Alevtina D. Zharikova

Quinol-based cyclic antioxidant mechanism in estrogen neuroprotection

Substantial evidence now exists that intrinsic free-radical scavenging contributes to the receptor-independent neuroprotective effects of estrogens. This activity is inherently associated with the presence of a phenolic A-ring in the steroid. We report a previously unrecognized antioxidant cycle that maintains the “chemical shield” raised by estrogens against the most harmful reactive oxygen species, the hydroxyl radical (•OH) produced by the Fenton reaction. In this cycle, the capture of •OH was shown to produce a nonphenolic quinol with no affinity to the estrogen receptors. This quinol is then rapidly converted back to the parent estrogen via an enzyme-catalyzed reduction by using NAD(P)H as a coenzyme (reductant) and, unlike redox cycling of catechol estrogens, without the production of reactive oxygen species. Due to this process, protection of neuronal cells against oxidative stress is also possible by quinols that essentially act as prodrugs for the active hormone. We have shown that the quinol obtained from a 17β-estradiol derivative was, indeed, able to attenuate glutamate-induced oxidative stress in cultured hippocampus-derived HT-22 cells. Estrone quinol was also equipotent with its parent estrogen in reducing lesion volume in ovariectomized rats after transient middle carotid artery occlusion followed by a 24-h reperfusion. These findings may establish the foundation for a rational design of neuroprotective antioxidants focusing on steroidal quinols as unique molecular leads.

Proteomic analysis of the synaptic plasma membrane fraction isolated from rat forebrain.

Mass spectrometry (MS) in conjunction with liquid chromatography and gel separation techniques has been utilized to identify synaptic plasma membrane (SPM) proteins isolated from rat forebrain and digested with the protease trypsin. Initial results employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation of the SPM protein mixture have shown that several membrane proteins were under-represented due to solubilization problems in the dimension of isoelectric-point focusing. Given the complexity of the SPM, multiple stages of separation were necessary prior to mass spectrometric detection in order to facilitate protein identification. This particular study involved several approaches using one-dimensional (1D) sodium dodecyl sulfate (SDS)-PAGE, strong cation-exchange (SCX) chromatography and capillary reversed-phase high performance liquid chromatography (HPLC) techniques. In addition to these gel and HPLC separation stages, complementary information was obtained by using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Data-dependent acquisition employing capillary HPLC-nanoESI/MS allowed for the detection of low-abundance tryptic peptides in the digested SPM fraction and identification of the corresponding proteins when product-ion information of a single or multiple peptides was used in protein database searching. The potential value of this subproteome methodology was exemplified by the identification of several proteins relevant to synaptic physiology which included various transporters, receptors, ion channels, and enzymes.

Electrospray ionization mass spectrometric and liquid chromatographic–mass spectrometric studies on the metabolism of synthetic dynorphin A peptides in brain tissue in vitro and in vivo

Metabolic stability of synthetic dynorphins [N-terminal fragments of dynorphin A (Dyn A)] were evaluated in vitro and in vivo. These peptides were applied at concentrations 100-1000 times higher than those of the endogenous dynorphins. Degradation kinetics of these peptides were studied in rat brain homogenate by using microbore gradient RP-LC assay, and limited information on their metabolism was obtained by electrospray ionization mass spectrometry (ESI-MS) of the isolated metabolites. In vivo cerebral microdialysis, in which the peptides were introduced via the probe placed in striatum region of the brain of the experimental animals, was used to circumvent contamination arising from autoproteolysis of brain during incubation of the samples in vitro. Metabolites of Dyn A (1-13) and Dyn A (1-11) were identified from electrospray ionization mass spectra of the microdialysates without chromatographic separation; the identification of peptides in the mixtures were supported by medium resolution ESI Fourier-transform ion cyclotron resonance MS. LC-MS was used to fully characterize the complex peptide mixture obtained after the striatal perfusion of Dyn A (1-12).

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.

Prodrugs to enhance central nervous system effects of the TRH-like peptide pGlu-Glu-Pro-NH2.

Potential prodrugs for the TRH-like tripeptide pGlu-Glu-Pro-NH(2) were synthesized either by esterifying the Glu side-chain of the parent peptide in solution with alcohols in the presence of resin-bound dicyclohexylcarbodiimide or by solid-phase peptide chemistry. Affinities of these ester prodrugs to lipid membranes as predictors of the transport across the blood-brain barrier were compared by immobilized artificial membrane chromatography, and prodrug activation was tested in the brain tissue of experimental animals. Esters of pGlu-Glu-Pro-NH(2) with long-chain primary alcohols emerged as potentially useful prodrugs to improve the central nervous system activity of pGlu-Glu-Pro-NH(2) upon systemic administration, as revealed by the enhancement of analeptic activity in mice.

Intermittent high-dose ethanol exposures increase motivation for operant ethanol self-administration: possible neurochemical mechanism.

