A phase 1 study of PF-06840003, an oral indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor in patients with recurrent malignant glioma.

Background PF-06840003 is a highly selective indoleamine 2, 3-dioxygenase (IDO1) inhibitor with antitumor effects in preclinical models. This first-in-human phase 1 study evaluated safety, pharmacokinetics/pharmacodynamics, and preliminary efficacy in recurrent malignant glioma to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Methods Patients (N = 17) received oral PF-06840003 in four dose-escalation groups: 125 mg once-daily (QD; n = 2); 250 mg QD (n = 4); 250 mg twice-daily (BID; n = 3); 500 mg BID (n = 8). A modified toxicity probability interval method determined the MTD. Results Four patients experienced serious adverse events (SAEs); one with treatment-related SAEs (grade 4 alanine and aspartate aminotransferase elevations). The dose-limiting toxicity (DLT) rate at 500 mg BID was 12.5% (n = 1/8); the MTD was not reached. Following PF-06840003 dosing, median time to maximum plasma concentration for the active enantiomer PF-06840002 was 1.5-3.0 hr and mean elimination half-life was 2 to 4 hr (Cycle 1 Day 1). Urinary recovery of PF-06840002 was low (< 1%). At 500 mg BID, maximum mean percentage inhibition of 13C10 kynurenine vs endogenous kynurenine was 75% vs 24%. PF-06840002 CSF-to-plasma ratio was 1.00. Disease control occurred in eight patients (47%). Mean duration of stable disease (SD) was 32.1 (12.1-72.3) weeks. Two patients with SD discontinued the study at 450 and 561 days and continued PF-06840003 on compassionate use. Conclusion PF­06840003 up to 500 mg BID was generally well tolerated with evidence of a pharmacodynamic effect and durable clinical benefit in a subset of patients with recurrent malignant glioma. ClinicalTrials.gov, NCT02764151, registered April 2016.

Spatio-temporal simulation of first pass drug perfusion in the liver.

The liver is the central organ for detoxification of xenobiotics in the body. In pharmacokinetic modeling, hepatic metabolization capacity is typically quantified as hepatic clearance computed as degradation in well-stirred compartments. This is an accurate mechanistic description once a quasi-equilibrium between blood and surrounding tissue is established. However, this model structure cannot be used to simulate spatio-temporal distribution during the first instants after drug injection. In this paper, we introduce a new spatially resolved model to simulate first pass perfusion of compounds within the naive liver. The model is based on vascular structures obtained from computed tomography as well as physiologically based mass transfer descriptions obtained from pharmacokinetic modeling. The physiological architecture of hepatic tissue in our model is governed by both vascular geometry and the composition of the connecting hepatic tissue. In particular, we here consider locally distributed mass flow in liver tissue instead of considering well-stirred compartments. Experimentally, the model structure corresponds to an isolated perfused liver and provides an ideal platform to address first pass effects and questions of hepatic heterogeneity. The model was evaluated for three exemplary compounds covering key aspects of perfusion, distribution and metabolization within the liver. As pathophysiological states we considered the influence of steatosis and carbon tetrachloride-induced liver necrosis on total hepatic distribution and metabolic capacity. Notably, we found that our computational predictions are in qualitative agreement with previously published experimental data. The simulation results provide an unprecedented level of detail in compound concentration profiles during first pass perfusion, both spatio-temporally in liver tissue itself and temporally in the outflowing blood. We expect our model to be the foundation of further spatially resolved models of the liver in the future.

Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues.

PURPOSE: Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the (125)iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer (125)I-GLP-1(7-36)amide. METHODS: Receptor autoradiography studies with (125)I-GLP-1(7-36)amide agonist or (125)I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. RESULTS: The antagonist (125)I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer (125)I-GLP-1(7-36)amide. For comparison, (125)I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. CONCLUSION: The GLP-1 receptor antagonist exendin(9-39) labelled with (125)I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients.

