A high concentration of flortaucipir (IC50 of 1.3 μM) was found to block binding of the MAO-B ligand safinamide to MAO-B on microsomes suggesting that, at micromolar concentrations, flortaucipir weakly binds to MAO-B in vitro. Conclusion These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [18F]flortaucipir.

Supplemental material and methods.

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Supplemental Methods. SI-Fig 1 HPLC chromatogram of [18F]4F-Glu and [18F]4F-Gln mixture. SI-Fig 2 Comparison of T/B values obtained by two readers. SI-Fig 3 Comparison of T/B ratios obtained from different tumor ROI methods. SI-Fig 4 Comparison of T/B ratios between single and multiROI approach. SI-Fig 5 Estimation of [18F]4F-Gln distribution volume (DV) by kinetic analyses of PET data.

Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [125I]KX1 as a PARP-1–selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo. The pharmacologic properties of the PARP radiotracer [125I]KX1 was characterized in multiple cell lines where single-agent sensitivity was correlated with [125I]KX1 binding to PARP-1. In vivo evaluation of [125I]KX1 verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [125I]KX1 correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516–24. ©2016 AACR.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476–84. ©2017 AACR.

SI-figure 1: "Chemical synthesis scheme for [125I]KX1." SI-figure 2: "Whole western blots and regions of interest generated around the protein of interest." SI-figure 3: "Kinetic analysis association and dissociation curves of [125I]KX1." SI-table 1: "Summary table for cell lines used in vitro to validate [125I]KX1 as a marker of PARP-1 expression." SI-table 2: "Competitive inhibition (Ki) Values are presented for each PARP inhibitor as measured in the different cell lines." SI-table 3: "Experimental association and dissociation values of [125I]KX1." SI-table 4: "Dissociation constants for [125I]KX1 in multiple cell lines." SI-table 5: " EC50 values for PARP inhibitors in cell lines evaluated."

[[Under stringent wash conditions, specific [These data suggest neither MAO-A nor MAO-B binding will contribute significantly to the PET signal in cortical target areas relevant to the interpretation of [

Introduction Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[18F]fluoroglutamine (4F-Gln) was reported as a glutamine-related metabolic imaging agent. To improve the in vivo kinetics of this radiotracer, two new dipeptides, [18F]Gly-(2S,4R)4-fluoroglutamine (Gly-4F-Gln) and [18F]Ala-(2S,4R)4-fluoroglutamine (Ala-4F-Gln) were investigated. Methods Radiolabeling was performed via 2-steps 18F-fluorination. Cell uptake studies of Gly-4F-Gln and Ala-4F-Gln were investigated in 9 L cell lines. In vitro and in vivo metabolism studies were carried out in Fisher 344 rats. Biodistribution and microPET imaging studies were performed in 9 L tumor-bearing rats. Results In vitro incubation of these [18F]dipeptides in rat and human blood showed a rapid conversion to (2S,4R)-4-[18F]fluoroglutamine (t1/2 = 2.3 and 0.2 min for [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, respectively for human blood). Biodistribution and PET imaging in Fisher 344 rats bearing 9 L tumor xenografts showed that these dipeptides rapidly localized in the tumors, comparable to that of (2S,4R)-4-[18F]fluoroglutamine (4F-Gln). Conclusions The results support that these dipeptides, [18F]Gly-4F-Gln and [18F]Ala-4F-Gln, are prodrugs, which hydrolyze in the blood after an iv injection. They appear to be selectively taken up and trapped by tumor tissue in vivo. The dipeptide, [18F]Ala-4F-Gln, may be suitable as a PET tracer for imaging glutaminolysis in tumors.

Despite the availability of PARP inhibitors for cancer therapy, a biomarker to clearly stratify patients for selection of this treatment remains lacking. Here we describe a radiotracer-based method that addresses this issue, using the novel compound [125I]KX1 as a PARP-1–selective radiotracer that can accurately measure PARP-1 expression in vitro and in vivo. The pharmacologic properties of the PARP radiotracer [125I]KX1 was characterized in multiple cell lines where single-agent sensitivity was correlated with [125I]KX1 binding to PARP-1. In vivo evaluation of [125I]KX1 verified in vitro results, validating PARP radiotracers to define PARP-1 enzyme expression as an in vivo biomarker. Notably, PARP-1 expression as quantified by [125I]KX1 correlated positively with the cytotoxic sensitivity of cell lines evaluated with PARP inhibitors. Overall, our results defined a novel technology with the potential to serve as a companion diagnostic to identify patients most likely to respond therapeutically to a PARP inhibitor. Cancer Res; 76(15); 4516–24. ©2016 AACR.

Triple-negative breast cancer (TNBC) is associated with high relapse rates and increased mortality when compared with other breast cancer subtypes. In contrast to receptor positive breast cancers, there are no approved targeted therapies for TNBC. Identifying biomarkers for TNBC is of high importance for the advancement of patient care. The sigma-2 receptor has been shown to be overexpressed in triple negative breast cancer in vivo and has been characterized as a marker of proliferation. The aim of the present study was to define the sigma-2 receptor as a target for therapeutic drug delivery and biomarker in TNBC.Three TNBC cell lines were evaluated: MDA-MB-231, HCC1937 and HCC1806. Sigma-2 compounds were tested for pharmacological properties specific to the sigma-2 receptor through competitive inhibition assays. Sigma-2 receptor expression was measured through radioligand receptor saturation studies. Drug sensitivity for taxol was compared to a sigma-2 targeting compound conjugated to a cytotoxic payload, SW IV-134. Cell viability was assessed after treatments for 2 or 48 h. Sigma-2 blockade was assessed to define sigma-2 mediated cytotoxicity of SW IV-134. Caspase 3/7 activation induced by SW IV-134 was measured at corresponding treatment time points.SW IV-134 was the most potent compound tested in two of the three cell lines and was similarly effective in all three. MDA-MB-231 displayed a statistically significant higher sigma-2 receptor expression and also was the most sensitive cell line evaluated to SW IV-134.Targeting the sigma-2 receptor with a cytotoxic payload was effective in all the three cell lines evaluated and provides the proof of concept for future development of a therapeutic platform for the treatment of TNBC.