We investigated the neurochemical mechanism of how high-dose ethanol exposure may increase motivation for ethanol consumption. First, we developed an animal model of increased motivation for ethanol using a progressive ratio (PR) schedule. Sprague-Dawley rats were trained to administer 10% ethanol-containing gelatin or plain gelatin (on alternate weeks) in daily 30-min sessions under different fixed ratio (FR) and PR schedules. During FR schedules, rats self-administered about 1 g/kg ethanol, which was decreased to 0.4+/-0.03 g/kg under PR10. Rats then received four pairs of either 3 g/kg ethanol or saline injections during the weeks when the reinforcer was plain gelatin. During subsequent ethanol gel sessions, breakpoints and ethanol consumption rose 40% in the high-dose ethanol group by the fourth set of injections with no change in plain gel responding. Alterations in amino acids in the ventral striatum (VS) during PR10 responding for 10% ethanol gelatin and plain gelatin were measured using microdialysis sampling coupled with capillary electrophoresis and laser-induced fluorescence detection. There was greater release of taurine, glycine and glutamate in the NAC of the high-dose ethanol rats during 10% ethanol-containing gelatin responding, compared to the control rats or during plain gel responding. An increase in the release of glycine in this same brain region has recently been shown to be involved with anticipation of a reward. Thus, it appears that intermittent high-dose ethanol exposure not only increases motivation for ethanol responding but may also change neurotransmitter release that mediates anticipation of reinforcement, which may play a key role in the development of alcoholism.

Determination of clodronate content in liposomal formulation by capillary zone electrophoresis

Liposome-mediated intracellular delivery of clodronate is reported to selectively deplete mononuclear phagocytic cells such as macrophages that are important effector cells involved in the pathogenesis of neuropathies associated with demyelination and destruction of neuronal cells. Application of liposome-encapsulated clodronate (dichloromethylenediphosphonic acid disodium salt) is a method of choice to deplete macrophages to prevent such a neurodegeneration. In the present work, a comparison of an ion-exchange chromatography (IEC) and a capillary zone electrophoresis (CZE) method with indirect UV detection was performed and, based on the results of this comparison, a CZE assay was developed for quantitation of clodronate in mannosylated liposomes. This CZE method employed Nitroso-R salt (1-nitroso-2-naphthol-3,6-disulphonic acid disodium salt) as background electrolyte with UV detection of the analyte at 254 nm. The assay for the determination of clodronate in mannosylated liposomes after their solubilization in 10 mM Triton X-100 showed acceptable within-day precision (repeatability), day-to-day precision (reproducibility) and linearity in the target quantitation range of 0.5-10.0 mg ml(-1). The method reported here can be used as part of the quality control during the preparation of liposome-encapsulated clodronate as a drug formulation for macrophage-mediated diseases.

Design, synthesis, and biological evaluation of novel, centrally-Acting thyrotropin-Releasing hormone analogues

Novel, metabolically stable and centrally acting TRH analogues with substituted pyridinium moieties replacing the [His(2)] residue of the endogenous peptide were prepared by solid-phase Zincke reaction. The 1,4-dihydropyridine prodrugs of these analogues obtained after reducing the pyridinium moiety were able to reach the brain and maintain a sustained concentration of the charged, degradation-resistant analogues formed after enzymatic oxidation of the prodrug, as manifested by the analeptic action measured in mice. Among the four analogues reported, compound 2a showed the highest potency and longest duration of action in reducing the pentobarbital-induced sleeping time compared to the parent TRH. No binding to the endocrine TRH-receptor was measured for 2a; thus, this compound emerged as a potent, centrally acting TRH analogue.

Cardiovascular effects of neuropeptide FF antagonists

The neuropeptide FF (NPFF) antagonist desaminotyrosyl-Phe-Leu-Phe-Gln-Pro-Gln-Arg-NH2 dose-dependently reversed NPFF-induced elevation of blood pressure in anesthetized rats after intravenous injection without causing a significant change of blood pressure and heart rate by itself. However, another antagonist dansyl-Pro-Gln-Arg-NH2 produced a significant drop of the mean arterial pressure only at a large dose (10 micromol/kg body weight), but reversal of the NPFF-induced hypertension was modest. Consequently and contrary to the conclusions of a previous study, NPFF antagonists cannot be identified simply by measuring the changes in the hemodynamic parameters upon the injection of the compounds alone and without a subsequent NPFF challenge.

Neuropharmacodynamic evaluation of the centrally active thyrotropin-releasing hormone analogue [Leu2]TRH and its chemical brain-targeting system.

The centrally active thyrotropin-releasing hormone (TRH) analogue pGlu-Leu-Pro-NH(2) ([Leu(2)]TRH) showed a significant increase in the extracellular acetylcholine concentration during its perfusion to the hippocampus in rats, and this effect was manifested upon the delivery of the analogue in much smaller quantities compared to TRH when measured by in vivo intracranial microdialysis. The neuropharmacodynamic efficacy of [Leu(2)]TRH upon intravenous administration was augmented by the use of a brain-targeting derivative in which the progenitor sequence of the mature peptide was embedded in a molecular architecture that promoted enhanced brain delivery, retention and in situ generation of the pharmacologically active molecule. Compared to the unmodified peptide, the targeting system significantly improved the cumulative effect of the treatment on extracellular acetylcholine levels in rats.