Adapted physical activity is beneficial on balance, functional mobility, quality of life and fall risk in community-dwelling older women: a randomized single-blinded controlled trial.

BACKGROUND: Exercise programmes have important role in prevention of falls, but to date, we have little knowledge about the effects of Adapted Physical Activity programme on balance of older women. AIM: The aim of this study was to investigate the effects of an Adapted Physical Activity programme on balance, risk of falls and quality of life in community-dwelling older women. DESIGN: This was a randomized controlled study. SETTING: Community, in a local sport centre. POPULATION: Older women aged over 60 years. METHODS: Seventy-six women were randomised to an exercise group providing Adapted Physical Activity programme for 25 weeks or a control group (in which they did not participate in any exercise programme). The one-leg stance test, Timed Up and Go test, incidence of fall and the quality of life (SF-36V2) were measured at baseline and after 25 weeks. RESULTS: The one-leg stance test and the Timed Up and Go test in the exercise group was significantly better than in the control group after the intervention period (P=0.005; P=0.001, respectively). The Physical Functioning, Vitality and General Health subdomains of quality of life were also significantly better in the exercise group compared to the control group (P=0.004; P=0.005; P=0.038, respectively). Relative risk was 0.40 (90% CI 0.174 to 0.920) and the number needed to treat was 5 (95% CI 2.3 to 23.3). CONCLUSION: This 25-week Adapted Physical Activity programme improves static balance, functional mobility, as well as Physical Functioning, Vitality and General Health subdomains of quality of life. CLINICAL REHABILITATION IMPACT: Based on our results, the Adapted Physical Activity programme may be a promising fall prevention exercise programme improving static balance and functional mobility for community-dwelling older women.

Novel gene C17orf37 in 17q12 amplicon promotes migration and invasion of prostate cancer cells.

C17orf37/MGC14832, a novel gene located on human chromosome 17q12 in the ERBB2 amplicon, is abundantly expressed in breast cancer. C17orf37 expression has been reported to positively correlate with grade and stage of cancer progression; however the functional significance of C17orf37 overexpression in cancer biology is not known. Here, we show that C17orf37 is highly expressed in prostate cancer cell lines and tumors, compared to minimal expression in normal prostate cells and tissues. Cellular localization studies by confocal and total internal reflection fluorescence microscopy revealed predominant expression of C17orf37 in the cytosol with intense staining in the membrane of prostate cancer cells. RNA-interference-mediated downregulation of C17orf37 resulted in decreased migration and invasion of DU-145 prostate cancer cells, and suppressed the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) transcription factor resulting in reduced expression of downstream target genes matrix metalloproteinase 9, urokinase plasminogen activator and vascular endothelial growth factor. Phosphorylation of PKB/Akt was also reduced upon C17orf37 downregulation, suggesting C17orf37 acts as a signaling molecule that increases invasive potential of prostate cancer cells by NF-kappaB-mediated downstream target genes. Our data strongly suggest C17orf37 overexpression in prostate cancer functionally enhances migration and invasion of tumor cells, and is an important target for cancer therapy.

Measurement of Hydroxyl-Radical Formation in the Rat Striatum by In Vivo Microdialysis and GC-MS.

A GC-MS method was developed for measuring hydroxyl-radical capture products of salicylic acid, a common trapping agent for this reactive oxygen species, in samples obtained by in vivo cerebral microdialysis experiments. The assay employed liquid-liquid extraction followed by derivatization of 2,3- and 2,5-dihydroxybenzoic acid, along with 3,5-dihydroxybenzoic acid added as an internal standard. Due to their simple electron ionization mass spectra featuring [M - 57](+) ions through the loss of tertiary alkyl group from the corresponding molecular ions, tert-butyldimethylsilyl (TBDMS) derivatives afforded straightforward method development based on selected-ion monitoring. In addition, tandem mass spectrometry probing collision-induced dissociation of [M - 57](+) ions obtained from the isomeric tert-butyldimethylsilyl derivatives revealed characteristic differences in the resultant product-ion spectra. Our work has demonstrated the applicability of GC-MS for the assay of microdialysates for 2,3- and 2,5-dihydroxybenzoic acid by confirming that local administration of the excitotoxic glutamate into the rat striatum significantly increased in vivo hydroxyl-radical production in this brain region and that subsequent systemic administration of α-phenyl-tert-butylnitrone reversed glutamate-induced oxidative stress.

Measurement of Acetylcholine in Rat Brain Microdialysates by LC - Isotope Dilution Tandem MS.

