Imaging Butyrylcholinesterase in Multiple Sclerosis.

PURPOSE: Molecular imaging agents targeting butyrylcholinesterase (BChE) have shown promise in other neurodegenerative disorders and may have utility in detecting changes to normal appearing white matter in multiple sclerosis (MS). BChE activity is present in white matter and localizes to activated microglia associated with MS lesions. The purpose of this study was to further characterize changes in the cholinergic system in MS pathology, and to explore the utility of BChE radioligands as potential diagnostic and treatment monitoring agents in MS. PROCEDURE: Cortical and white matter lesions were identified using myelin staining, and lesions were classified based on microglial activation patterns. Adjacent brain sections were used for cholinesterase histochemistry and in vitro autoradiography using phenyl 4-[123I]-iodophenylcarbamate (123I-PIP), a previously described small-molecule cholinesterase-binding radioligand. RESULTS: BChE activity is positively correlated with microglial activation in white matter MS lesions. There is no alteration in cholinesterase activity in cortical MS lesions. 123I-PIP autoradiography revealed uptake of radioactivity in normal white matter, absence of radioactivity within demyelinated MS lesions, and variable uptake of radioactivity in adjacent normal-appearing white matter. CONCLUSIONS: BChE imaging agents have the potential to detect MS lesions and subtle pathology in normal-appearing white matter in postmortem MS brain tissue. The possibility of BChE imaging agents serving to supplement current diagnostic and treatment monitoring strategies should be evaluated.

Comparison of dimethylated and methylchlorinated amylose stationary phases, coated and covalently immobilized on silica, for the separation of some chiral compounds in supercritical fluid chromatography.

The chromatographic properties of a new coated amylose tris(3-chloro-5-methylphenylcarbamate) were evaluated in supercritical fluid chromatography for the separation of enantiomers of chiral 1-aryl-5-aryl-pyrrolidin-2-one derivatives, potential anticancer agents, and some commercial drugs. The mobile phase consisted of CO2-modifier mixtures with 30% of either methanol or ethanol, the flow rate was 3 mL/min. The column oven temperature was 40 °C and the outlet pressure was 15 MPa, in order to limit the compressibility of the CO2, thus limiting density variation along the column. The obtained results were then compared to those observed toward 3 other stationary phases: the coated amylose tris(3,5-dimethylphenylcarbamate), the immobilized amylose tris(3,5-dimethylphenylcarbamate) and the coated amylose tris(5-chloro-2-methylphenylcarbamate). It was shown that the new coated amylose tris(3-chloro-5-methylphenylcarbamate) was the most retentive column whatever the studied compounds, particularly for thalidomide and omeprazole with retention factors up to 73.3 and 29.5for the second enantiomer, respectively. Concerning the enantioselectivity, even most of the compounds are separated on all the four columns, the coated amylose tris(3-chloro-5-methylphenylcarbamate) allows the best resolution for most of the ten studied analytes (except omeprazole for which the resolution values are equal to 7.8 and 9.7 on the coated amylose tris(3-chloro-5-methylphenylcarbamate) and amylose tris(3,5-dimethylphenylcarbamate), respectively). Acting in complementary ways, the two chlorinated stationary phases permitted the complete separation of enantiomers of nine compounds out of the ten.

Design, synthesis and evaluation of rivastigmine and curcumin hybrids as site-activated multitarget-directed ligands for Alzheimer's disease therapy.

A series of novel 2-methoxy-phenyl dimethyl-carbamate derivatives were designed, synthesized and evaluated as site-activated MTDLs based on rivastigmine and curcumin. Most of them exhibited good to excellent AChE and BuChE inhibitory activities with sub-micromolar IC50 values. Among all the compounds, 6a demonstrated the most potent AChE inhibition with IC50 value of 0.097µM, which is about 20-fold than that of rivastigmine. In addition, the three selected compounds 5a, 6a and 6e demonstrated inhibitory activity against Aß self-aggregation similar to cucurmin in TEM assay, which is obviously different from the weak activity of rivastigmine. Moreover, the hydrolysate of 6a (compound 7) also showed potent ABTS(+) scavenging and moderate copper ion chelating activity in vitro.

A synthetic decursin analog with increased in vivo stability suppresses androgen receptor signaling in vitro and in vivo.

Targeting androgen receptor (AR) signaling with agents distinct from current antagonist drugs remains a rational approach to the prevention and treatment of prostate cancer (PCa). Our previous studies have shown that decursin and isomer decursinol angelate (DA), isolated from the Korean medicinal herb Angelica gigas Nakai, interrupt AR signaling and possess anti-PCa activities in vitro. In the LNCaP PCa cell model, these pyranoccoumarin compounds exhibit properties distinct from currently used antagonists (e.g., Casodex). However, both are rapidly de-esterified to decursinol, a partial AR agonist. We report here that a synthetic decursin analog, decursinol phenylthiocarbamate (DPTC), has greater in vivo stability than the parent compounds. DPTC-decursinol conversion was undetectable in mice. Furthermore, in LNCaP cells, DPTC decreased prostate specific antigen (PSA) expression, down-regulated AR abundance and mRNA and inhibited AR nuclear translocation. The effect of DPTC on AR and PSA mRNA and protein abundance was also observed in VCaP cells expressing wild type AR. DPTC inhibited growth of both PCa cell lines through G(1) cell cycle arrest and apoptosis, as did decursin and DA. Furthermore, i.p. administration of DPTC for 3 weeks suppressed the expression of AR target genes probasin and Nkx3.1 in mouse prostate glands. Overall, our data suggest that DPTC represents a prototype lead compound for development of in vivo stable and active novel decursin analogs for the prevention or therapy of PCa.