We report the synthesis and biological evaluation of a series of indole-based σ2 receptor ligands derived from siramesine. In vitro competition binding assays showed that these analogues possessed high to moderate affinity and selectivity for σ2 receptors. Structure–affinity relationship analyses of these indole-based σ2 receptor ligands were performed. In the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 1a and 1b displayed significant and comparable antiproliferative activity in DU145, MCF7 and C6 cells to siramesine. In cell cycle analyses, compounds 1a, 1b and siramesine were found to induce a G1 phase cell cycle arrest in DU145 cells using flow cytometry. The combination of 5,6-dimethoxyisoindoline scaffold and N-(4-fluorophenyl)indole moiety was identified as a new σ2 receptor ligand deserving further investigation as an antitumor agent.

Glutamine is an essential source of energy, metabolic substrates, and building block for supporting tumor proliferation. Previously, (2S,4R)-4-[Radiolabeling was performed via 2-stepsIn vitro incubation of these [The results support that these dipeptides, [

There is often overlap in the diagnostic features of common pathologic processes such as infection, sterile inflammation, and cancer both clinically and using conventional imaging techniques. Here, we report the development of a positron emission tomography probe for live bacterial infection based on the small-molecule antibiotic trimethoprim (TMP). [18F]fluoropropyl-trimethoprim, or [18F]FPTMP, shows a greater than 100-fold increased uptake in vitro in live bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) relative to controls. In a rodent myositis model, [18F]FPTMP identified live bacterial infection without demonstrating confounding increased signal in the same animal from other etiologies including chemical inflammation (turpentine) and cancer (breast carcinoma). Additionally, the biodistribution of [18F]FPTMP in a nonhuman primate shows low background in many important tissues that may be sites of infection such as the lungs and soft tissues. These results suggest that [18F]FPTMP could be a broadly useful agent for the sensitive and specific imaging of bacterial infection with strong translational potential.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476–84. ©2017 AACR.

There is a need for improved methods to image genetically engineered cells, including immune cells used for cell-based therapy. Given the genetic manipulation inherent to gene therapy, the use of a reporter protein is a logical solution and positron emission tomography (PET) can provide the desired sensitivity and spatial localization. We developed a broadly applicable PET imaging strategy based on the small bacterial protein E. coli dihydrofolate reductase (Ec dhfr) and its highly specific small molecule inhibitor, trimethoprim (TMP). The difference in TMP affinity for bacterial compared to mammalian DHFR suggests that a TMP radioligand would have a low background in unmodified mammalian tissues and high retention in Ec dhfr engineered cells, providing high contrast imaging. Here, we describe the in vitro properties of [11C]TMP and show over 10-fold increased signal in transgenic Ec dhfr cells compared to control. In a mouse xenograft model, [11C]TMP rapidly accumulated in Ec dhfr carrying cells within minutes of intravenous administration. Moreover, [11C]TMP can identify less than a million xenografted cells in a small volume in tissues other than the abdominal compartment. This limit of detection is a clinically relevant number and bodes well for clinical translation especially given that [11C]TMP is an isotopologue of clinically approved antibiotic.

Although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. In particular, tumor cells with the upregulated c-Myc gene are generally reprogrammed to utilize glutamine. We have developed new 3-fluoropropyl analogs of glutamine, namely [(18)F](2S,4R)- and [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. Optically pure isomers labeled with (18)F and (19)F (2S,4S) and (2S,4R)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (≥95%). Cell uptake studies of both isomers showed that they were taken up efficiently by 9L tumor cells with a steady increase over a time frame of 120 min. At 120 min, their uptake was approximately two times higher than that of l-[(3)H]glutamine ([(3)H]Gln). These in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. Yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [(18)F](2S,4R)-4-(3-fluoropropyl)glutamine, 3. We, therefore, focused on [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4. The in vitro cell uptake studies suggested that the new probe, [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the LAT transport system, followed by System N and ASC transporters. A dual-isotope experiment using l-[(3)H]glutamine and the new probe showed that the uptake of [(3)H]Gln into 9L cells was highly associated with macromolecules (>90%), whereas the [(18)F](2S,4S)-4-(3-fluoropropyl)glutamine, 4, was not (

Introduction O -(2-[ 18 F]fluoroethyl)-l-tyrosine (FET, [ 18 F] 1 ) is a useful amino-acid-based imaging agent for brain tumors. This paper reports the synthesis and evaluation of three FET prodrugs, O -(2-[ 18 F]fluoroethyl)-l-tyrosyl-l-glycine (FET-Gly, [ 18 F] 2 ), O -(2-[ 18 F]fluoroethyl)-l-tyrosyl-l-alanine (FET-Ala, [ 18 F] 3 ) and N -acetyl O -(2-[ 18 F]fluoroethyl)-l-tyrosine (AcFET, [ 18 F] 4 ), which could be readily hydrolyzed to FET in vivo for tumor imaging. We investigated their metabolism in the blood and imaging properties in comparison to FET ([ 18 F] 1 ). Methods Three new [ 18 F]FET derivatives, 2 – 4 , were prepared from their corresponding tosylate-precursors through nucleophilic fluorination and subsequent deprotection reactions. In vitro uptake studies were carried out in 9L glioma cancer cell lines. In vitro and in vivo hydrolysis studies were conducted to evaluate the hydrolysis of FET prodrugs in blood and in Fisher 344 rats. Biodistribution and PET imaging studies were then performed in rats bearing 9L tumors. Results New FET prodrugs were prepared with 3–28% decay corrected radiochemical yields, good enantiomeric purity (>95%) and high radiochemical purity (>95%). FET-Gly ([ 18 F] 2 ), FET-Ala ([ 18 F] 3 ), and AcFET ([ 18 F] 4 ) exhibited negligible uptake in comparison to the high uptake of FET ([ 18 F] 1 ) in 9L cells. Metabolism studies of FET-Gly ([ 18 F] 2 ), FET-Ala ([ 18 F] 3 ), and AcFET ([ 18 F] 4 ) in rat and human blood showed that FET-Ala ([ 18 F] 3 ) was hydrolyzed to FET ([ 18 F] 1 ) faster than FET-Gly ([ 18 F] 2 ) or AcFET ([ 18 F] 4 ). Most of the FET-Ala (79%) was converted to FET ([ 18 F] 1 ) within 5min in blood in vivo . Biodistribution studies demonstrated that FET-Ala ([ 18 F] 3 ) displayed the highest tumor uptake. The tumor-to-background ratios of FET-Ala ([ 18 F] 3 ) and FET ([ 18 F] 1 ) were comparable and appeared to be better than those of FET-Gly ([ 18 F] 2 ) and AcFET ([ 18 F] 4 ). PET imaging studies showed that both FET ([ 18 F] 1 ) and FET-Ala ([ 18 F] 3 ) could visualize tumors effectively, and that they share similar imaging characteristics. Conclusions FET-Ala ([ 18 F] 3 ) demonstrated promising properties as a prodrug of FET ([ 18 F] 1 ), which could be used in PET imaging of tumor amino acid metabolism.

