Bioanalytical Method Development and Validation of Dapoxetine Hydrochloride in Human Plasma by RP-HPLC

A simple, accurate and rapid Bioanalytical reverse phase high performance liquid chromatography (RPHPLC) method for determination of Dapoxetin hydrochloride in human plasma was validated as per ICH guideline. Dapoxetin hydrochloride is significantly superior in premature ejaculation and more active against serotonin transport inhibitor than any other drug in class. The total analysis was carried out on using stationary phase symmetry C1 (4.6mm X 250mm, 5µm) with Mobile Phase Acetonitrile: Buffer (60:40) pH adjusted to3.5 flow rate was 1.0 ml/min, injection volume of 10 ppm and detection wavelength was 293nm at ambient temperature with total run time of 10 minutes. Retention time of spiked plasma and dapoxetine hydrochloride were found to be 2.153 min and 4.442 min, r2 value were 0.995 and 0.999 and linearity range was 5ppm to 25ppm for both. The method was developed for accuracy, linearity, precision, recovery and stability in complies and stability in complies with CDER and ICH guideline.

A model of dopamine and serotonin-kynurenine metabolism in cortisolemia: Implications for depression

A major factor contributing to the etiology of depression is a neurochemical imbalance of the dopaminergic and serotonergic systems, which is caused by persistently high levels of circulating stress hormones. Here, a computational model is proposed to investigate the interplay between dopaminergic and serotonergic-kynurenine metabolism under cortisolemia and its consequences for the onset of depression. The model was formulated as a set of nonlinear ordinary differential equations represented with power-law functions. Parameter values were obtained from experimental data reported in the literature, biological databases, and other general information, and subsequently fine-tuned through optimization. Model simulations predict that changes in the kynurenine pathway, caused by elevated levels of cortisol, can increase the risk of neurotoxicity and lead to increased levels of 3,4-dihydroxyphenylaceltahyde (DOPAL) and 5-hydroxyindoleacetaldehyde (5-HIAL). These aldehydes contribute to alpha-synuclein aggregation and may cause mitochondrial fragmentation. Further model analysis demonstrated that the inhibition of both serotonin transport and kynurenine-3-monooxygenase decreased the levels of DOPAL and 5-HIAL and the neurotoxic risk often associated with depression. The mathematical model was also able to predict a novel role of the dopamine and serotonin metabolites DOPAL and 5-HIAL in the ethiology of depression, which is facilitated through increased cortisol levels. Finally, the model analysis suggests treatment with a combination of inhibitors of serotonin transport and kynurenine-3-monooxygenase as a potentially effective pharmacological strategy to revert the slow-down in monoamine neurotransmission that is often triggered by inflammation.

Chronic d-ribose and d-mannose overload induce depressive/anxiety-like behavior and spatial memory impairment in mice

The effects of different forms of monosaccharides on the brain remain unclear, though neuropsychiatric disorders undergo changes in glucose metabolism. This study assessed cell viability responses to five commonly consumed monosaccharides—D-ribose (RIB), D-glucose, D-mannose (MAN), D-xylose and L-arabinose—in cultured neuro-2a cells. Markedly decreased cell viability was observed in cells treated with RIB and MAN. We then showed that high-dose administration of RIB induced depressive- and anxiety-like behavior as well as spatial memory impairment in mice, while high-dose administration of MAN induced anxiety-like behavior and spatial memory impairment only. Moreover, significant pathological changes were observed in the hippocampus of high-dose RIB-treated mice by hematoxylin-eosin staining. Association analysis of the metabolome and transcriptome suggested that the anxiety-like behavior and spatial memory impairment induced by RIB and MAN may be attributed to the changes in four metabolites and 81 genes in the hippocampus, which is involved in amino acid metabolism and serotonin transport. In addition, combined with previous genome-wide association studies on depression, a correlation was found between the levels of Tnni3k and Tbx1 in the hippocampus and RIB induced depressive-like behavior. Finally, metabolite–gene network, qRT-PCR and western blot analysis showed that the insulin-POMC-MEK-TCF7L2 and MAPK-CREB-GRIN2A-CaMKII signaling pathways were respectively associated with RIB and MAN induced depressive/anxiety-like behavior and spatial memory impairment. Our findings clarified our understanding of the biological mechanisms underlying RIB and MAN induced depressive/anxiety-like behavior and spatial memory impairment in mice and highlighted the deleterious effects of high-dose RIB and MAN as long-term energy sources.