Laurate permeabilizes the paracellular pathway for small molecules in the intestinal epithelial cell model HT-29/B6 via opening the tight junctions by reversible relocation of claudin-5. [Corrected].

PURPOSE: To mechanistically analyze effects of the medium-chain fatty acid laurate on transepithelial permeability in confluent monolayers of the intestinal epithelial cell line HT-29/B6, in context with an application as an absorption enhancer improving transepithelial drug permeation. METHODS: Transepithelial resistance and apparent permeability for paracellular flux markers was measured using Ussing-type chambers. Two-path impedance spectroscopy was employed to differentiate between transcellular and paracellular resistance, and confocal imaging and Western blotting was performed. RESULTS: Laurate resulted in a substantial and reversible decrease in transepithelial resistance by 50% which was attributed to a decrease in paracellular resistance. Simultaneously, an increase in permeability for fluorescein (330 Da) was detected, while permeabilities for 4 kDa FITC-dextran and sulpho-NHS-SS-biotin (607 Da) remained unaltered. Confocal laser-scanning microscopy revealed a marked reduction of claudin-5, while other tight junction proteins including tricellulin, a protein preventing the paracellular passage of macromolecules, were not affected. CONCLUSIONS: Laurate induces an increase in paracellular permeability for molecules up to a molecular mass of 330 Da by retrieval of claudin-5 from tight junctions without affecting tricellular contacts and the paracellular passage of macromolecules. We hereby provide, for the first time, a mechanistical explanation of laurate-induced permeability enhancement on molecular level.

Oxalic acid supported Si-18F-radiofluorination: one-step radiosynthesis of N-succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for protein labeling.

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)⊂2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)⊂2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a ß-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.

N-Succinimidyl 4-[(18)F]-fluoromethylbenzoate-labeled dimeric RGD peptide for imaging tumor integrin expression.

RGD peptides, radiolabeled with (18)F, have been used in the clinic for PET imaging of tumor angiogenesis in cancer patients. RGD peptides are typically labeled using a prosthetic group such as N-succinimidyl 4-[(18)F]-fluorobenzoate ([(18)F]SFB) or 4-nitrophenyl 2-[(18)F]-fluoropropionate ([(18)F]NPFP). However, the complex radiosynthetic procedures have impeded their broad application in clinical studies. We previously radiolabeled proteins and peptides with the prosthetic group, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB), which was prepared in a simple one-step procedure. In this study, we labeled a PEGylated cyclic RGD peptide dimer, PEG(3)-E[c(RGDyK)](2) (PRGD2), using [(18)F]SFMB and evaluated for imaging tumor αvß3 integrin expression with positron emission tomography (PET). [(18)F]SFMB was prepared in one step using [(18)F]fluoride displacement of a nitrobenzenesulfonate leaving group under mild reaction conditions followed by HPLC purification. The (18)F-labeled peptide, [(18)F]FMBPRGD2 was prepared by coupling PRGD2 with [(18)F]SFMB in pH 8.6 borate buffer and purified with HPLC. The direct labeling on BMBPRGD2 was also attempted. A Siemens Inveon PET was used to image the uptake of the [(18)F]FMBPRGD2 into a U87MG xenograft mouse model. [(18)F]FMBPRGD2, was prepared with a 15% overall radiochemical yield (uncorrected) in a total synthesis time of 90 min, which was considerably shorter than the preparation of [(18)F]SFB- and [(18)F]NPFP-labeled RGD peptides. The direct labeling, however, was not successful. High quality microPET images using [(18)F]FMBPRGD2 clearly visualized tumors by 15 min with good target to background ratio. Early tracer accumulation in the bladder suggests fast renal clearance. No obvious bone uptake can be detected even at 4-h time point indicating that fluorine attachment is stable in mice. In conclusion, N-succinimidyl 4-[(18)F]-fluoromethylbenzoate ([(18)F]SFMB) prosthetic group can be a good alternative for labeling RGD peptides to image αvß3 integrin expression and for labeling other peptides.