Introduction Biological evaluation of [ 125 I]FlipIDAM (2-((2-((dimethylamino)methyl)-4-iodophenyl)thio)phenyl)methanol ([ 125 I]4), a new single-photon emission computed tomography (SPECT) radioligand for imaging the serotonin transporter (SERT) which displayed improved in vivo kinetics for mapping SERT binding sites in the brain. Methods In vitro binding studies of [ 125 I]4 were performed with membrane homogenates of LLC-PK1 cells stably transfected and overexpressing one of the monoamine transporter (SERT, DAT or NET) and rat cortical homogenates. Biodistribution and ex vivo autoradiography studies were carried out in rats. In vivo competition experiments were evaluated to determine the SERT selectivity of [ 125 I]4 vs. [ 125 I]IDAM ([ 125 I]1). Results In vitro binding studies of 4 showed excellent binding affinity (K i,SERT =0.90±0.05nM) and excellent selectivity over the other monoamine transporters (100 fold and >4000 fold for NET and DAT respectively). Scatchard analysis of saturation binding of [ 125 I]4 to rat cortical homogenates gave a K d value of 0.5±0.09nM and a B max value of 801.4±58.08fmol/mg protein. The biodistribution study showed rapid high brain uptake (3.09±0.11% dose/organ at 2min) and a good target to non-target ratio (hypothalamus to cerebellum) at 30min (2.62) compared to [ 125 I]1 (2.19). Ex vivo autoradiography showed that FlipIDAM localizes in accordance with SERT distribution patterns in the brain. In vivo and ex vivo competition experiments with specific and non-specific SERT compounds also showed that [ 125 I]4 binds specifically to SERT rich regions. Conclusions The biological evaluation of [ 125 I]4 demonstrates that [ 123 I]4 would be a good candidate for SPECT imaging of SERT.

The sigma-1 receptor (S1R) has attracted a great deal of attention as a prospective drug target due to its involvement in numerous neurological disorders and, more recently, for its therapeutic potential in neuropathic pain. As there was no crystal structure of this membrane-bound protein reported until 2016, ligand generation was driven by pharmacophore refinements to the general model suggested by Glennon and co-workers. The generalised S1R pharmacophore comprises a central region where a basic amino group is preferred, flanked by two hydrophobic groups. Guided by this pharmacophore, S1R ligands containing piperazines, piperazinones, and ethylenediamines have been developed. In the current work, we systematically deconstructed the piperazine core of a prototypic piperazine S1R ligand (vide infra) developed in our laboratories. Although we did not improve the affinity at the S1R compared to the lead, we identified several features important for affinity and selectivity. These included at least one basic nitrogen atom, conformational flexibility and, for S1R, a secondary or tertiary amine group proximal to the anisole. Furthermore, S2R selectivity can be tailored with functional group modifications of the N-atom proximal to the anisole.

Introduction Nine novel analogues were synthesized including a 6-carbon spacer analogue of ISO-1 ( 7 ). They have moderate binding affinity for sigma-2 ( σ 2 ) receptors and high selectivity for σ 2 receptors relative to sigma-1 ( σ 1 ) receptors. Methods ([ 18 F] 7 ) was synthesized and evaluated as a candidate ligand for positron emission (PET) imaging of the σ 2 receptor in tumors. Radioligand [ 18 F] 7 was radiolabeled with 18 F via displacement of the corresponding mesylate precursor with [ 18 F]fluoride. Cellular uptake study of [ 18 F] 7 was performed in EMT-6 tumor cell, and in vivo biodistribution study of [ 18 F] 7 and microPET imaging study of [ 18 F] 3 and [ 18 F] 7 carried out in female Balb/c mice bearing EMT-6 tumors. Results [ 18 F] 7 had a respectable tumor uptake (1.55%ID/g at 60 min post-injection) and high tumor/muscle ratios at 60 and 120 min post-injection. MicroPET imaging of [ 18 F] 7 in tumor-bearing mice as above showed significant tumor localization and a high tumor/muscle ratio as well. Conclusions These results are similar to or better than [ 18 F]ISO-1 ([ 18 F] 3 ), which indicates that [ 18 F] 7 has potential for imaging the σ 2 receptor status of solid tumors.

Background PARP inhibitors (PARPi) have the potential to impact cancer therapy in a selective patient population; however, despite current patient selection methods clinical trials have shown mixed response rates. It is therefore clinically useful to determine which patients will respond prior to receiving PARPi therapy. One essential biomarker is to measure the level of PARP enzyme expression in tumors. Small molecule radiotracers have been developed to accurately quantify PARP-1 expression in vitro and in vivo . [ 125 I] KX-02-019 is the first report of a radioiodinated analogue of the benzimidazole class of PARPi. Herein, we studied the pharmacological properties of [ 125 I] KX-02-019 as well as the in vivo biodistribution. Methods [ 125 I] KX-02-019 was evaluated in both cancer and non-cancer cell lines. We evaluated the pharmacologic properties of [ 125 I] KX-02-019 in live cells by measuring enzyme association and dissociation kinetics, saturation, and specificity. In addition, competitive inhibition experiments were carried out with commercially available PARPi. Protein expression was analyzed by Western blot to compare PARP-1 and PARP-2 expression across cell lines studied. The biodistribution was studied in a mouse EMT6 tumor model at time points of 0.5, 1, 2, 4 and 6h. Results [ 125 I] KX-02-019 showed subtle differences in pharmacological properties in the absence of PARP-2. In addition, [ 125 I] KX-02-019 was competitively displaced by clinical PARPi. In vivo biodistribution studies showed an increasing tumor to muscle ratio over 6h as well as fast clearance from healthy tissues. Conclusion [ 125 I] KX-02-019 has binding sites in both PARP1 KO cells as well as PARP2 KO cells showing higher affinity for PARP-2. This observation is supported by a decrease in binding affinity in PARP2 KO cells compared to PARP1 KO cells. The pharmacologic and biological properties of [ 125 I] KX-02-019 studied in vitro and in vivo showed that this analogue may be useful in determining pharmacokinetic and pharmacodynamic properties of clinical PARPi.

