Vien Nguyen

17β-Estradiol eye drops protect the retinal ganglion cell layer and preserve visual function in an in vivo model of glaucoma

Neuroprotection in glaucoma as a curative strategy complementary to current therapies to lower intraocular pressure (IOP) is highly desirable. This study was designed to investigate neuroprotection by 17β-estradiol (E2) to prevent retinal ganglion cell (RGC) death in a glaucoma model of surgically elevated IOP in rats. We found that daily treatment with E2-containing eye drops resulted in significant E2 concentration in the retina with concomitant profound neuroprotective therapeutic benefits, even in the presence of continually elevated IOP. The number of apoptotic cells in the RGC layer was significantly decreased in the E2-treated group, when compared to the vehicle-treated controls. Deterioration in visual acuity in these animals was also markedly prevented. Using mass spectrometry-based proteomics, beneficial changes in the expression of several proteins implicated in the maintenance of retinal health were also found in the retina of E2-treated animals. On the other hand, systemic side effects could not be avoided with the eye drops, as confirmed by the measured high circulating estrogen levels and through the assessment of the uterus representing a typical hormone-sensitive peripheral organ. Collectively, the demonstrated significant neuroprotective effect of topical E2 in the selected animal model of glaucoma provides a clear rationale for further studies aiming at targeting E2 into the eye while avoiding systemic E2 exposure to diminish undesirable off-target side effects.

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.

Separation of dansylated 17β-estradiol, 17α-estradiol and estrone on a single HPLC column for simultaneous quantitation by LC-MS/MS

AbstractWe show here that baseline separation of dansylated estrone, 17β-estradiol, and 17α-estradiol can be done, contrary to previous reports, within a short run time on a single RP-LC analytical column packed with particles bonded with phenyl-hexyl stationary phase. The chromatographic method coupled with isotope dilution tandem MS offers a simple assay enabling the simultaneous analysis of these analytes. The method employs 13C-labeled estrogens as internal standards to eliminate potential matrix effects arising from the use of deuterated estrogens. The assay also offers adequate accuracy and sensitivity to be useful for biological samples. The practical applicability of the validated method is demonstrated by the quantitative analyses of in vivo samples obtained from rats treated with Premarin®. FigureQuantification of estrogens from rat samples by LC–MS/MS

The prodrug DHED selectively delivers 17β-estradiol to the brain for treating estrogen-responsive disorders

The bioprecursor prodrug 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED) allows 17β-estradiol to form in the female brain for neuroprotection and to treat neurological and psychiatric symptoms of menopause. Estrogen prodrug protects the brain Although estrogen is considered to heal the brain of many neurological and psychiatric symptoms, targeting the hormone to the brain only—and avoiding activity in other tissues—is no trivial feat. Prokai et al. discovered a “prodrug,” called DHED (10β, 17β-dihydroxyestra-1,4-dien-3-one), that is selectively converted to one main estrogen, 17β-estradiol, only in the brain. Similar to 17β-estradiol itself, DHED protected menopausal female rodents from neurological symptoms of estrogen deprivation, and also provided neuroprotection to rats after stroke, but without the negative systemic (specifically uterotrophic and cancerous) effects of the free hormone. With an efficacious and safe profile in vivo, this prodrug has the opportunity to provide positive estrogenic effects on the brain and relieve symptoms in patients with a broad range of central nervous system diseases. Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17β-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17β-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17β-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

Exploratory neuropharmacological evaluation of a conformationally constrained thyrotropin-releasing hormone analogue

A conformationally constrained peptidomimetic derived from the endocrine and neuroactive tripeptide thyrotropin-releasing hormone (pGlu-His-Pro-NH(2)) was synthesized by convenient solid-phase organic chemistry and evaluated as a potential central nervous system agent. While this ethylene-bridged peptide analogue has been reported to lack the hormonal effect of the native peptide, we have shown in animal models that it possesses central nervous system activity characteristic of thyrotropin-releasing hormone. Compared to control, the peptidomimetic showed significant analeptic and antidepressant-like potencies. Moreover, an enhanced selectivity in antidepressant-like effect was measured when compared to that of the native peptide. Immobilized artificial membrane chromatography and in vitro metabolic stability studies also revealed that this constrained peptidomimetic has higher affinity to the blood-brain barrier than the native peptide and is metabolically stable. Consequently, this structure may be used as a template to design centrally selective and metabolically stable thyrotropin-releasing hormone analogues as potential neuropharmaceutical agents.

Treatment with an orally bioavailable prodrug of 17β-estradiol alleviates hot flushes without hormonal effects in the periphery.