Diacetylene copolymers for fingermark development.

In 1979, Miller and Patel showed that a solution containing two diacetylene monomers, 2,4-hexadiyne-1,6-bis(phenylurethane) (HDDPU) and 2,4-hexadiyne-1,6-bis(p-chlorophenylurethane) (HDDCPU) could be used to develop latent fingermarks on a non-porous surface. In the current work, the same mixture (HDDPU:HDDCPU=10:1, in acetone solution) was used to develop fingermarks on a wide variety of surfaces, both non-porous and porous, including paper. An airbrush system was optimized for the application of the reagent solution. Once the solution evaporates on a surface, the monomers co-crystallize in different ways, depending upon a number of factors, including the surface residue. "Active" co-crystallization leads (with heat or radiation) to the formation of purple polymer, while "inactive" crystallization results in a non-polymerizable white deposit. Fingermark contrast was achieved as a result of active co-crystallization (giving purple polymer) in either the ridges or the furrows, depending upon the surface and other factors. A general observation (supported by spot tests with linseed oil, salt and amino acid solutions) was that on paper, oily materials are more likely to lead to the formation of the purple polymer, while the presence of water inhibits polymerization. However, these observations are not consistent across all other substrates. It is hypothesized that water disrupts hydrogen bonding between diacetylene molecules, and thus prevents the topochemical polymerization of the diacetylenes, which occurs in the solid state between favourably aligned monomers. An interesting observation was the development of fingermarks deposited on paper that had already been treated with the diacetylene reagent.

Enantiomeric separation of FMOC-amino acids by nano-LC and CEC using a new chiral stationary phase, cellulose tris(3-chloro-4-methylphenylcarbamate).

A novel polysaccharide-based chiral stationary phase (CSP), cellulose tris(3-chloro-4-methylphenylcarbamate), also known as Sepapak-2 or Lux Cellulose-2, has been evaluated for the enantiomeric separation of FMOC derivatives of amino acids. After mobile-phase optimization in nano liquid chromatography (nano-LC) the column enabled the enantiomeric separation of 19 out of 23 amino acids tested, indicating the high chiral recognition power of this new CSP. Subsequently, a comparison of the driving force employed (pressure or voltage) was carried out comparing nano-LC and CEC under the same conditions. Better peak efficiencies and resolution were observed by using CEC experiments, which enabled the chiral discrimination of 20 out of 23 amino acids tested. Finally, in order to show the potential of this new CSP, the determination of the content and the enantiomeric purity of the non-protein amino acid citrulline in food supplements was performed. For that purpose, the method was optimized, evaluated and applied to different commercial samples.

Novel carbamate cholinesterase inhibitors that release biologically active amines following enzyme inhibition.

Conjugation of the phenol derived from rivastigmine with amphetamines gave access to novel carbamate cholinesterase inhibitors. All compounds possessed increased affinity and selectivity for AChE compared to rivastigmine and were orally bioavailable. Compound 4a, incorporating d-amphetamine, caused significant inhibition of cholinesterase in vivo at doses that were well tolerated. Release of amphetamine from 4a was demonstrated following in vitro and in vivo inhibition of cholinesterase. Compound 4a was also effective in alleviating scopolamine induced amnesia in a rat passive avoidance model.

Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography-mass spectrometry.

The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (approximately 60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC-MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC-MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC-MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.

A study of rivastigmine liposomes for delivery into the brain through intranasal route.

The present study is mainly aimed at delivering a drug into the brain via the intranasal route using a liposomal formulation. For this purpose, rivastigmine, which is used in the management of Alzheimer's disease, was selected as a model drug. Conventional liposomes were formulated by the lipid layer hydration method using cholesterol and soya lecithin as lipid components. The concentration of rivastigmine in brain and plasma after intranasal liposomes, free drug and per oral administration was studied in rat models. A significantly higher level of drug was found in the brain with intranasal liposomes of rivastigmine compared to the intranasal free drug and the oral route. Intranasal liposomes had a longer half-life in the brain than intranasally or orally administered free drug. Delivering rivastigmine liposomes through the intranasal route for the treatment of Alzheimer's disease might be a new approach to the management of this condition.

Enantioseparation of N-fluorenylmethoxycarbonyl alpha-amino acids on polysaccharide-derived chiral stationary phases by reverse mode liquid chromatography.