An LC-MS/MS method was developed for measuring acetylcholine (ACh) in an aqueous medium using reversed-phase ion-pair chromatography, electrospray ionization on a quadrupole ion trap instrument and a tetradeuterated analogue (ACh-1,1,2,2-d(4)) as an internal standard. A rapid separation was achieved on a 5-cm long octadecylsilica column (2.1 mm i.d.) by employing heptafluorobutyric acid (0.1% v/v) as an ion-pairing agent and requiring 10% v/v acetonitrile in 20 mM ammonium formate buffer under isocratic elution at 200 µl/min flow rate. The instrument's response was calibrated with samples containing known mole ratios of ACh and ACh-1,1,2,2-d(4) in an artificial cerebrospinal fluid, which afforded the conclusion that analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with the known molar concentration of the ACh-1,1,2,2-d(4) added. The rapid and simple assay was tested by measuring the basal neurotransmitter concentration in rat brain microdialysates without the use of a cholinesterase inhibitor upon sample collection.

The utility of oligopeptidase in brain-targeting delivery of an enkephalin analogue by prodrug design.

In a brain-targeting prodrug approach for a metabolically stable enkephalin analogue DADLE, specific enzymes are utilized for in vivo prodrug activation. Prolyl oligopeptidase (POP) may be especially useful in this regard. In vitro metabolic stability of the putative metabolites of prodrugs having various "spacers" has shown that POP provides significantly faster release of DADLE from conjugates having dipeptidyl spacer (specifically Xaa-Pro or Xaa-Ala) than alternative peptidases utilized when single amino acids are used as spacers. In vitro half-lives measured in rat brain homogenate showed excellent correlation with CNS-mediated analgesia using the tail-flick model in rats providing, thus, an in vivo substantiation of the prodrug approach relying on POP as the peptidase to release DADLE.

A pyridinium-substituted analog of the TRH-like tripeptide pGlu-Glu-Pro-NH2 and its prodrugs as central nervous system agents.

A metabolically stable and centrally acting analog of pGlu-Glu-Pro-NH2 ([Glu2]TRH, a tripeptide structurally related to TRH (thyrotropin-releasing hormone)) was designed by replacing the amino-terminal pyroglutamyl residue with a pyridinium moiety. The analeptic action of the analog was used to optimize the efficacy of this novel CNS agent when administered intravenously in its CNS-permeable prodrug forms obtained via the reduction of the pyridinium moiety to the nonionic dihydropyridine and esterifying the central Glu with various alcohols. The maximum effect in antagonizing pentobarbital-induced narcosis in mice was achieved with the hexyl ester that was used subsequently for a comparative evaluation with a prodrug of the parent neuropeptide in the Porsolt swim test as a paradigm for antidepressant effect. The novel analog maintained its antidepressant potency but showed reduced analeptic action compared to [Glu2]TRH; thus, an increase in the selectivity of CNS-action was obtained by the incorporation of the pyridinium moiety.

Integration of mass spectrometry into early-phase discovery and development of central nervous system agents.

The early-phase discovery and development of useful central nervous system (CNS) agents present ample opportunities to exploit mass spectrometry and provide detailed compound/mixture characterization, or to make the process faster and/or more economic. Neuropeptide FF antagonists and centrally active thyrotropin-releasing hormone analogues were used as specific examples in this work. We evaluated the characterization of focused libraries of peptide derivatives by electrospray ionization, tandem mass spectrometry and liquid chromatography/tandem mass spectrometry on a quadrupole ion trap and nanoelectrospray on a Fourier transform ion cyclotron resonance mass spectrometer. Immobilized artificial-membrane chromatography was employed as a model to predict/rank new agents against lead compounds for their potential to reach the central nervous system in pharmacologically significant amounts. Measuring brain concentrations in rodents after the intravenous administration of test compounds was used as an in vivo approach, and we took advantage of microdialysis sampling that furnished samples without interfering tissue matrix and afforded the estimation of extracellular concentrations in a localized part of the brain. Overall, making atmospheric-pressure ionization mass spectrometry an integral part of the process has played a major role in increasing throughput, selectivity, specificity and detection sensitivity and thereby providing useful information about the extent or mechanism of transport and metabolic activation/inactivation in early-phase discovery and development of CNS agents.

Membrane fluidity effects of estratrienes.