Interpretation of cellular proliferation data: avoid the panglossian.

There are several statistics that may be calculated to characterize a cellular proliferation experiment. By far, the most commonly-reported statistic is the percent of cells in the final culture that have divided; however, this statistic has significant limitations. Other statistics provided by software modeling provide a much richer characterization of the biological response; however, their use also comes with caveats. Here, I discuss the practical application of these statistics, including their limitations and interdependencies, using hypothetical data. The goal of this perspective is to prevent the blind reliance or overly optimistic ("panglossian") interpretation of the statistics generated by software, so that researchers and reviewers have a more-informed basis for drawing conclusions from the data.

Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention.

Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated.

Interaction and uptake of exosomes by ovarian cancer cells.

BACKGROUND: Exosomes consist of membrane vesicles that are secreted by several cell types, including tumors and have been found in biological fluids. Exosomes interact with other cells and may serve as vehicles for the transfer of protein and RNA among cells. METHODS: SKOV3 exosomes were labelled with carboxyfluorescein diacetate succinimidyl-ester and collected by ultracentrifugation. Uptake of these vesicles, under different conditions, by the same cells from where they originated was monitored by immunofluorescence microscopy and flow cytometry analysis. Lectin analysis was performed to investigate the glycosylation properties of proteins from exosomes and cellular extracts. RESULTS: In this work, the ovarian carcinoma SKOV3 cell line has been shown to internalize exosomes from the same cells via several endocytic pathways that were strongly inhibited at 4°C, indicating their energy dependence. Partial colocalization with the endosome marker EEA1 and inhibition by chlorpromazine suggested the involvement of clathrin-dependent endocytosis. Furthermore, uptake inhibition in the presence of 5-ethyl-N-isopropyl amiloride, cytochalasin D and methyl-beta-cyclodextrin suggested the involvement of additional endocytic pathways. The uptake required proteins from the exosomes and from the cells since it was inhibited after proteinase K treatments. The exosomes were found to be enriched in specific mannose- and sialic acid-containing glycoproteins. Sialic acid removal caused a small but non-significant increase in uptake. Furthermore, the monosaccharides D-galactose, α-L-fucose, α-D-mannose, D-N-acetylglucosamine and the disaccharide ß-lactose reduced exosomes uptake to a comparable extent as the control D-glucose. CONCLUSIONS: In conclusion, exosomes are internalized by ovarian tumor cells via various endocytic pathways and proteins from exosomes and cells are required for uptake. On the other hand, exosomes are enriched in specific glycoproteins that may constitute exosome markers. This work contributes to the knowledge about the properties and dynamics of exosomes in cancer.

Noninvasive in vivo monitoring of the biofate of 195 kDa poly(vinyl alcohol) by multispectral fluorescence imaging.

A comprehensive knowledge of the in vivo fate of polymers is essential for their potential application in humans. In this study, the body distribution, accumulation, and elimination processes of intraperitoneally (ip) administered poly(vinyl alcohol) (PVA) in mice were investigated in detail. Two derivatives of PVA (195 kDa) having covalently bound fluorescent dye labels were synthesized and used to follow PVA in vivo by noninvasive multispectral fluorescence imaging over several months. Detailed ex vivo fluorescence imaging was performed additionally and combined with tissue accumulation studies using confocal microscopy. Filtration and confocal imaging at appropriate synthetic membranes, used as models for glomerular filtration, confirmed a considerable PVA permeation. This investigation yields new scientific findings about the fate of PVA in vivo. PVA accumulated in fat tissue at high levels, which suggests that PVA is suitable not only for abdominal surgeries but also for controlled release applications after ip or subcutaneous injection.

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model.

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

Quantitative analysis of Her2 receptor expression in vivo by near-infrared optical imaging.

Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancers is associated with poor prognosis and resistance to therapy. Current techniques for estimating this important characteristic use ex vivo assays that require tissue biopsies. We suggest a novel noninvasive method to characterize HER2 expression in vivo, using optical imaging, based on HER2-specific probes (albumin-binding domain-fused-(ZHER2:342)2-Cys Affibody molecules [Affibody AB, Solna, Sweden], labeled with Alexa Fluor 750 [Molecular Probes, Invitrogen, Carlsbad, CA]) that could be used concomitantly with HER2-targeted therapy. Subcutaneous tumor xenografts, expressing different levels of HER2, were imaged with a near-infrared fluorescence small-animal imaging system at several times postinjection of the probe. The compartmental ligand-receptor model was used to calculate HER2 expression from imaging data. Correlation between HER2 amplification/overexpression in tumor cells and parameters, directly estimated from the sequence of optical images, was observed (eg, experimental data for BT474 xenografts indicate that initial slope, characterizing the temporal dependence of the fluorescence intensity detected in the tumor, linearly depends on the HER2 expression, as measured ex vivo by an enzyme-linked immunosorbent assay for the same tumor). The results obtained from tumors expressing different levels of HER2 substantiate a similar relationship between the initial slope and HER2 amplification/overexpression. This work shows that optical imaging, combined with mathematical modeling, allows noninvasive monitoring of HER2 expression in vivo.

Development of double-stranded siRNA labeling method using positron emitter and its in vivo trafficking analyzed by positron emission tomography.

Pharmacokinetic study of small interfering RNA (siRNA) is an important issue for the development of siRNAs for use as a medicine. For this purpose, a novel and favorable positron emitter-labeled siRNA was prepared by amino group-modification using N-succinimidyl 4-[fluorine-18] fluorobenzoate ([(18)F]SFB), and real-time analysis of siRNA trafficking was performed by using positron emission tomography (PET). Naked [(18)F]-labeled siRNA or cationic liposome/[(18)F]-labeled siRNA complexes were administered to mice, and differential biodistribution of the label was imaged by PET. The former was cleared quite rapidly from the bloodstream and excreted from the kidneys; but in contrast, the latter tended to accumulate in the lungs. We also confirmed the biodistribution of fluorescence-labeled naked siRNA and cationic liposome/siRNA complexes by use of a near-infrared fluorescence imaging system. As a result, a similar biodistribution was observed, although quantitative data were obtained only by planar positron imaging system (PPIS) analysis but not by fluorescence in vivo imaging. Our results indicate that PET imaging of siRNA provides important information for the development of siRNA medicines.

Chromatographic and computational screening of anisotropic lipophilicity and pharmacokinetics of newly synthesized 1-aryl-3-ethyl-3-methylsuccinimides.

The present study is focused on a series of newly synthesized 1-aryl-3-ethyl-3-methylsuccinimide derivatives, as potential anticonvulsants. The retention behavior of eleven succinimide derivatives was determined by using reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase high performance thin layer chromatography (RP-HPTLC). The estimated retention behavior was correlated with partition (logP) and distribution coefficients (logD). These high correlations pointed out that the determined retention parameters (logk0 and RM0) can be considered chromatographic (anisotropic) lipophilicity of the studied succinimide derivatives. The structural properties, which dominantly affect the chromatographic lipophilicity, were determined as well. The significant correlations between the chromatographic lipophilicity and plasma protein binding (PPB), Madin-Darby Canine Kidney (MDCK) cells permeability, volume of distribution (Vd) and absorption constant (Ka) indicate the strong influence of lipophilicity on pharmacokinetics of 1-aryl-3-ethyl-3-methylsuccinimide derivatives. These derivatives have also been tested applying Comprehensive Medicinal Chemistry (CMC) drug-like rules which confirmed their drug-like properties. Besides, their blood-brain penetration (BBB) ability has been estimated applying the set of Clark's rules and by using Pre-ADMET software. Regarding toxicity, it was predicted that only one compound from the set might have toxic effects by blocking the hERG potassium channel. The present study reveals which molecular features in the structure of novel succinimide derivatives could be crucial for their lipophilicity, and consequently for their pharmacokinetic properties. The results indicate that the newly synthesized series of succinimide derivatives should be further considered in design of novel anticonvulsants.

