Effects of Gamma-Tocotrienol on Intestinal Injury in a GI-Specific Acute Radiation Syndrome Model in Nonhuman Primate.

The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.

DRD1 agonist A-68930 improves mitochondrial dysfunction and cognitive deficits in a streptozotocin-induced mouse model.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by irreversible cognitive deficits and memory dysfunction. Dopamine is the most abundant catecholaminergic neurotransmitter in the brain which regulates motivation, reward, movement, and cognition. Recently, increasing evidences have shown that dopaminergic system is disturbed in AD conditions, and pharmacological interventions targeting dopamine D1 receptor (DRD1) exhibit certain therapeutic benefits in AD models. However, the underlying link between DRD1 and AD remains elusive. This study sought to test whether the selective DRD1 agonist A-68930 could improve streptozotocin (STZ)-induced cognitive impairment in mice. Here we found that A-68930 treatment through intraperitoneal injection efficiently alleviated STZ-induced cognitive deficits in mice. Moreover, our mechanism researches revealed that the DRD1 signaling induced by A-68930 significantly rescued STZ-induced mitochondrial biogenesis deficit, mitochondrial dysfunction, Aß overexpression, and tau phosphorylation in mice hippocampus and cortex and SH-SY5Y cells, which may be mediated through stimulating AMPK/PGC-1α pathway. This study indicates that DRD1 agonist A-68930 can improve STZ-induced cognitive deficits and mitochondrial dysfunction in vivo and in vitro, and DRD1 may represent an appropriate target candidate for AD drug development.

Dopamine D1 receptor agonist A-68930 ameliorates Aß1-42-induced cognitive impairment and neuroinflammation in mice.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by progressive cognitive dysfunction and memory impairment. Dopamine is an important catecholaminergic neurotransmitter that controls movement, reward, motivation, and cognition. Recently, dopamine receptors were reported to regulate immune system in both periphery and central nervous system. However, whether dopamine D1 receptor (DRD1) activation could improve neuroinflammation in AD conditions remains unknown. The present study aimed to investigate the therapeutic effects and underlying mechanisms of a potent and selective DRD1 agonist A-68930 on Aß1-42-induced mice. Here we showed that intraperitoneal injection of A-68930 significantly ameliorated Aß1-42-induced cognitive dysfunction in mice. Moreover, both in vivo and in vitro data showed that A-68930-induced DRD1 activation significantly inhibited NLRP3 inflammasome-dependent neuroinflammation induced by Aß1-42, and this effect may be mediated by the activation of AMPK/autophagy signaling pathway, which enhanced NLRP3 inflammasome degradation and thus decreased the secretion of IL-1ß and IL-18. The present study suggests that A-68930-induced DRD1 signaling efficiently alleviates Aß1-42-induced cognitive impairment and neuroinflammation in mice and BV2 cells, and DRD1 may become a promising therapeutic target for AD.

Dopamine D1 receptor agonist A68930 attenuates acute kidney injury by inhibiting NLRP3 inflammasome activation.

Renal ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI), characterized by tubulointerstitial inflammation. Currently, progress in developing effective therapies to prevent or ameliorate AKI by anti-inflammation remains slow. Emerging studies have suggested that NLRP3 (the NOD-, LRR- and pyrin domain-containing 3) inflammasome plays a key role in a wide spectrum of kidney disease models including I/R injury. In this study, we investigated the renal protective effects of A68930, a specific agonist for the D-1 dopamine receptor (DRD1), which was recently recognized to downregulate NLRP3 inflammasome via DRD1 signaling. AKI was induced by renal I/R injury and A68930 was intraperitoneally injected 3 times after renal reperfusion. We showed that A68930 significantly ameliorated renal dysfunction. Meanwhile, A68930 markedly reduced macrophages and T cells infiltration, renal pro-inflammatory cytokines production (TNF-α, IL-6, IL-1ß), serum pro-inflammatory cytokine (TNF-α and IL-6) and NLRP3 inflammasome activation. Additionally, A68930 attenuated I/R-induced mitochondria injury, which was observed by transmission electron microscopy. In summary, our results demonstrated that activation of DRD1 by A68930 inhibited renal and systematic inflammation, and improved kidney function in I/R induced AKI model, which was probably related to the inhibition of the NLRP3 inflammasome activation.

Discovery of Potent, Selective, and State-Dependent NaV1.7 Inhibitors with Robust Oral Efficacy in Pain Models: Structure-Activity Relationship and Optimization of Chroman and Indane Aryl Sulfonamides.

Voltage-gated sodium channel NaV1.7 is a genetically validated target for pain. Identification of NaV1.7 inhibitors with all of the desired properties to develop as an oral therapeutic for pain has been a major challenge. Herein, we report systematic structure-activity relationship (SAR) studies carried out to identify novel sulfonamide derivatives as potent, selective, and state-dependent NaV1.7 inhibitors for pain. Scaffold hopping from benzoxazine to chroman and indane bicyclic system followed by thiazole replacement on sulfonamide led to identification of lead molecules with significant improvement in solubility, selectivity over NaV1.5, and CYP2C9 inhibition. The lead molecules 13, 29, 32, 43, and 51 showed a favorable pharmacokinetics (PK) profile across different species and robust efficacy in veratridine and formalin-induced inflammatory pain models in mice. Compound 51 also showed significant effects on the CCI-induced neuropathic pain model. The profile of 51 indicated that it has the potential for further evaluation as a therapeutic for pain.