Estrogens have demonstrable neuroprotective effects. This fact has lead to the proposed use of estrogens for the prevention and/or treatment of Alzheimer's disease. The exact protective mechanism estrogens provide is not fully understood. In this report, a potential non-genomic mechanism for estratrienes involving alterations in membrane fluidity was studied. Steroids, such as estrogen, are known to be membrane-active and can alter the lipid packing. In this study we used fluorescent methodologies to address the effect of naturally occurring steroids (17alpha and 17beta-estradiol, testosterone, and progesterone) and new estratriene analogs on membrane fluidity using liposomes and HT-22 hippocampal cells. The study's results indicate steroids, based on the estratriene nucleus, can modulate lipid packing as evidenced by (1) decreased membrane fusion events and (2) decreased membrane fluidity. The effects on the membrane were both time and concentration dependent. It was also demonstrated through rational design estratriene analogs can be synthesized with enhanced membrane effects. Finally, in a glutamate-induced toxicity HT-22 model, we also demonstrated cellular protection with the estratriene-based molecules and analogs. The data suggest the plethora of cellular actions of estrogens may relate to or be influenced by membrane effects of the steroid.

Combinatorial lead optimization of a neuropeptide FF antagonist.

The tripeptide Pro-Gln-Arg-NH2, derivatized at the secondary amino group of the proline residue with 5-(dimethylamino)-1-naphthalenesulfonyl (dansyl-PQR-NH2), antagonizes the central anti-opioid action of neuropeptide FF in animals after systemic administration and, therefore, is a therapeutic lead to treat opiate withdrawal. For a combinatorial optimization to improve potency, libraries focused on the possible replacement of the proline and glutamine residues of this lead compound were obtained by a solid-phase split-and-mix method using coded amino acids (excluding cysteine) as building blocks. After screening for competitive binding against a radioiodinated neuropeptide FF analogue, 5-(dimethylamino)-1-naphthalenesulfonyl-Gly-Ser-Arg-NH2 (dansyl-GSR-NH2) has emerged as one of the compounds in the library with high affinity to the NPFF receptor and even with a moderate increase compared to dansyl-PQR-NH2 in its predicted ability to penetrate the central nervous system.

Synthesis and biological evaluation of 17beta-alkoxyestra-1,3, 5(10)-trienes as potential neuroprotectants against oxidative stress.

17beta-O-Alkyl ethers (methyl, ethyl, propyl, butyl, hexyl, and octyl) of estradiol were obtained from 3-O-benzyl-17beta-estradiol with sodium hydride/alkyl halide, followed by the removal of the O-benzyl protecting group via catalytic transfer hydrogenation. An increase compared to estradiol in the protection of neural (HT-22) cells against oxidative stress due to exposure of glutamate was furnished by higher (C-3 to C-8) alkyl ethers, while methyl and ethyl ethers decreased the neuroprotective effect significantly. Lipophilic (butyl and octyl) ethers blocking the phenolic hydroxyl (3-OH) of A-ring were inactive.

Exploratory pharmacokinetics and brain distribution study of a neuropeptide FF antagonist by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

Dansyl-Pro-Gln-Arg-NH(2), an N-terminally modified tripeptide amide and a putative neuropeptide FF antagonist, was amenable to both positive-ion ESI and APCI. The protonated molecule yielded several fragment ions upon collision-induced dissociation in a quadrupole ion trap instrument for the development of LC/MS/MS assay methods. ESI clearly outperformed APCI in limits of detection, and was the method of choice for coupling with narrow-bore reversed-phase liquid chromatography to assess the pharmacokinetic profile and brain concentration of the neuropeptide FF antagonist in experimental animals. While plasma could be analyzed after rapid sample preparation, brain tissue required cleanup (solid phase extraction) and preconcentration before injection, and the assay was prone to matrix interference. This study indicated a rapid disappearance of dansyl-Pro-Gln-Arg-NH(2) from the plasma and the brain, and modest CNS bioavailability after intravenous administration to rats.

Targeting drugs to the brain by redox chemical delivery systems.