Introduction This paper reports the synthesis and labeling of 18 F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine–serine–cysteine preferring (ASC) transporter system. Methods Three new 18 F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labeling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[ 18 F]fluoromethyl)-L-alanine (L-[ 18 F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). Results New 18 F alanine derivatives were prepared with 7%–34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[ 18 F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than that observed for the other two alanine derivatives and [ 18 F]FDG in the first 1h. Inhibition of cell uptake studies suggested that L-[ 18 F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[ 18 F]FMA remained stable and was not incorporated into protein within 2h. In vivo biodistribution studies demonstrated that L-[ 18 F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[ 18 F]FMA in both 9L rat and transgenic mouse. Conclusion L-[ 18 F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor proliferation.

Recently, there has been a renewed interest in the study of tumor metabolism above and beyond the Warburg effect. Studies on cancer cell metabolism have provided evidence that tumor-specific activation of signaling pathways, such as the upregulation of the oncogene myc, can regulate glutamine uptake and its metabolism through glutaminolysis to provide the cancer cell with a replacement of energy source. Methods: We report a convenient procedure to prepare l-[5-11C]-glutamine. The tracer was evaluated in 9L and SF188 tumor cells (glioma and astrocytoma cell lines). The biodistribution of l-[5-11C]-glutamine in rodent tumor models was investigated by dissection and PET. Results: By reacting 11C-cyanide ion with protected 4-iodo-2-amino-butanoic ester, the key intermediate was obtained in good yield. After hydrolysis with trifluoroacetic and sulfonic acids, the desired optically pure l-[5-11C]-glutamine was obtained (radiochemical yield, 5% at the end of synthesis; radiochemical purity, >95%). Tumor cell uptake studies showed maximum uptake of l-[5-11C]-glutamine reached 17.9% and 22.5% per 100 μg of protein, respectively, at 60 min in 9L and SF188 tumor cells. At 30 min after incubation, more than 30% of the activity appeared to be incorporated into cellular protein. Biodistribution in normal mice showed that l-[5-11C]-glutamine had significant pancreas uptake (7.37 percentage injected dose per gram at 15 min), most likely due to the exocrine function and high protein turnover within the pancreas. Heart uptake was rapid, and there was 3.34 percentage injected dose per gram remaining at 60 min after injection. Dynamic small-animal PET studies in rats bearing xenografted 9L tumors and in transgenic mice bearing spontaneous mammary gland tumors showed a prominent tumor uptake and retention. Conclusion: The data demonstrated that this tracer was favorably taken up in the tumor models. The results suggest that l-[5-11C]-glutamine might be useful for probing in vivo tumor metabolism in glutaminolytic tumors.

A novel fluorine-18 prosthetic ligand, 5-(1,3-dioxolan-2-yl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)pyridine [(18)F]2, has been synthesized. The prosthetic ligand is formed in high radiochemical yield (rcy = 71 ± 2%, n = 3) with excellent radiochemical purity (rcp = 99 ± 1%, n = 3) in a short reaction time (10 min). [(18)F]2 is a small, neutral, organic complex, easily synthesized in four steps from a readily available starting material. It can be anchored onto a target molecule containing an aminooxy functional group under acidic conditions by way of an oxime bond. We report herein two examples [(18)F]23 and [(18)F]24, potential imaging agents for β-amyloid plaques, which were labeled with this prosthetic group. This approach could be used for labeling proteins and peptides containing an aminooxy group. Biodistribution in male ICR mice for both oxime labeled complexes [(18)F]23 and [(18)F]24 were compared to that of the known β-amyloid plaque indicator, [(18)F]-AV-45, florbetapir 1. Oximes [(18)F]23 and [(18)F]24 are larger in size and therefore should reduce the blood-brain barrier (BBB) penetration. The brain uptake for oxime [(18)F]23 appeared to be reduced, but still retained some capability to cross the BBB. Oxime [(18)F]24 showed promising results after 2 min post injection (0.48% dose/gram); however, the uptake increased after 30 min post injection (0.92% dose/gram) suggesting an in vivo decomposition/metabolism of compound [(18)F]24. We have demonstrated a general protocol for the fluoride-18 labeling with a new prosthetic ligand [(18)F]2 that is tolerant toward several functional groups and is formed via chemoselective oxime coupling.

Introduction Amino acids based tracers represent a promising class of tumor metabolic imaging agents with successful clinical applications. Two new phenylalanine derivatives, p -(2-[ 18 F]fluoroethyl)-l-phenylalanine (FEP, [ 18 F] 2 ) and p -(3-[ 18 F]fluoropropyl)-l-phenylalanine (FPP, [ 18 F] 3 ) were synthesized and evaluated in comparison to clinically utilized O -(2-[ 18 F]fluoroethyl)-l-tyrosine (FET, [ 18 F] 1 ). Methods FEP ([ 18 F] 2 ) and FPP ([ 18 F] 3 ) were successfully synthesized by a rapid and efficient two-step nucleophilic fluorination of tosylate precursors and deprotection reaction. In vitro cell uptake studies were carried out in 9L glioma cells. In vivo studies, 9L tumor xenografts were implanted in Fisher 344 rats. Results FEP ([ 18 F] 2 ) and FPP ([ 18 F] 3 ) could be efficiently labeled within 90 min with good enantiomeric purity (>95%), good yield (11–37%) and high specific activity (21–69 GBq/μmol). Cell uptake studies showed FEP had higher uptake than FPP as well as reference ligand FET ([ 18 F] 1 ). Uptake mechanism studies suggested that FEP is a selective substrate for system L and prefers its subtype LAT1. In vivo biodistribution studies demonstrated FEP had specific accumulation in tumor cells and tumor to background ratio reached 1.45 at 60 min. Small animal positron emission tomography (PET) imaging studies showed FEP was comparable to FET for imaging rats bearing 9L tumor model. FEP had high uptake in 9L tumor compared to surrounding tissue and was quickly excreted through urinary tract. Conclusion Biological evaluations indicate that FEP ([ 18 F] 2 ) is a potential useful tracer for tumor imaging with PET.