The enantioseparation of N-protected fluorenylmethoxycarbonyl (N-FMOC) alpha-amino acids was carried out on three polysaccharide-derived chiral stationary phases, such as cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethyl-phenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ), and the influence of acetonitrile composition and pH of the eluents on the enantioseparation in reverse mode chromatography was examined. The best separation of the enantiomers was achieved with 40% acetonitrile in 50mM phosphate buffer at pH 2. However, increasing the composition of acetonitrile to 50% on Chiralcel OD yielded a considerable decrease of retention time with minimum loss of resolution. The elution order of N-FMOC alpha-amino acid enantiomers on Chiralcel OD and OJ were quite different, indicating that both phases could be used in a complementary manner for the separation of the enantiomers of N-FMOC alpha-amino acids. The positive relationship between the capacity factor of N-FMOC alpha-amino acids and the hydrophobicity of amino acids indicated that hydrophobicity plays an important role on the retention of the N-FMOC alpha-amino acids in the reverse mode.

HPLC separation of naringin, neohesperidin and their C-2 epimers in commercial samples and herbal medicines.

Flavanone glycosides, such as naringin and neohesperidin, are distributed in some Citrus species and have a chiral center in the C-2 position of the flavanone moiety. Naringin and neohesperidin (2S-form) were separated from the corresponding C-2 epimers (2R-epi-form) by normal-phase HPLC using a polysaccaride-derived chiral stationary phases (CSPs), CHIRALPAK IB. The analyses of commercial samples of naringin revealed that the relative ratios of naringin to the C-2 epimer were 29-89%. In the case of a commercial sample of neohesperidin, the relative ratio of the neohesperidin (2S-form) is 84%. The HPLC application to Citrus species used as crude drugs in Japan (Kijitsu, Kikoku and Tohi) showed that the relative ratios of naringin to the C-2 epimer were 75-93% in Kijitsu, 74-79% in Kikoku and 54-64% in Tohi. However, there is a quite small ratio of the (2R)-epi-neohesperidin in Citrus. This result suggested that the averages of relative ratio of (2S)-naringin in Citrus species reduced according to the maturity of fruits (Kijitsu

Cellulose tris(3,5-dichlorophenylcarbamate) immobilised on silica: a novel chiral stationary phase for resolution of enantiomers.

CHIRALPAK IC is a new chiral stationary phase (CSP) made by immobilising cellulose tris(3,5-dichlorophenylcarbamate) on silica gel. The chiral selector is distinct from any other commercially available polysaccharide-based CSPs. Apart from its compatibility with the whole series of solvents; this CSP is able to operate under various chromatographic conditions and bring about new characteristics in enantiomeric recognition. It can afford many large and specific enantiomeric separations. It exhibits complementary properties with regard to the existing immobilised chiral packing materials of the same category.

In situ-forming oleogel implant for rivastigmine delivery.

PURPOSE: To provide a simplified dosing schedule and potentially reduce side effects associated to peak plasma concentrations, an in situ-forming oleogel implant was studied for the sustained-release of rivastigmine. MATERIALS AND METHODS: The gel was prepared by dissolving 5-10% (w/w) N-stearoyl L: -alanine methyl ester (SAM) organogelator in safflower oil containing either dissolved rivastigmine or its dispersed hydrogen tartrate salt. Rheological analysis, differential scanning calorimetry, and infrared spectroscopy were carried out to assess the impact of drug incorporation on the oleogel; this was followed by in vitro and in vivo release studies. RESULTS: A weakening of intermolecular interactions was suggested by gel-sol transition temperature drops of 10-15 degrees C upon incorporation of dissolved drug. Meanwhile, the dispersed drug salt induced minimal or no changes in transition temperature. Gels containing dispersed rivastigmine had the lowest burst in vitro (<15% in 24 h). In vivo, the 10% SAM formulation containing dispersed rivastigmine provided prolonged drug release within the therapeutic range for 11 days, with peak plasma levels well below the toxic threshold and up to five times lower than for the control formulation. CONCLUSIONS: This study established SAM gels to be a promising option for sustained-release formulations in the treatment of Alzheimer's Disease.

Synthesis of new OBAN's and further studies on positioning of the catalytic group.

Two new zinc ion dependent oligonucleotide based artificial nucleases (OBAN's) have been synthesized. These consist of 2'-O-methyl modified RNA oligomers conjugated to 5-amino-2,9-dimethylphenanthroline (neocuproine)via a urea linker. OBAN 4 carries the catalytic group on a linker extending from the C-4 of an internal cytosine moiety. OBAN 5 has two neocuproine units attached, each to linkers extending from the C-5 position of uridine moieties, one placed internally and the other at the at the 5'-end of the oligonucleotide. The key step in the synthesis of the OBAN systems is conjugation of the catalytic group to the respective amino linkers of the modified oligonucleotides. This is achieved by first converting the 5-amino-2,9-dimethylphenanthroline to the phenylcarbamate. The reaction of this neocuproine phenylcarbamate with the oligonucleotide carrying one or two primary aliphatic amines in aqueous buffer (at pH 8.5) leads to nearly quantitative formation of the urea-linked conjugates. Both OBAN systems were found to cleave RNA in the bulged out regions formed from the non-complementary part of the target sequences, in the presence of Zn(II) ions. Differences in efficiency between these and previously reported systems are discussed.