Novel radiopharmaceutical (Technetium-99m)-(DOTA-NHS-ester)-Methionine as a SPECT-CT tumor imaging agent.

Breast cancer is the most common type of cancer in women worldwide. There have been many efforts for early breast cancer detection and among them molecular imaging have been extremely of high importance. Single-photon emission computed tomography (SPECT/CT) is a kind of imaging technique able to reveal crucial information with using radiopharmaceuticals. In this study, Technetium-99m-(DOTA-NHS-ester)-Methionine radiopharmaceutical was synthesized. Between 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-HNS ester) (MACROCYCLICS, DOTA-NHS ester, Plano, Texas, USA) and methionine(marker) were conjugated. The DOTA-HNS ester-Methionine was labeled with Technetium-99m (Inter-Medical, Technetium-99m, Bergamo, Italy). The synthesized radiopharmaceutical was used in SPECT/CT imaging for breast cancer diagnosis. For radiopharmaceutical evaluation, MTT assay for cellular toxicity, biodistribution, cellular uptake and radiochemical purity were employed.Technetium-99m-(DOTA-NHS-ester)-Methionine radiochemical had less cellular toxicity in human embryonic kidney cells 293 cell line (HEK293). Cellular uptake was indicated higher percent with use of Methionine as a marker, and radiochemical purity was high. Based on the results Technetium-99m-(DOTA-NHS-ester)-Methionine radiochem may be a better option for early detection of breast cancer. Further study is recommended to confirm these findings in clinical practice.

Imaging and pharmacokinetics of (64)Cu-DOTA-HB22.7 administered by intravenous, intraperitoneal, or subcutaneous injection to mice bearing non-Hodgkin's lymphoma xenografts.

PURPOSE: The aim of the study is to compare the tumor-specific targeting, pharmacokinetics, and biodistribution of (64)Cu-DOTA-HB22.7 when administered to xenograft-bearing mice intravenously (IV), intraperitoneally (IP), and subcutaneously (SQ). PROCEDURES: Mice bearing human non-Hodgkin's lymphoma (NHL) xenografts were injected IV, IP, or SQ with (64)Cu-DOTA-HB22.7. Xenograft targeting was evaluated by micro positron emission tomography (microPET) and confirmed by organ biodistribution studies. Blood measurements of (64)Cu were performed to determine the pharmacokinetics and clearance of (64)Cu-DOTA-HB22.7. RESULTS: (64)Cu-DOTA-HB22.7 demonstrated equivalent tumor targeting within 24-48 h, regardless of the route of administration. Organ biodistribution confirmed tumor-specific targeting. Blood pharmacokinetics demonstrated that (64)Cu-DOTA-HB22.7 accessed the bloodstream after IP and SQ administration to a similar degree as IV administration, albeit at a slower rate. CONCLUSIONS: These findings establish (64)Cu-DOTA-HB22.7 as a potential radioimmunotherapeutic and/or NHL-specific imaging agent. These findings provide evidence that IP and SQ administration can achieve results equivalent to IV administration and may lead to more efficient, reproducible treatment plans for antibody-based therapeutics.

[A comparison on radiochemical behavior and biological property of antisense oligonucleotide labeled with technetium-99m by two methods: NHS-MAG3 versus SHNHP].