A versatile synthetic route to prepare all four stereoisomeric 4-fluoro-glutamines was developed by exploiting a Passerini three-component reaction. The skeleton of 4-substituted glutamine derivatives was efficiently constructed. Subsequent four-step reactions, highlighted by a "neutralized" TASF fluorination, provided the desired products with high yields and excellent optical purity. The optically pure fluorine-18 labeled 4-fluoroglutamines were also successfully prepared using either a 18-crown-6/KHCO(3) or K[222]/K(2)CO(3) catalysis system. Preliminary cell uptake and inhibition studies using the 9L tumor cells and SF188(Bcl-xL) tumor cells (a glutamine addicted tumor derived from glioblastoma) provided strong evidence for their potential application in conjunction with positron emission tomography (PET) for in vivo imaging of tumors, which use glutamine as an alternative energy source.

Introduction The utility of [ 18 F]FPBM [2-(2′-((dimethylamino)methyl)-4′-(3-[ 18 F]-fluoropropoxy)phenylthio)benzenamine], a selective serotonin transporter (SERT) tracer, and [ 18 F]AV-133 [(+)-2-Hydroxy-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-1,2,3,4,6,7-hexahydro-11bH-benzo[a]quinolizine], a selective vesicular monoamine transporter 2 (VMAT2) tracer, were tested in the 6-hydroxydopamine (6-OHDA) unilateral lesioned rat model. Methods Positron emission tomography (PET) imaging of three 6-OHDA unilateral lesioned male Sprague Dawley rats (Rats 1–3) were performed with [ 18 F]FPBM and [ 18 F]AV-133 to examine whether changes in SERT and VMAT2 binding, respectively, could be detected in the brain. The brains of the three rats were then removed and examined by in vitro autoradiography with [ 18 F]FPBM and the dopamine transporter ligand, [ 125 I]IPT [ N -(3′-[ 125 I]-iodopropen-2′-yl)-2-beta-carbomethoxy-3-beta-(4-chloro phenyl) tropane, for confirmation. Biodistribution of [ 18 F]FPBM in a separate group of p-chloroamphetamine (PCA) treated rats were also performed. Results PET image analysis showed varying levels of SERT binding reduction (Rat 1=-11%, Rat 2=-4%, Rat 3=-43%; n =2) and a clear and definitive loss of VMAT2 binding (Rat 1=-87%, Rat 2=-72%, and Rat 3=-91%; n =1) in the left striatum when compared to the right (non-lesioned side) striatum. The results from PET imaging were corroborated with quantitative in vitro autoradiography. Rats treated with a selective serotonin toxin (p-chloroamphetamine) showed a significant reduction of [ 18 F]FPBM uptake in the cortex and hypothalamus regions of the brain. Conclusion The preliminary data suggest that [ 18 F]FPBM and [ 18 F]AV-133 may be useful for the examination of serotonergic and dopaminergic neuron integrity, respectively, in the living brain.

The promise of human embryo stem cells (hESCs) for regenerative medicine is offset by the ethical and practical challenges involved in sourcing eggs and embryos for this objective. In this study we sought to isolate an hESC line from clinically failed eggs, the usage of which would not conflict with donor interests to conceive. A total of 8 blastocysts were allocated for hESC derivation from a pool of 579 eggs whose fertilization had been clinically assessed to have occurred abnormally (i.e., three pronuclei) or failed (i.e., no pronuclei) following in vitro insemination or intracytoplasmic sperm injection (ICSI). The latter were subjected to a recovery intervention consisting of either reinsemination by ICSI or parthenogenetic stimulation. One hESC line (RCM1) was obtained from a failed-to-fertilize inseminated egg recovered by parthenogenetic activation. Standard in vitro and in vivo characterization revealed this line to possess all of the properties attributed to a normal euploid hESC line. Whole-genome single-nucleotide polymorphism analysis further revealed that the line was biparental, indicating that sperm penetration had occurred, although parthenogenetic stimulation was required for activation. Our results demonstrate the viability of an alternative strategy to generate normal hESC lines from clinically failed eggs, thereby further minimizing the potential to conflict with donor reproductive interest to conceive.

Introduction The preliminary in vivo evaluation of novel 5-[ 18 F]fluoroalkyl-2′-deoxyuridines ([ 18 F]FPrDU, [ 18 F]FBuDU, [ 18 F]FPeDU; [ 18 F] 1a−c , respectively) and 2′-fluoro-2′-deoxy-5-[ 18 F]fluoroalkyl-1-β-d-arabinofuranosyl uracils ([ 18 F]FFPrAU, [ 18 F]FFBuAU, [ 18 F]FFPeAU; [ 18 F] 1d−f , respectively) as probes for imaging herpes simplex virus type 1 thymidine kinase (HSV1- tk ) gene expression is described. Methods [ 18 F] 1a−f were successfully synthesized by a rapid and efficient two-step one-pot nucleophilic fluorination reaction using 5- O -mesylate precursors and [ 18 F]F − . For in vivo studies, tumor xenografts were grown in nude mice by implanting RG2 cells stably expressing HSV1- tk (RG2TK+) and wild-type cells (RG2). Results Biodistribution studies at 2 h pi revealed that the uptake of [ 18 F] 1a−b and [ 18 F] 1d−e in RG2TK+ tumors was not significantly different from control tumors. However, [ 18 F] 1c and [ 18 F] 1f had an average 1.6- and 1.7-fold higher uptake in RG2TK+ tumors than control RG2 tumors. Blood activity curves for [ 18 F] 1c and [ 18 F] 1f highlight rapid clearance of radioactivity in the blood. Dynamic small animal PET (A-PET) imaging studies of tumor-bearing mice with [ 18 F] 1c and [ 18 F] 1f showed higher initial uptake (3.5- and 1.4-fold, respectively) in RG2TK+ tumors than in control tumors, with continued washout of activity from both tumors over time. Conclusions Biological evaluations suggest that [ 18 F] 1c and [ 18 F] 1f may have limited potential for imaging HSV1- tk gene expression due to fast washout of activity from the blood, thus significantly decreasing sensitivity and specificity of tracer accumulation in HSV1- tk -expressing tumors.