Chemical delivery systems (CDSs) based on the redox conversion of a lipophilic dihydropyridine to an ionic, lipid-insoluble pyridinium salt have been developed to improve the access of therapeutic agents to the central nervous system. A dihydropyridinium-type CDS or a redox analog of the drug is sufficiently lipophilic to enter the brain by passive transport, then undergoes an enzymatic oxidation to an ionic pyridinium compound, which promotes retention in the CNS. At the same time, peripheral elimination of the entity is accelerated due to facile conversion of the CDS in the body. This review discusses chemical, physicochemical, biochemical, and biological aspects in relation to the principles and practical implementation of the redox brain-targeting approach to various classes of drugs. Representative examples to the brain-enhanced delivery of neurotransmitters, steroids, anticonvulsants, antibiotics, antiviral, anticancer and antidementia agents, and neuropeptides and their analogs are presented in detail. In vivo and in vitro studies and preliminary clinical data of several novel derivatives have been promising, which could lead to a practical use of the redox CDSs after proper pharmaceutical development. The investigations accentuate the need for considering physicochemical, metabolic, and pharmacokinetic properties in designing of carrier systems that are able to target drugs into the central nervous system.

Brain-targeted chemical delivery of [Leu2, Pip3]-TRH: synthesis and biological evaluation.

A chemical targeting system for [Leu2, Pip3]-TRH (Gln,Leu,Pip) was synthesized in order to allow its specific delivery to the central nervous system (CNS). Sequential metabolism of the obtained 'packaged' chemical delivery system, (CDS), DHT-Pro-Pro-Gln-Leu-Pip-OCh, should yield a 'locked-in' precursor following the oxidative conversion of the dihydrotrigonellyl (DHT) to the trigonellyl (T+) moiety, followed by removal of the cholesteryl function and cleavage of the T+-Pro-Pro by prolyl endopeptidase. The antagonism of barbiturate-induced sleeping time was used to assess the activity of the CDS. The sleeping time after administration of vehicle and [Leu2]-TRH was 100.5 +/- 6.3 min, and 78.2 +/- 4.7 min, respectively. The [Leu2, Pip3]-TRH-CDS showed a significant decrease in sleeping time (58.2 +/- 3.4 min) compared to the vehicle or [Leu2]-TRH. These results indicate successful brain delivery of the precursor construct, and an effective release of the active GlnLeuPip in the brain.

Metabolism-based brain-targeting system for a thyrotropin-releasing hormone analogue.

Gln-Leu-Pro-Gly, a progenitor sequence for the thyrotropin-releasing hormone (TRH) analogue [Leu(2)]TRH (pGlu-Leu-Pro-NH(2)), was covalently and bioreversibly modified on its N- and C-termini (by a 1,4-dihydrotrigonellyl and a cholesteryl group, respectively) to create lipoidal brain-targeting systems for the TRH analogue. The mechanism of targeting and the recovery of the parent peptide at the target site involve several enzymatic steps, including the oxidation of the 1,4-dihydropyridine moiety. Due to the lipid insolublity of the peptide pyridinium conjugate obtained after this reaction, one of the rudimentary steps of brain targeting (i.e., trapping in the central nervous system) can be accomplished. Our design also included spacer amino acid(s) inserted between the N-terminal residue of the progenitor sequence and the dihydrotrigonellyl group to facilitate the posttargeting removal of the attached modification. The release of the TRH analogue in the brain is orchestrated by a sequential metabolism utilizing esterase/lipase, peptidyl glycine alpha-amidating monooxygenase (PAM), peptidase cleavage, and glutaminyl cyclase. In addition to in vitro experiments to prove the designed mechanism of action, the efficacy of brain targeting for [Leu(2)]TRH administered in the form of chemical-targeting systems containing the embedded progenitor sequence was monitored by the antagonistic effect of the peptide on the barbiturate-induced anesthesia (measure of the activational effect on cholinergic neurons) in mice, and considerable improvement was achieved over the efficacy of the parent peptide upon using this paradigm.

Metabolism-based drug design and drug targeting.

Even at the early stages of drug discovery and structure-based drug design, the pharmacokinetic, pharmacodynamic and toxicological consequences of drug metabolism cannot be ignored. Drug metabolism is also of interest to medicinal chemists in the design of drugs with controlled, predictable deactivation after achieving the therapeutic objective in prodrug design and in chemical-enzymatic drug targeting. In this review, the authors provide an overview of concepts that can be utilized from drug discovery to pharmaceutical development to overcome problems associated with drug metabolism, or that may be used to take advantage of 'designed-in' metabolic activation to achieve drug targeting.

In vitro and in vivo study of water-soluble prodrugs of dexanabinol.