This study was undertaken to explore and compare the radiochemical behavior and biological property of antisense oligonucleotide (ASON) labeled with Technetium-99m using two methods: N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) versus hydrazino nicotinamide derivative (SHNH). After SHNH and NHS-MAG3 were synthesized, ASON was labeled with Technetium-99m using SHNH and NHS-MAG3 as a bifunctional chelator, separately. The stability in vivo and in vitro, the combination with plasma albumen of rabbit, the biodistribution in BALB/ C mice and the HT29 cellular uptake were compared between labeled compound 99mTc-SHNH-ASON, using SHNH as a bifunctional complex reagent, and 99mTc-MAG3-ASON, using NHS-MAG3 as a bifunctional chelator. The results revealed that the labeling rate and the stability of 99mTc-MAG3-ASON were evidently higher than that of 99mTc-SHNH-ASON (P < 0.05), the combination rate of 99mTc-MAG3-ASON with plasma albumen was markedly lower than that of 99mTc-SHNH-ASON (P < 0.05); the biodistribution of 99mTc-MAG3-ASON was markedly lower than that of 99mTc-SHNH-ASON in blood, heart, stomach and intestines (P < 0.05), slightly lower than that of 99mTc-SHNH-ASON in liver and spleen (P > 0.05), and markedly higher than that of 99mTc-SHNH-ASON in kidney (P < 0.05); the HT29 cellular uptake rates of 99mTc-MAG3-ASON was markedly higher than that of 99mTc-SHNH-ASON (P < 0.05). Therefore, the radiochemical behavior and biological property of 99mTc-MAG3-ASON labeled using NHS-MAG3 is better than that of 99mTc-SHNH-ASON labeled using SHNH.

Biodistribution of 211At labeled HER-2 binding affibody molecules in mice.

The size of affibody molecules makes them suitable as targeting agents for targeted radiotherapy with the alpha-emitter 211At, since their biokinetic properties match the short physical half-live of 211At. In this study, the potential for this approach was investigated in vivo. Two different HER-2 binding affibody molecules were radiolabeled with 211At using both the linker PAB (N-succinimidyl-para-astatobenzoate) and a decaborate-based linker, and the biodistribution in tumor-bearing nude mice was investigated. The influence of L-lysine and Na-thiocyanate on the 211At uptake in normal tissues was also studied. Based on the biokinetic information obtained, the absorbed dose was calculated for different organs. Compared with a previous biodistribution with 125I, the 211At biodistribution using the PAB linker showed higher uptake in lungs, stomach, thyroid and salivary glands, indicating release of free 211At. When the decaborate-based linker was used, the uptake in those organs was decreased, but instead, high uptake in kidneys and liver was found. The uptake, when using the PAB linker, could be significantly reduced in some organs by the use of L-lysine and/or Na-thiocyanate. In conclusion, affibody molecules have suitable blood-kinetics for targeted radionuclide therapy with 211At. However, the labeling chemistry affects the distribution in normal organs to a high degree and needs to be improved to allow clinical use.

Evaluation of in silico pharmacokinetic properties and in vitro cytotoxic activity of selected newly synthesized N-succinimide derivatives.

Design of a new drug entity is usually preceded by analysis of quantitative structure activity (properties) relationships, QSA(P)R. Six newly synthesized succinimide derivatives have been determined for (i) in silico physico-chemical descriptors, pharmacokinetic and toxicity predictors, (ii) in vitro biological activity on four different carcinoma cell lines and on normal fetal lung cells and (iii) lipophilicity on liquid chromatography. All compounds observed were predicted for good permeability and solubility, good oral absorption rate and moderate volume of distribution as well as for modest blood brain permeation, followed by acceptable observed toxicity. In silico determined lipophilicity, permeability through jejunum and aqueous solubility were correlated with experimentally obtained lipophilic constants (by use of high pressure liquid chromatography) and linear correlations were obtained. Absorption rate and volume of distribution were predicted by chromatographic lipophilicity measurements while permeation through blood bran barrier was predicted dominantly by molecular size defined with molecular weight. Five compounds have demonstrated antiproliferative activity toward cervix carcinoma HeLa cell lines; three were cytotoxic against breast carcinoma MCF-7 cells, while one inhibited proliferation of colon carcinoma HT-29 cell lines. Only one compound was cytotoxic toward normal cell lines, while other compounds were proven as safe. Antiproliferative potential against HeLa cells was described as exponential function of lipophilicity. Based on obtained results, lead compounds were selected.

Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1H-indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure-activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.