Objectives Development of imaging agents for pancreatic beta cell mass may provide tools for studying insulin-secreting beta cells and their relationship with diabetes mellitus. In this paper, a new imaging agent, [ 18 F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1- a ]isoquinoline [ 18 F](+) 4 , which displays properties targeting vesicular monoamine transporter 2 (VMAT2) binding sites of beta cells in the pancreas, was evaluated as a positron emission tomography (PET) agent for estimating beta cell mass in vivo. The hydrolyzable epoxide group of (+) 4 may provide a mechanism for shifting biodistribution from liver to kidney, thus reducing the background signal. Methods Both 18 F- and 19 F-labeled (+) and (−) isomers of 4 were synthesized and evaluated. Organ distribution was carried out in normal rats. Uptake of [ 18 F](+) 4 in pancreas of normal rats was measured and correlated with blocking studies using competing drugs, (+)dihydrotetrabenazine [(+)-DTBZ] or 9-fluoropropyl-(+)dihydro tetrabenazine [FP-(+)-DTBZ, (+) 2 ]. Results In vitro binding study of VMAT2 using rat brain striatum showed a K i value of 0.08 and 0.15 nM for the (+) 4 and (±) 4 , respectively. The in vivo biodistribution of [ 18 F](+) 4 in rats showed the highest uptake in the pancreas (2.68 %ID/g at 60 min postinjection). In vivo competition experiments with cold FP-(+)-DTBZ, (+) 2 , (3.5 mg/kg, 5 min iv pretreatment) led to a significant reduction of pancreas uptake (85% blockade at 60 min). The inactive isomer [ 18 F](−) 4 showed significantly lower pancreas uptake (0.22 %ID/g at 30 min postinjection). Animal PET imaging studies of [ 18 F](+)4 in normal rats demonstrated an avid pancreatic uptake in rats. Conclusion The preliminary results suggest that the epoxide, [ 18 F](+) 4 , is highly selective in binding to VMAT2 and it has an excellent uptake in the pancreas of rats. The liver uptake was significantly reduced through the use of the epoxide group. Therefore, it may be potentially useful for imaging beta cell mass in the pancreas.

Recent studies on gene expression of β-cell mass (BCM) in the pancreas showed that vesicular monoamine transporter 2 (VMAT2) is highly expressed in the BCM (mainly in the islets of Langerhans). Imaging pancreatic BCM may provide an important tool for understanding the relationship between loss of insulin-secreting β-cells and onset of diabetes mellitus. In this article, 9-fluoropropyl-(+)-dihydrotetrabenazine (FP-(+)-DTBZ), which is a VMAT2 imaging agent, was evaluated as a PET agent for estimating BCM in vivo. Methods: Organ biodistribution after an intravenous injection of 18F-FP-(+)-DTBZ (active isomer) and 18F-FP-(−)-DTBZ (inactive isomer) was evaluated in normal rats. The specificity of uptake of 18F-FP-(+)-DTBZ was assessed by a pretreatment (3.8 mg of (+)-DTBZ per kilogram and 3.5 mg of FP-(+)-DTBZ per kilogram, intravenously, 5 min prior) or coadministration (2 mg of (+)-DTBZ per kilogram). PET studies were performed in normal rats. Results: The in vivo biodistribution of 18F-FP-(+)-DTBZ in rats showed the highest uptake in the pancreas (5% dose/g at 30 min after injection), whereas 18F-FP-(−)-DTBZ showed a very low pancreas uptake. Rats pretreated with FP-(+)-DTBZ displayed a 78% blockade of pancreas uptake. PET studies in normal rats demonstrated an avid pancreas uptake of 18F-FP-(+)-DTBZ. Conclusion: The preliminary data obtained with 18F-FP-(+)-DTBZ suggest that this fluorinated derivative of DTBZ shows good pancreas specificity and has the potential to be useful for quantitative measurement of VMAT2 binding sites reflecting BCM in the pancreas.

Introduction A new 18 F ligand, 2-(2′-((dimethylamino)methyl)-4′-(3-[ 18 F]fluoropropoxy)-phenylthio)benzenamine ([ 18 F] 1 ), for positron emission tomography (PET) imaging of serotonin transporters (SERT) was evaluated. Methods Binding affinity was determined through in vitro binding assays with LLC-PK1 cells overexpressing SERT, NET or DAT (LLC-SERT, LLC-NET and LLC-DAT) and with rat cortical homogenates. Localization and selectivity of [ 18 F] 1 binding in vivo were evaluated by biodistribution, autoradiography and A-PET imaging studies in rats. Results This compound displayed excellent binding affinity for SERT in vitro with K i =0.33 and 0.24 nM in LLC-SERT and rat cortical homogenates, respectively. Biodistribution studies with [ 18 F] 1 showed good brain uptake (1.61% dose/g at 2 min postinjection), high uptake into the hypothalamus (1.22% dose/g at 30 min) and a high target-to-nontarget (hypothalamus to cerebellum) ratio of 9.66 at 180 min postinjection. Pretreatment with a SERT selective inhibitor considerably inhibited [ 18 F] 1 binding in biodistribution studies. Ex vivo autoradiography reveals [ 18 F] 1 localization to brain regions with high SERT density, and this binding was blocked by pretreatment with SERT selective inhibitors. Small animal PET (A-PET) imaging in rats provided clear images of tracer localization in the thalamus, midbrain and striatum. In A-PET chasing experiments, injecting a SERT selective inhibitor 75 min post-tracer injection causes a dramatic reduction in regional radioactivity and the target-to-nontarget ratio. Conclusion The results of the biological studies and the ease of radiosynthesis with moderately good radiochemical yield (RCY=10–35%) make [ 18 F] 1 an excellent candidate for SERT PET imaging.

