Fluorescent analogs of peptoid-based HDAC inhibitors: Synthesis, biological activity and cellular uptake kinetics.

Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.

Combinatorial Dansyl Library and its Applications to pH-Responsive Probes.

Herein, we report the first 48-membered, dansyl-based, combinatorial fluorescent library. From the electronic and structural properties of the probes, we analyzed their optical properties and chemical yields, with an average of 49 %. The molecules were examined for their pH responses, and DS-2 and DS-45 showed blue-shifts, whereas DS-7 and DS-40 showed red-shifts in wavelength with increasing pH. Finally, cell permeability was investigated by treating SNU-2292 cells. Our results demonstrate the potential application of this library in biosensors, bio-imaging and pH indicators.

Biotransformation of magnetic nanoparticles as a function of coating in a rat model.

Long-term in vivo studies in murine models have shown that DMSA-coated nanoparticles accumulate in spleen, liver and lung tissues during extended periods of time (at least up to 3 months) without any significant signs of toxicity detected. During that time, nanoparticles undergo a process of biotransformation either by reducing the size or the particle aggregation or both. Using a rat model, we have evaluated the transformations of magnetic nanoparticles injected at low doses. Particles with two different coatings, dimercaptosuccinic acid (NP-DMSA) and polyethylene glycol (NP-PEG-(NH2)2) have been administered to animals, to evaluate the role of coating in the degradation of the particles. We have found that low doses of magnetic nanoparticles are quickly metabolized by the animals. In fact, using a nanoparticle dose four times lower than in previous experiments, NP-DMSA were not observed 24 h after the administration either in the liver or in the lungs. Interestingly, an increased amount of ferritin, the iron storage protein, was observed in liver tissues from rats that were treated with the low dose of NP-DMSA in comparison with the control ones, suggesting a rapid metabolization of the particles into ferritin iron. On the other side we have found that, NP-PEG-(NH2)2 are still detectable in several organs 24 h after their administration at low doses. Probably, due to the longer circulation times of the NP-PEG-(NH2)2, there is a delay in the arrival of the particles to the tissue and this is the reason why we are able to see the particles 24 h post-administration. PEG coating could also be protecting the nanoparticles from rapid degradation of the reticuloendothelial system. Knowledge on the biodistribution, circulation time and degradation processes is required to gain a better understanding of the safety evaluation of this kind of nanomaterial for biomedical applications.

Quantifying the CDK inhibitor VMY-1-103's activity and tissue levels in an in vivo tumor model by LC-MS/MS and by MRI.

The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma.

Investigations on the use of fluorescence dyes for labeling dendrimers: cytotoxicity, accumulation kinetics, and intracellular distribution.

Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO(2)-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.

Synthesis and uptake of fluorescence-labeled Combi-molecules by P-glycoprotein-proficient and -deficient uterine sarcoma cells MES-SA and MES-SA/DX5.

Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells. Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative effects of 4 and 6 were independent of P-gp status of the cells.

Synthesis, biological evaluation and radiochemical labeling of a dansylhydrazone derivative as a potential imaging agent for apoptosis.

To develop a small molecule-based tracer for in vivo apoptosis imaging, dansylhydrazone (DFNSH) was synthesized in 93% yield in less than 30 min. The biological evaluation showed that DFNSH selectively binds to paclitaxel-induced apoptotic cancer cells. The high magnification fluorescent images demonstrate that DFNSH is localized within the cytoplasm of cells that bound Alexa 488 labeled annexin V on the plasma membrane. [(18)F]-DFNSH ([(18)F]-3) was synthesized and isolated in 50-60% radiochemical yields, based on [K/K(222)](18)F, with a synthesis time of 50 min (EOB). The straightforward preparation of fluorine-18 labeled 3 makes it a promising tracer for PET imaging of apoptosis.

Neonatal blood is more resistant to oxidative stress induced by stable nitroxide radicals than adult blood.

Neonate erythrocytes are more susceptible to oxidizing drugs than adults; however, there are controversial reports in the literature regarding the total antioxidant capacity of neonate blood. Stable nitroxide radicals (NRs) are reduced by blood and some other biological materials to the corresponding hydroxylamines. The kinetics of the nitroxide's disappearance using electron paramagnetic resonance (EPR) spectroscopy, provides useful biochemical and biophysical information about the antioxidant properties of biological systems. In order to investigate the antioxidant properties in the newborn's blood, we applied this novel method on 38 umbilical vein blood samples and 40 healthy adults. The NR, 5-Dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6,-tetramethyl-piperidine-oxyl (R), was used for this purpose. Ascorbate is the only known natural antioxidant that reduces R. We found that the reduction rates of R in neonate's whole blood are significantly higher (P < 0.001) than the reduction rates of R in adult's whole blood. However, there were no significant differences in the antioxidant capacity between the two groups. Newborn's blood has significantly higher ability to deal with oxidative stress, caused by R, in comparison with adult blood. We suggest that the system that responds to the recycling of ascorbate is more efficient in neonate blood than in adult's blood.

Determination of endothelin antagonist BMS182874 in plasma by high-performance liquid chromatography.

A simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of BMS182874 (BMS) in mouse plasma. The drug was extracted from plasma by a liquid-liquid extraction process. The method consists of reversed-phase chromatography using a Thermo Hypersil-Keystone RP-18 5 microm, 250 x 2.1 mm column and UV spectrophotometer detection at 255 nm. The mobile phase consists of 45% (v/v) acetonitrile: 55% (v/v) trifluoroacetic acid (0.015% v/v; pH 3.0) at a flow rate of 0.6 ml/min. Validity of the method was studied and the method was precise and accurate with a linearity range from 100 ng/ml to 1000 ng/ml. The extraction efficiency was found to be 81, 84 and 87% for 100, 500 and 1000 ng/ml, respectively for spiked drug in plasma. The limit of quantification and limit of detection were found to be 50 and 10 ng/ml, respectively in plasma. Within-day and between-day precision expressed by relative standard deviation was less than 4% and inaccuracy did not exceed 4%. The assay was also used to analyze samples collected during animal studies. The suitability and robustness of the method for in vivo samples were confirmed by analysis of BMS from mouse plasma and tissues dosed with BMS.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis.

PURPOSE: Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. METHODS: Using both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis. RESULTS: ApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining. CONCLUSION: ApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.

A novel method of imaging lysosomes in living human mammary epithelial cells.

Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs) was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2). The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.

Pharmacokinetics and metabolism of endothelin receptor antagonist: contribution of kidneys in the overall in vivo N-demethylation.

In vivo clearance of BMS-182874 was primarily due to metabolism via stepwise N-demethylation. Despite in vivo clearance approached ca 50% of the total liver plasma flow, BMS-182874 was completely bioavailable after oral administration in rats. Saturable first-pass metabolism and the role of extrahepatic tissue were evaluated as possible reasons for complete oral bioavailability despite extensive metabolic clearance. Pharmacokinetic parameters were obtained after an intravenous and a range of oral doses of BMS-182874 in rats. Bile and urine were collected from bile-duct cannulated (BDC) rats and the in vivo metabolic pathways of BMS-182874 were evaluated. Pharmacokinetics of BMS-182874 were also compared in nephrectomized (renally impaired) vs. sham-operated control rats. Oral bioavailability of BMS-182874 averaged 100%, indicating that BMS-182874 was completely absorbed and the first-pass metabolism (liver or intestine) was negligible. The AUC and Cmax values increased dose-proportionally, indicating kinetics were linear within the oral dose range of 13 to 290 mmole/kg. After intravenous administration of BMS-182874 to BDC rats, about 2% of intact BMS-182874 was recovered in excreta, indicating that BMS-182874 was cleared primarily via metabolism in vivo. The major metabolite circulating in plasma was the mono-N-desmethyl metabolite and the major metabolite recovered in excreta was the di-N-desmethyl metabolite. In vivo clearance of BMS-182874 was significantly reduced in nephrectomized rats. These observations suggest saturable first-pass metabolism is unlikely to be a mechanism for complete oral bioavailability of BMS-182874. Reduced clearance observed in the nephrectomized rats suggests that extrahepatic tissues (e.g., kidneys) may play an important role in the in vivo clearance of xenobiotics that are metabolized via N-demethylation.

Interaction mechanism between indoxyl sulfate, a typical uremic toxin bound to site II, and ligands bound to site I of human serum albumin.

PURPOSE: The study was performed for clarifying the mechanism of interaction between indoxyl sulfate (IS), a typical uremic toxin bound to site II, and site I-ligands when bound to human serum albumin (HSA). The effect of the N to B transition on the interactions was also examined. METHODS: Quantitative investigation of the relations between ligands bound to HSA was performed by equilibrium dialysis, and the binding data were analyzed on the basis of a theoretical model for simultaneous binding of two ligands. RESULTS: The high-affinity binding constants for the site I-ligands warfarin (WF) and dansyl-L-asparagine (DNSA) increased with increasing pH, whereas those for the site II-ligands IS and dansylsarcosine (DNSS) were hardly affected by pH. Mutual displacement experiments showed that even though IS binds to site II it influenced binding of DNSA at the azapropazone binding area in site I. By contrast, it is unlikely that IS affects the WF binding area of site I. Furthermore, pH-profiles showed that the interaction between IS and DNSA was very sensitive to the N to B transition: "competitive-like" strong allosteric regulation was observed for binding of the two ligands to the N conformer (pH 6.5), whereas in the B conformation (pH 8.5) binding of these molecules was nearly "independent". CONCLUSIONS: The present data provide useful information for elucidating a potential mechanism of interaction between drugs and endogenous substances including uremic toxins.

Electrostatic interactions regulate desensitization of the nicotinic acetylcholine receptor.

To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.

The effects of an alpha hydroxy acid (glycolic acid) on hairless guinea pig skin permeability.

