Design, stereoselective synthesis, configurational stability and biological activity of 7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide

Chiral 5-arylbenzothiadiazine derivatives have recently attracted particular attention because they exhibit an interesting pharmacological activity as AMPA receptor (AMPAr) positive modulators. However, investigations on their configurational stability suggest a rapid enantiomerization in physiological conditions. In order to enhance configurational stability, preserving AMPAr activity, we have designed the novel compound (R,S)-7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide bearing a pyrrolo moiety coupled with the 5-(furan-3-yl) substituent on benzothiadiazine core. A stereoselective synthesis was projected to obtain single enantiomer of the latter compound. Absolute configuration was assigned by X-ray crystal structure. Patch clamp experiments evaluating the activity of single enantiomers as AMPAr positive allosteric modulator showed that R stereoisomer is the active component. Molecular modeling studies were performed to explain biological results. An on-column stopped-flow bidimensional recycling HPLC procedure was applied to obtain on a large scale the active enantiomer with enantiomeric enrichment starting from the racemic mixture of the compound

JLB: a flexible and effective device in critical patients. Review of clinical cases

JLB catheter (Deltamed Inc) is an alternative way to manage difficult venous access; it is placed under US-guidance in large bore veins, with an easy-sterile approach. Internal jugular vein (IJV) is the first choice for cannulation, followed by subclavian or deep upper-arm veins. The catheter is available in different lengths and gauges, it allows high flow rates and can be left in place up to 30 days. From June 2015 to March 2017, JLB has been positioned in 409 patients: in 354 as primary access in IJV, brachial or subclavian vein; in 55 cases JLB became an introducing line for the Seldinger guidewire and further CVC positioning. All clinical cases were reviewed selecting those with greater clinical relevance. We report 8 cases in which JLB resulted determinant for the patient treatment: a 16 years old obese girl born with perinatal distress, a 78 years old obese woman with hemorrhagic shock caused by gastrointestinal bleeding, a 40 years old man with severe hypokalemia, a 30 years old man with severe sepsis, a 40 years old man with Becker’s muscular dystrophy and severe sepsis, a 40 years old man with multiple myeloma who had to carry out cycles of chemotherapy, a 76 years old man with CMV pancolitis and myelofibrosis who needed parenteral nutrition, antiviral therapy and frequent blood and platelets transfusion. Moreover, it has been useful in elderly patients who needed to carry out palliative care for seniority or cancer lasting up to 30 days . In our experience the JLB catheter is safe, easy to place, quick and cost –effective. It is a valid solution either in unstable patients requiring an immediate access in emergency and stable patients with difficult venous access, in which invasive devices can be considered an over-treatment

IS PREGNENOLONE SULFATE AN ENDOGENOUS MODULATOR OF NICOTINIC ACH RECEPTOR?

Pregnenolone sulfate (PregS) is an important endogenous modulator of brain activity. PregS reduces the inhibitory activity of GABAA receptors, increases excitatory neurotransmission potentiating NMDA receptors function, trafficking and glutamate release. These latter actions are mediated by PregS at the level of sigma I receptor, calcium and TRP channels [1]. The peleiotropic mechanism of action of PregS could explain some of the pharmacological effects of the steroid: in preclinical studies PregS is neuroprotective, it potentiates synaptic LTP and improve spatial cognitive performance in rats eventhough the pro-mnestic effect of PregS likely depends on the mode of administration and behavioral model being tested [2]. Because cholinergic transmission is fundamental for cognitive functions such as learning and memory we studied the effect of PregS on the activity of nicotinic Ach receptors (nAchR). Using the patch-clamp technique in the whole-cell configuration we tested PregS on the currents mediated by nAChRs expressed in SH-SY5Y cells and in neurons grown in primary cultures. In SH-SY5Y application of PregS reduced NIC (10 μM) - evoked currents with an IC50 in the low micromolar range (14+9 μM) and a maximal effect of 58+10 %. To investigate the mechanism of PregS antagonism we applied the same concentration (10 μM) of the neurosteroid together with different concentrations of NIC (from 5 to 100 μM). PregS effect was not dependent on agonist concentration suggesting a non competitive mechanism of action. The analysis of the dose-response curves of PregS effect at 10 and 100 μM NIC evidentiates that potency and efficacy of the steroid is unchanged. PregS reduces nAchR-mediated currents by increasing receptor desensitization and this effect is more pronounced at high agonist concentrations. Experiments in primary cultures of cortical and cerebellar neurons have confirmed the antagonistic effect of PregS on NIC-evoked current. The inhibitory effect of PregS of nAchR- mediated current cannot support its activity as cognitivie enhancer. To find a rationale it has to be taken into account an additional complexity resulting from the activity of PregS on a neuronal network: it is possible that the reduction of cholinergic activity be limited to specific inhibitory circuits and that the overall effect will be an improved excitatory neurotransmission. It is also conceivable that PregS effect could be served as a compensatory mechanis