Alterations in serotonin and norepinephrine neuronal functions have been observed in patients with major depression. Several antidepressants bind to both serotonin transporters and norepinephrine transporters (NET). The ability to image NET in the human brain would be a useful step toward understanding how alterations in NET relate to disease. In this study, we report the synthesis and characterization of a new series of derivatives of iodonisoxetine, a known radioiodinated probe. The most promising, ( R )- N -methyl-3-(3-iodopyridin-2-yloxy)-3-phenylpropylamine (PYINXT), displayed a high and saturable binding to NET, with a K d value of 0.53±0.03 nM. Biodistribution studies of ( R )- N -methyl-3-(3- 125 I-pyridin-2-yloxy)-3-phenylpropan-1-amine in rats showed moderate initial brain uptake (0.54% dose/organ at 2 min) with a relatively fast washout from the brain (0.16% dose/organ at 2 h) as compared to [ 125 I]INXT. The hypothalamus (a NET-rich region)-to-striatum (a region devoid of NET) ratio was found to be 2.14 at 4 h after intravenous injection. Preliminary results suggest that this improved iodinated ligand, when labeled with 123 I, may be useful for mapping NET-binding sites with single photon emission computed tomography in the living human brain.

The nuclear enzyme PARP1 plays a central role in sensing DNA damage and facilitating repair. Tumors with BRCA1/2 mutations are highly dependent on PARP1 as an alternative mechanism for DNA repair, and PARP inhibitors generate synthetic lethality in tumors with BRCA mutations, resulting in cell cycle arrest and apoptosis. Zhou et al. recently synthesized an (18)F-labeled PARP1 inhibitor ([(18)F]FluorThanatrace) for PET, and demonstrated high specific tracer uptake in a xenograft model of breast cancer [1]. In the current study, we characterize the level of baseline PARP expression and activity across multiple human breast cancer cell lines, including a BRCA1 mutant line. PARP expression and activity, as measured by levels of PAR and PARP1, is correlated with in vitro [(18)F]FluorThanatrace binding as well as tracer uptake on PET in a xenograft model of breast cancer. Radiotracer uptake in genetically-engineered mouse fibroblasts indicates [(18)F]FluorThanatrace is selective for PARP1 versus other PARP enzymes. This motivates further studies of [(18)F]FluorThanatrace as an in vivo measure of PARP1 expression and activity in patients who would benefit from PARP inhibitor therapy.

Rationale The sigma-2 receptor is a protein with a Heme binding region and is capable of receptor-mediated endocytosis. It is overexpressed in many cancers making it a potential vector for therapeutic drug delivery. Our objective was to introduce an alpha-emitting radionuclide, astatine-211, into a selective sigma-2 ligand moiety to provide cytotoxic capabilities without adversely altering the pharmacological characteristics. In this study we investigated the in vitro/in vivo tumor targeting and estimated dosimetry of alpha-emitting sigma-2 ligand, 5-(astato- 211 At)-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2,3-dimethoxybenzamide ( 211 At-MM3), in a pre-clinical human breast cancer model. Methods Astatine-211 was produced in a cyclotron and isolated by dry distillation. Radiosynthesis of 211 At-MM3 was performed using a tin precursor through radioastatodestannylation. In vitro sigma-2 binding experiments using 211 At-MM3 were carried out in live EMT6 and MDA-MB-231 breast cancer cells and liver homogenate tissue. In vivo biodistribution experiments were performed using EMT6 mouse breast cancer cells in BALB/c female mice. Approximately 370kBq of 211 At-MM3 was administered intravenously and at time points of 5min, 1, 2, 4, 8, and 24h organs/tissue were harvested. Estimated human dosimetry was extrapolated from biodistribution data using OLINDA/EXM (VU e-Innovations). Results Astatine-211 was successfully produced and isolated in quantities suitable for in vitro and small animal in vivo experiments. Radiosynthesis of 211 At-MM3 was reproducible with high radiochemical purity. Astatine-211-MM3 exhibited picomolar affinity to the sigma-2 receptor in contrast to the iodinated analog that had nanomolar affinity. Prolonged tumor targeting was measured through biodistribution studies with a maximal tumor to muscle ratio of 9.02 at 4h. Estimated human dosimetry revealed doses of up to 370MBq in an adult female patient were below organ radiation limits with the potential to provide a high therapeutic dose to tumors. Conclusion The sigma-2 receptor could serve as a suitable targeting platform for designing radiotherapeutics. 211 At-MM3 showed tumor targeting properties in vitro/in vivo and favorable estimated human dosimetry establishing the proof of concept for future development as a radiotherapeutic for the treatment of breast cancer.

Objective To compare the effect of prophylactic laparoscopic salpingectomy versus division of the fallopian tubes on ovarian response to gonadotropins in women undergoing IVF. Design Retrospective study. Setting National Health Service-based tertiary referral center for reproductive medicine. Patient(s) One hundred sixty-eight women with tubal factor infertility. Sixty-five women with hydrosalpinges had either salpingectomy (n = 40, group A) or proximal tubal division (n = 25, group B), while the remaining women with tubal disease but without hydrosalpinges acted as the control group (n = 103, group C). Intervention(s) Prophylactic laparoscopic salpingectomy or proximal division of the fallopian tubes and ovarian stimulation with gonadotropins for IVF. Main Outcome Measure(s) Day 2 serum FSH levels before surgery and 3 months after surgery but before ovarian stimulation, ovarian response assessed as total dose of hMG administered, serum E 2 concentrations on day 3 and day 8 of stimulation and on the day of hCG injection, number of follicles, and number of oocytes retrieved and fertilized. Result(s) In group A, baseline FSH levels were significantly raised after surgery compared with before surgery. Postsurgery FSH concentrations were significantly higher in group A compared with group B. The number of follicles (15–20 mm) was significantly lower in group A compared with group B and group C. The serum E 2 levels on day 8 of stimulation were lower in group A compared with group B, and on the day of hCG injection it was significantly reduced in group A compared with groups B and C. The number of oocytes retrieved per cycle was significantly lower in group A compared with group B. There were no significant differences in pregnancy rates and miscarriage rates among the three groups. Conclusion(s) These findings suggest that prophylactic salpingectomy in women with hydrosalpinx may compromise ovarian response to stimulation without affecting pregnancy rates. A randomized control trial is recommended to determine the most appropriate laparoscopic procedure in the management of hydrosalpinx before IVF.