Trialkylammonium acetoxymethyl esters of dexanabinol were synthesized and evaluated as water-soluble prodrugs. Syntheses were performed by conventional methods; solubility in water and stability in buffers and human plasma were determined by HPLC, and in vivo tissue distribution studies were performed in a rat model. Most of the new derivatives were soluble in water (approximately 50 mg/mL). They were relatively stable in water, while rapidly hydrolyzed in human plasma. Distribution studies indicated that peak concentrations of drug both in blood (30 microg/mL) and brain (2 microg/mL) were rapidly (5 min) achieved after iv administration of a selected prodrug to rats. The blood concentration decreased faster than brain levels which were detectable even after 24 h. Some of the examined esters could be further developed as water soluble prodrugs of dexanabinol.

Microcolumn size-exclusion chromatography coupled with electrospray ionization mass spectrometry.

Microcolumn (250 x 0.5 mm I.D.) size-exclusion chromatography was implemented for the separation of polydisperse mixtures prior to electrospray ionization (ESI) mass spectrometric detection. An improved separation, compared to conventional-bore SEC, was demonstrated upon coupling with ESI quadrupole ion-trap mass spectrometry and a Fourier transform ion cyclotron resonance instrument for the separation of individual oligomers present in octylphenoxypoly(ethoxy)ethanol.

Gel permeation chromatography coupled to fourier transform mass spectrometry for polymer characterization.

We report an on-line coupling of gel permeation chromatography (GPC) to Fourier transform mass spectrometry (FTMS) using a modified commercial electrospray ionization (ESI) interface. Selected oligomer profiles for the sodiated (1+ through 5+ charge states) oligomer ions of a narrow-molecular-weight poly(methyl methacrylate) were generated and used for obtaining a calibration curve. Using the MS-generated calibration curve and the refractive index response for quantification, an accurate molecular weight distribution was calculated and showed an excellent agreement with the value specified by the supplier. GPC/ESI/FTMS also allowed for an unequivocal end-group determination and characterization of a secondary distribution due to the formation of cyclic reaction products. We analyzed a glycidyl methacrylate/butyl methacrylate copolymer with a broad molecular weight distribution, where fractionation and high resolving power were required for adequate characterization. Molecular weight distribution data showed the advantage of coupling high-resolution MS and GPC to overcome the difficulty of analyzing polydisperse polymers with MS alone.

Identification of synaptic metabolites of dynorphin A (1-8) by electrospray ionization and tandem mass spectrometry.

Synaptic metabolism of the endogenous opioid octapeptide dynorphin (Dyn) A (1-8) (Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile) was studied in vitro upon its incubation with synaptosomes and with synaptosomal plasma membranes isolated from rat brain tissue. Electrospray ionization (ESI) and tandem mass spectromety were performed using an ion trap instrument, and afforded the identification of Dyn A (2-8), Dyn A (1-6) and leucine enkephalin [Dyn A (1-5)] as major metabolites. Preliminary quantitative data on the kinetics of Dyn A (1-8) degradation and metabolite formation was obtained by size-exclusion chromatography/ESI tandem mass spectrometry, and revealed an apparent involvement of several enzymes in the metabolism upon incubation with synaptosomes. Predominant formation of Dyn A (1-6) was observed with the synaptosomal plasma membranes.

Strategies to target kyotorphin analogues to the brain.

The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound contains the active peptide in a packaged, disguised form, flanked between the lipophilic cholesteryl ester on the C-terminus and the 1, 4-dihydrotrigonellyl redox targetor, attached to the N-terminus through strategically selected L-amino acid(s) spacer. It was found that for successful brain targeting, the epsilon-amine of Lys needs to be also converted to a lipophilic function. Through sequential enzymatic bioactivation, the Tyr-Lys dipeptide is released in a sustained manner, producing significant and prolonged analgesic activity as demonstrated by the rat tail latency test. An alternate strategy was also employed. Lys was replaced by a redox amino acid pair, Nys+ left and right arrow Nys, the nicotinamide left and right arrow 1,4-dihydronicotinamide analogues of Lys (Nys+ is 2-amino-6-(3-carbamoyl-1-pyridiniumyl)hexanoic acid). The Nys form is lipophilic and facilitates delivery in addition to the C- and N-terminal lipophilic functions. Enzymatic oxidation to Nys+ provides the lock-in, followed by removal of the lipophilic groups, releasing Tyr-Nys+ from the brain-targeted analogue (BTRA). Nys+ was shown to be an effective substitution for Arg or Lys. The activities of the CDS and BTRA, respectively, were antagonized by naloxone, supporting the designed brain-targeted processes. The most potent compound is the two-proline spacer containing CDS (CDS-PP), followed by the BTRA.