Estrogen deprivation has a profound effect on the female brain. One of the most obvious examples of this condition is hot flushes. Although estrogens relieve these typical climacteric symptoms, many women do not want to take them owing to unwanted side-effects impacting, for example, the uterus, breast and blood. Therefore, there is a need for developing safer estrogen therapies. We show here that treatment with 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), a novel brain-targeting bioprecursor prodrug of the main human estrogen, 17β-estradiol, alleviates hot flushes in rat models of thermoregulatory dysfunction of the brain. Oral administration of DHED elicits a significant reduction of tail skin temperature (TST) rise representing hot flushes in the morphine-dependent ovariectomized rat model and results in the restoration of estrogen deprivation-induced loss of diurnal rhythm in TST. These beneficial effects occur without detrimental peripheral hormonal exposure; thus, the treatment avoids potentially harmful stimulation of estrogen-sensitive peripheral organs, including the uterus and the anterior pituitary, or the proliferation of MCF-7a breast cancer cell xenografts. Our promising preclinical assessments warrant further considerations of DHED for the development of a brain-selective 17β-estradiol therapy to relieve hot flushes without undesirable peripheral side-effects.

Evidence for interplay between thyrotropin-releasing hormone (TRH) and its structural analogue pGlu-Glu-Pro-NH2 ([Glu2]TRH) in the brain: An in vivo microdialysis study

Local perfusion of pGlu-Glu-Pro-NH2, an endogenous peptide structurally related to thyrotropine-releasing hormone (TRH), via in vivo microdialysis into the rat hippocampus did not change the basal level of extracellular acetylcholine. However, co-perfusion of pGlu-Glu-Pro-NH2 with TRH in equimolar concentrations yielded a significant attenuation of TRH-induced acetylcholine release. The results have supported the studys hypothesis that pGlu-Glu-Pro-NH2 opposes the cholinergic effect of TRH in the mammalian central nervous system. The enantiomer pGlu-d-Glu-Pro-NH2 affected neither basal extracellular nor TRH-induced increase of acetylcholine levels.

Design and Exploratory Neuropharmacological Evaluation of Novel Thyrotropin-Releasing Hormone Analogs and Their Brain-Targeting Bioprecursor Prodrugs

Efforts to take advantage of the beneficial activities of thyrotropin-releasing hormone (TRH) in the brain are hampered by its poor metabolic stability and lack of adequate central nervous system bioavailability. We report here novel and metabolically stable analogs that we derived from TRH by replacing its amino-terminal pyroglutamyl (pGlu) residue with pyridinium-containing moieties. Exploratory studies have shown that the resultant compounds were successfully delivered into the mouse brain after systemic administration via their bioprecursor prodrugs, where they manifested neuropharmacological responses characteristic of the endogenous parent peptide. On the other hand, the loss of potency compared to TRH in a model testing antidepressant-like effect with a simultaneous preservation of analeptic activity has been observed, when pGlu was replaced with trigonelloyl residue. This finding may indicate an opportunity for designing TRH analogs with potential selectivity towards cholinergic effects.

[Glu2]TRH dose-dependently attenuates TRH-evoked analeptic effect in mice

Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH(2)) and the structurally related [Glu(2)]TRH (pGlu-Glu-Pro-NH(2)) are endogenous peptides with a plethora of actions in the central nervous system. Many centrally-mediated effects of TRH are shared with those of [Glu(2)]TRH, although the involvement of different receptors is presumed. The analeptic action is the best-known TRH-related central nervous system effect. While [Glu(2)]TRH itself is analeptic, its co-administration with TRH into mice produced a dose-dependent attenuation of TRH-evoked reversal of barbiturate-induced sleeping time. This finding is in agreement with our previous observations that [Glu(2)]TRH significantly attenuates TRH-induced hippocampal extracellular acetylcholine release. Taken together, [Glu(2)]TRH may be considered as a negative modulator for the cholinergic effect of TRH in the mouse brain.

Mass spectrometric analysis of carisoprodol and meprobamate in rat brain microdialysates

We report the evaluation of several mass spectrometry-based methods for the determination of carisoprodol and meprobamate in samples obtained from the rat brain by in vivo intracranial microdialyis. Among the techniques that aspire to perform analyses without chromatographic separation and thereby increase throughput, chip-based nanoelectrospray ionization and the use of an atmospheric pressure solids analysis probe fell short of requirements because of insufficient detection sensitivity and hard ionization, respectively. Although direct analysis in real time provided the required soft ionization, shortcomings of a tandem mass spectrometry-based assay also included inadequate detection sensitivity and, in addition, poor quantitative reproducibility. Therefore, liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry was developed to determine carisoprodol and meprobamate from artificial cerebrospinal fluid as the medium. No desalting and/or extraction of the samples was necessary. The assay, combined with in vivo sampling via intracranial microdialyis, afforded time-resolved concentration profiles for the drug and its major metabolite from the nucleus accumbens region of the brain in rats after systemic administration of carisoprodol. Copyright