Objective To assess the implantation, pregnancy, and live birth rates after the transfer of frozen-thawed embryos (FET) in a natural or hormonal control cycle. Design Retrospective study. Setting National Health Service tertiary referral center for reproductive medicine in Manchester, United Kingdom. Patient(s) Two comparable groups of women with regular menstrual cycles: Group A (n = 212) had FET in a natural cycle after spontaneous ovulation; group B (n = 205) had FET in a pituitary-desensitized hormonally controlled cycle. Intervention(s) In group B, GnRH agonist was commenced in the midluteal phase of the previous cycle and discontinued 3 days before embryo transfer. Oral estradiol valerate and vaginal progesterone pessary were used to prepare the endometrium. Embryo transfer was carried out 3 days after detection of the endogenous LH surge in group A and on day 3 of progesterone administration in group B. Main Outcome Measure(s) Implantation, pregnancy, and live birth rates per cycle and per embryo transfer (ET). Result(s) In the 212 women who had natural-cycle FET, 172 ETs were performed and 247 embryos replaced. The implantation rate was 14.1% (35/247). Twenty clinical pregnancies (20/172, 11.6%) were achieved. In the 205 women who had down-regulated hormone replacement–cycle FET, 173 embryo transfers were performed and 243 embryos replaced. The implantation rate was 13.5% (33/243). Eighteen clinical pregnancies (18/173, 10.2%) were achieved. There were no significant differences between the two groups with regard to the implantation, clinical pregnancy, or live birth rates per cycle or per ET. Conclusion(s) These findings suggest that both FET protocols are equally effective in terms of implantation rate and pregnancy outcome in women with regular menstrual cycles.

Ovarian hyperstimulation syndrome (OHSS) is a serious and potentially life-threatening complication following ovarian stimulation for in vitro fertilization (IVF). Coasting is the practice whereby the gonadotrophins are withheld and the administration of human chorionic gonadotrophin (hCG) is delayed until serum oestradiol (E2) has decreased to what is considered to be a safe level, to prevent the onset of OHSS. This study aimed to assess the length of coasting on the reproductive outcome in women at risk of developing OHSS. Coasting was undertaken when the serum E2 concentrations wereor = 17000 pmol/L but21000 pmol/L. Daily E2 measurements were performed and hCG was administered when hormone levels decreased to17000 pmol/L. Eighty-one women who had their stimulation cycles coasted were grouped according to the number of coasting days. Severe OHSS occurred in one case, which represented 1.2% of patients who underwent coasting because of an increased risk of developing the syndrome. No difference was found between cycles coasted for 1 - 3 days and cycles coasted foror = 4 days in terms of oocyte maturity, fertilization and embryo cleavage rates. Women in whom coasting lasted foror = 4 days had significantly fewer oocytes retrieved (P0.05) and decreased implantation rate (P0.05) compared to those coasted for 1 - 3 days. Pregnancy rate/embryo transfer and live birth rate did not differ between groups. In conclusion, coasting appears to decrease the risk of OHSS without compromising the IVF cycle pregnancy outcome. Prolonged coasting is, however, associated with reduced implantation rates, perhaps due to the deleterious effects on the endometrium rather than the oocytes.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.

Between 1978 and 1990, 122 men underwent semen analysis before starting sterilising chemotherapy for Hodgkin's disease. Eighty-one (66%) had semen quality within the normal range, 25 were oligospermic (

The major stumbling block for egg donation lies in the recruitment of sufficient suitable donors. This study ascertained the views of egg donors in the UK by analysing 113 completed questionnaires that asked questions about demographics, stimulus to donate, support network available, ethics, the 'process' of donation and payment. Ideas for future recruitment were also sought. The mean age of donors was 31.7 years, and most donors were donating for the first time. Ninety-one per cent of donors were Caucasian and 93% had children of their own. Ninety-six (85%) donors felt fully supported in their decision to become an egg donor and 60 (53%) discussed their donation with their GP. Information regarding egg donation came from many sources. The main motivation to donate was a desire to help childless couples. Many respondents had themselves suffered, or knew of couples with, infertility. Eighty-three (73.5%) respondents felt that expenses alone should be paid to egg donors, and many expressed concerns that large financial incentives may attract the 'wrong women' to donate. Forty-nine (43%) respondents found the procedure painful or stressful in some way, although 95% had no regrets concerning their donation, and 72% would donate again. A common reason for donors not wishing to donate again was age restriction. Respondents were asked their opinion with regard to recruitment and the enthusiasm they expressed needs to be harnessed if the current shortcomings in available donors are to be overcome. Specific recommendations to achieve this are made.

A series of tetrahydroindazole derivatives were synthesized and evaluated for their affinities for both sigma-1 and sigma-2 receptors. These compounds are hybrid structures of a tetrahydroindazole substituted benzamide and a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety or a 9-azabicyclo[3.3.1]nonan-3-yl-amine moiety. These newly synthesized hybrid analogs showed various affinities for sigma-2 receptor without any significant affinities for sigma-1 receptor. In particular, compounds 12, 15b, 15c, and 15d, demonstrated moderate affinity and excellent selectivity for sigma-2 receptor. It is interesting to note that 3–5 carbon units between the tetrahydroindazole substituted benzamide and the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety are appropriate for sigma-2 receptor binding. No substitution on the 9-aza nitrogen is proper for sigma-2 affinity in the 2-(9-azabicyclo[3.3.1]nonan-3-yl-amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide analogs.