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.

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.

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.

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.

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.

Synthesis, Biological Evaluation and Pharmacokinetic Studies of Mefenamic Acid - N-Hydroxymethylsuccinimide Ester Prodrug as Safer NSAID.

BACKGROUND: Non steroidal anti-inflammatory drugs are the most widely prescribed drugs to manage pain and inflammatory conditions, but their long term use is associated with gastrointestinal toxicity. OBJECTIVES: The study aimed to synthesize an ester-based prodrug of a non steroidal anti-inflammatory agent, mefenamic acid in order to improve the therapeutic index vis a vis to overcome the side effects such as gastrointestinal irritation and bleeding associated with the use of mefenamic acid. METHODS: The ester prodrug (MA-NH) was prepared by condensing mefenamic acid with N-hydroxymethylsuccinimide in the presence of Phosphorus oxychloride. The pharmacokinetic profile, including stability and release of mefenamic acid and N-hydroxymethylsuccinimide from the ester prodrug (MA-NH) was studied by RP- HPLC in acidic medium (pH 1.2), basic medium (pH 7.4), 80 % v/v human plasma, 10 % w/v rat intestinal homogenate and 10 % w/v rat liver homogenate (pH 7.4). RESULTS: The chemical structure of the title compound was characterized by using modern spectroscopic techniques. The prodrug was found to be stable in acid medium, but it hydrolyzed and released sufficient quantities of the drug in alkaline medium. The prodrug produced lesser number of ulcers and showed improved analgesic and anti-inflammatory activity as compared to the parent drug. CONCLUSION: The results indicate that the synthesized prodrug (MA-NH) is better in terms of analgesic and antiinflammatory activities and with less GI toxicity than the parent drug.

Tracking cells and monitoring proliferation.

In many areas of immunology it is desirable to be able to track particular cells throughout an assay, whether it be in vivo or in vitro. There are two classes or reagents used for this purpose-general protein labels (reactive compounds that form random covalent bonds with amino groups on cellular proteins) and general membrane labels (lipophilic compounds that partition stably but non-covalently into the plasma membrane). The fluorescein derivative, carboxyfluorescein diacetate succinimidyl ester, CFDA-SE (general protein label), has been found to be particularly well suited for the purpose of cell tracking. Once labeled, cells can be tracked both in vivo and in vitro. Moreover, due to the randomness of the labeling, cells undergoing division maintain half of the staining intensity of the parent cell. This halving of the staining intensity additionally allows for monitoring of cells undergoing division for 6-8 consecutive cycles.

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.

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.

Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties.

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

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.

18F-radiolabeled GLP-1 analog exendin-4 for PET/CT imaging of insulinoma in small animals.

BACKGROUND: Insulinoma is a neuroendocrine tumor derived from the ß cells of pancreatic islets. They are usually relatively inaccessible for surgical intervention. High expression levels of glucagon-like peptide-1 (GLP-1) receptor have been detected in insulinoma. AIM: The aim of the study was to evaluate the potential of F-radiolabeled GLP-1 analog exendin-4 for the diagnosis of insulinoma using PET/computed tomography imaging. MATERIALS AND METHODS: The GLP-1 receptor-specific molecular probe [F]FB-exendin-4 was prepared by the conjugation of exendin-4 and N-succinimidyl-4-[F] fluorobenzoate ([F]SFB). High expression of GLP-1 by the RIN-m5f insulinoma line and GLP-1 receptor specificity were evaluated by determining the saturation curve for in-vitro binding of I-radiolabeled exendin-4 and by investigation of the competitive binding between I-radiolabeled and unlabeled exendin-4. Further, the in-vivo biodistribution and micro-PET/computed tomography images of insulinoma-bearing mice were studied. RESULTS: An overall radiochemical yield of 35.6±2.3% (decay corrected, n=5) and specific radioactivity of around 30 GBq/µmol were achieved for [F]FB-exendin-4, and the radiochemical purity was over 98%. Both in-vitro and in-vivo studies confirmed the specificity of [F]FB-exendin-4 to insulinoma cells. CONCLUSION: [F]FB-exendin-4 has been found to be an effective molecular imaging probe for detecting insulinomas.

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.

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.

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.

Experimental studies and modeling of drug release from a tunable affinity-based drug delivery platform.

An affinity-based drug delivery platform for controlling drug release is analyzed by a combination of experimental studies and mathematical modeling. This platform has the ability to form selective interactions between a therapeutic agent and host matrix that yields advantages over systems that employ nonselective methods. The incorporation of molecular interactions in drug delivery can increase the therapeutic lifetime of drug delivery implants and limit the need for multiple implants in treatment of chronic illnesses. To analyze this complex system for rational design of drug delivery implants, we developed a mechanistic mathematical model to quantify the molecular events and processes. With a ß-cyclodextrin hydrogel host matrix, defined release rates were obtained using a fluorescent model drug. The key processes were the complexation between the drug and cyclodextrin and diffusion of the drug in the hydrogel. Optimal estimates of the model parameters were obtained by minimizing the difference between model simulation and experimentally measured drug release kinetics. Model simulations could predict the drug release dynamics under a wide range of experimental conditions.

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.

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.