Serotonin stimulates Echinococcus multilocularis larval development

Background Serotonin is a phylogenetically ancient molecule that is widely distributed in most metazoans, including flatworms. In addition to its role as a neurotransmitter, serotonin acts as a morphogen and regulates developmental processes. Although several studies have focused on the serotonergic nervous system in parasitic flatworms, little is known on the role of serotonin in flatworm development. Methods To study the effects of serotonin on proliferation and development of the cestode Echinococcus multilocularis, we cloned the genes encoding the E. multilocularis serotonin transporter (SERT) and tryptophan hydroxylase (TPH), analyzed gene expression by transcriptome analysis and whole mount in situ hybridization (WMISH) and performed cell culture experiments. Results We first characterized orthologues encoding the SERT and TPH, the rate-limiting enzyme in serotonin biosynthesis. WMISH and transcriptomic analyses indicated that the genes for both SERT and TPH are expressed in the parasite nervous system. Long-term treatment of parasite stem cell cultures with serotonin stimulated development towards the parasite metacestode stage. Mature metacestode vesicles treated with serotonin showed increased rates of incorporation of the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), indicating stimulated cell proliferation. In contrast, treatment with the selective serotonin reuptake inhibitor paroxetine strongly affected the viability of parasite cells. Paroxetine also caused structural damage in metacestode vesicles, suggesting that serotonin transport is crucial for the integrity of parasite vesicles. Conclusions Our results indicate that serotonin plays an important role in E. multilocularis development and proliferation, providing evidence that the E. multilocularis SERT and TPH are expressed in the nervous system of the protoscolex. Our results further suggest that the E. multilocularis SERT has a secondary role outside the nervous system that is essential for parasite integrity and survival. Since serotonin stimulated E. multilocularis metacestode development and proliferation, serotonin might also contribute to the formation and growth of the parasite in the liver.

Chemical and structural investigation of the paroxetine-human serotonin transporter complex

Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

Chemical and structural investigation of the paroxetine-human serotonin transporter complex

ABSTRACTAntidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand the binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation. Such modifications to SERT have led to questions about the relationships of these structures to the bona fide conformation and inhibitor binding poses of the wild-type transporter. To address these concerns, we characterized wild-type SERT with truncated N- and C-termini and thermostabilized variants of SERT bound with paroxetine using x-ray crystallography, single particle cryo-EM and biochemical techniques. Moreover, using a C–H functionalization approach to synthesize enantiopure analogues, we replaced the halide of the fluorophenyl group in paroxetine with either bromine or iodine. We then exploited the anomalous scattering of Br and I to define the pose of the respective paroxetine analog. These structures provide mutually consistent insights into how paroxetine and its analogs bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.

Serotonin transport in the 21st century

Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides cytoplasmic 5-HT for refilling of synaptic vesicles. SERT is the target for many antidepressant medications as well as psychostimulants such as cocaine and ecstasy (3,4-methylenedioxymethamphetamine). SERT belongs to the SLC6 family of ion-coupled transporters and is structurally related to several other transporter families. SERT was studied in the 1970s and 1980s using membrane vesicles isolated from blood platelets. These studies led to a proposed stoichiometry of transport that has been challenged by high-resolution structures of SERT and its homologues and by studies of SERT electrophysiology. Here, we review the original evidence alongside more recent structural and electrophysiological evidence. A self-consistent picture emerges with surprising insights into the ion fluxes that accompany 5-HT transport.