The barrier integrity of hairless guinea pig skin after treatment with an alpha hydroxy acid was assessed through in vivo topical application of an oil-in-water emulsion containing 5 or 10% glycolic acid at pH 3.0. The control was a commercial moisturizing lotion, pH 7.8. A dosing regimen for the glycolic acid formulations that was tolerated by the hairless guinea pigs and significantly decreased stratum corneum turnover time was determined using the dansyl chloride staining technique. Once-daily dosing of hairless guinea pig skin for 3 weeks with the glycolic acid formulations resulted in approximately a 36-39% decrease in stratum corneum turnover time compared with the control lotion. After this treatment, hairless guinea pigs were sacrificed for the in vitro measurement of the percutaneous absorption of [14C]hydroquinone and [14C]musk xylol. No significant differences in the 24-hour absorption of either test compound were found for skin treated with the control lotion or the glycolic acid formulations. There were also no significant differences found in the absorption of [3H]water through skin from the different treatment groups. Although no increase in skin penetration occurred after treatment with the glycolic acid formulations, histology revealed approximately a twofold increase in epidermal thickness. Also the number of nucleated cell layers nearly doubled in skin treated with 5% and 10% glycolic acid compared with the control lotion and untreated skin. These studies demonstrate that substantial changes in the structure of hairless guinea pig epidermis can occur without significant effect on skin permeability of two model compounds.

Influence of non-steroidal anti-inflammatory drugs on the binding kinetics of dansylsarcosine to human serum albumin. Stereoselectivity, steric and inductive effects.

The effect of a series of non-steroidal anti-inflammatory drugs (NSAIDs) on the binding kinetics of dansylsarcosine (CAS 72517-44-3, DS), a marker ligand for the benzodiazepine binding site, and human serum albumin (HSA) was studied using the stopped-flow method. Both native (7% glycated) and 25% glycated HSA were used. The binding parameters were determined on the basis of the consecutive model. The DS association rate constant (k2) was 649 +/- 84 s-1 and 375 +/- 13 s-1 for 7% and 25% glycated HSA, respectively. These values were substantially influenced by addition of NSAIDs (molar ratio HSA:NSAID = 2:1), depending on the structure of NSAIDs. The calculated DS dissociation rate constant (k-2) was approximately 20 s-1. this value did not show marked dependence on the degree of glycation or on the presence of NSAIDs at the concentration used. The values were similar to estimates of kd (the displacement rate constant of DS) with the exception of diclofenac (CAS 15307-86-5) where kd was significantly lower, reaching 4.8 +/- 0.4 s-1 and 4.8 +/- 0.6 s-1 vs. k-2 parameters of 14 +/- 2.8 s-1 and 15 +/- 3.7 s-1 for 7% and 25% glycated HSA, respectively. A comparison of the enantiomers R- and S-ibuprofen (CAS 15687-27-1) and the regioisomers fenbufen (CAS 36330-85-5) and ketoprofen (CAS 22071-15-4) showed slight or no stereoslectivity of effects on the DS binding kinetics. However, the binding was influenced by bulk and nature of substituents at the aryl rest of propionic acid. The results obtained for mefenamic acid (CAS 61-68-7) suggest that this NSAID binds to a site of human serum albumin other than site II. Increased concentrations of glycoalbumin, as observed in diabetic patients, are not presumed to have inhibitory effects additional to that of NSAIDs which interact differentially with drugs at site II of HSA.

A general, wide-rage spectrofluorometric method for measuring the site-specific affinities of drugs toward human serum albumin.

Binding of drugs to serum albumin is one of the most important pharmacokinetic determinants and the design of drugs should take advantage of this property. In the present work, the fluorescent ligands Warfarin and dansylsulfonamide were used as probes of IIA site of human albumin and dansylsarcosine as the probe of the IIIA site. From the changes in fluorescence upon binding at 37 degrees C, pH 7.4, the following dissociation constants were determined: Warfarin, 3.43 +/- 0.69 microM; dansylsulfonamide, 7.57 +/- 0.88 microM; and dansylsarcosine, 6.06 +/- 1.09 microM. Nonfluorescent ligands displace these probes competitively and the type of probe displaced identifies the site specificity of the ligands. Nonlinear least-squares analysis of the decrease in fluorescence accompanying the displacement yields the stoichiometry and the dissociation constant may also be estimated rapidly from displacement at a single competitor concentration. The method yields reliable Kd values for at least the range of 0.2 to 100 microM. Representative dissociation constants for the IIA site-specific ligands are as follows: phenylbutazone, 1.9 +/- 0.3 microM; U-99,499, 1.8 +/- 0.2 microM; U-96,988, 5.3 +/- 1.5 microM; and U-105,665, 42 +/- 7 microM. For the IIIA site we find the following Kd values: oxazepam, 27.7 +/- 2.1 microM; diazepam, 7.7 +/- 1.0 microM; and ibuprofen, 2.7 +/- 1.2 microM. The method is eminently suitable for large-scale screening.

Microscopic evaluation of the dansyl chloride test.

The classical dansyl chloride test relies on visual assessment of the extinction of fluorescence in time. We introduce a microscopic and morphometric rating of this test. We show that soaps may remove the fluorescing dye from corneocytes. Therefore caution should be taken in interpreting fluorescence extinction only as an estimate of the stratum corneum renewal.