Simultaneous determination of pregnenolone sulphate, dehydroepiandrosterone and allopregnanolone in rat brain areas by liquid chromatography-electrospray tandem mass spectrometry

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain

Modulatory effects of neurosteroids and thyroid hormones on GABA-evoked currents in cultured dorsal root ganglion cells

Neurosteroids (NSs) and Thyroid hormones (THs, T3, triiodothyronine and T4, thyroxine) are important endogenous modulators of GABAA receptor (GABAAR) function (Puia and Losi, 2011). The involvement of NSs in several physiological and pathological processes has been largely acknowledged, among them pain transmission. Several studies revealed the antinociceptive properties of some NSs and demonstrated that they can induce a potent peripheral analgesia via a direct GABAAR allosteric modulation (Poisbeau et al., 2014). Very little is known instead of how THs affect synaptic transmission in structures devoted to pain transmission. Pain sensitivity is related to the thyroid status, indeed hyperthyroidism confers greater sensitivity to thermal noxious stimuli (Edmondson et al., 1990) and alters the nociceptive responses in rats (Bruno et al., 2005). Dorsal root ganglion (DRG) cells are primary sensory neurons playing important roles in pain transmission between periphery and CNS. By using the patch clamp technique in the whole cell configuration we analyzed the effect of THs and of some NSs (Pregnenolone Sulfate, PS, and Allopregnenolone, ALLO) on GABA-evoked currents in rat DRG cells grown in primary cultures T3, T4 and PS (from 500 nM to 50 µM) reduce GABA-evoked currents with an IC50 of 0.8±0.3μM for T3 (eff max =-42±9%), of 1.4±0.7 μM for T4 (eff max =-41±6%) and of 4.3±1.2μM (eff max =-60±8%) for PS. ALLO potentiates GABA-evoked current in DRG neurons with an IC50 of 1.3±0.8 μM and a maximal effect of 110±20%. To investigate the mechanism of action of THs and PS we applied increasing concentrations of GABA (5, 10 and 50 µM) to the same concentration of modulator (10 µM). The effect of T3, T4 and PS was not dependent on the GABA concentration used suggesting that they act in a non-competitive way. The modulatory activity of THs and PS on sIPSCs amplitude and frequency measured in lamina II neurons of acutely dissociated spinal cord slices was also investigated. In conclusion, since DRG neurosteroidogenesis is a physiologically relevant process (Schaeffer et al., 2010), our findings suggest that NSs modulation of GABAAR in this cells could play an important role in pain transmission from periphery to spinal cord. Furthermore the decreased GABAAR activity induced by T3 and T4 result in a reduced inhibitory neurotransmission that could contribute to the increased pain sensitivity detected in hyperthyroid animals

An unexpected reversal in the pharmacological stereoselectivity of benzothiadiazine AMPA positive allosteric modulators

Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure-activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD

Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50 = 26 ± 6 μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets

7-Chloro-5-(furan-3-yl)-3-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-Dioxide as Positive Allosteric Modulator of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor. The End of the Unsaturated-Inactive Paradigm?

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1