Enantiomer of the novel flexible heteroarotinoid, SL-1-09, blocks cell cycle progression in breast cancer cells.

Flexible heteroarotinoids (Flex-Hets) are compounds with promising anti-cancer activities. SHetA2, a first-generation Flex-Het, has been shown to inhibit the growth of cervical, head and neck, kidney, lung, ovarian, prostate, and breast cancers. However, SHetA2's high lipophilicity, limited selectivity, low oral bioavailability, and complicated synthesis has led to the development of second-generation compounds, such as 1-(1-(naphthalen-1-yl)ethyl)-3-(4-nitrophenyl) thiourea or SL-1-09. Results from our lab show that SL-1-09 exhibits anti-cancer activities against ERα+ and ERα- breast cancer cells at micromolar concentrations. SL-1-09 is a mixture of two enantiomers, R and S. The objective of this study was to further analyze these enantiomers to determine their individual anti-cancer activities. Cell cycle analysis demonstrated that the percentage of cells in S-phase is reduced significantly when breast cancer cell lines MCF-7, T47D and MDA-MB-453 cells are treated with 5.0 µM of the S enantiomer. Consistent with this finding, treatment of these cells with the S enantiomer resulted in lower expression levels of cell cycle proteins. Overall, our data indicate that the S enantiomer shows greater growth inhibitory effects than the R form against ERα+ (MCF7 and T47D) and ERα- (MDA-MB-453) breast cancer cells, suggesting that the activity observed in SL-1-09 is most likely due to the ability of the S enantiomer to block cell cycle progression.

CGK062, a small chemical molecule, inhibits cancer upregulated gene 2­induced oncogenesis through NEK2 and ß­catenin.

The mechanisms through which cancer­upregulated gene 2 (CUG2), a novel oncogene, affects Wnt/ß­catenin signaling, essential for tumorigenesis, are unclear. In this study, we aimed to elucidate some of these mechanisms in A549 lung cancer cells. Under the overexpression of CUG2, the protein levels and activity of ß­catenin were evaluated by western blot analysis and luciferase assay. To examine a biological consequence of ß­catenin under CUG2 overexpression, cell migration, invasion and sphere formation assay were performed. The upregulation of ß­catenin induced by CUG2 overexpression was also accessed by xenotransplantation in mice. We first found that CUG2 overexpression increased ß­catenin expression and activity. The suppression of ß­catenin decreased cancer stem cell (CSC)­like phenotypes, indicating that ß­catenin is involved in CUG2­mediated CSC­like phenotypes. Notably, CUG2 overexpression increased the phosphorylation of ß­catenin at Ser33/Ser37, which is known to recruit E3 ligase for ß­catenin degradation. Moreover, CUG2 interacted with and enhanced the expression and kinase activity of never in mitosis gene A­related kinase 2 (NEK2). Recombinant NEK2 phosphorylated ß­catenin at Ser33/Ser37, while NEK2 knockdown decreased the phosphorylation of ß­catenin, suggesting that NEK2 is involved in the phosphorylation of ß­catenin at Ser33/Ser37. Treatment with CGK062, a small chemical molecule, which promotes the phosphorylation of ß­catenin at Ser33/Ser37 through protein kinase C (PKC)α to induce its degradation, reduced ß­catenin levels and inhibited the CUG2­induced features of malignant tumors, including increased cell migration, invasion and sphere formation. Furthermore, CGK062 treatment suppressed CUG2­mediated tumor formation in nude mice. Taken together, the findings of this study suggest that CUG2 enhances the phosphorylation of ß­catenin at Ser33/Ser37 by activating NEK2, thus stabilizing ß­catenin. CGK062 may thus have potential for use as a therapeutic drug against CUG2­overexpressing lung cancer cells.

(3R)­5,6,7­trihydroxy­3­isopropyl­3­methylisochroman­1­one attenuates cardiac dysfunction via the apelin/APJ signaling pathway.

Myocardial infarction (MI) is associated with a high risk of mortality and is a major global health concern. The present study aimed to investigate the protective effects of (3R)­5,6,7­trihydroxy­3­isopropyl­3­methylisochroman­1­one (TIM) against MI induced by isoproterenol (ISO) in a rat model and the underlying mechanisms. Wistar rats were assigned to 4 groups (n=10): The control group received saline treatment; the ISO group received an intraperitoneal injection of ISO (100 mg/kg); and the TIM (low) and TIM (high) groups received an intraperitoneal injection of ISO, plus a 1 and 2 mg/kg dose of TIM orally, respectively. TIM rats were treated with TIM daily for 12 days and received ISO injections on the final 2 days to induce MI. Cardiac function, apoptosis index and protein expression were subsequently determined. The levels of oxidative stress markers were determined by ELISAs, whereas DNA damage was detected using a Cell Death Detection ELISA kit. Gene and protein expression were determined via reverse transcription­quantitative polymerase chain reaction and western blot analyses, respectively. Following treatment with ISO, the maximum left ventricular contraction/relaxation velocity and left ventricular systolic pressure were significantly decreased, whereas the left ventricular end­diastolic pressure was increased; however, treatment with TIM significantly ameliorated ISO­induced cardiac dysfunction. Additionally, TIM treatment significantly decreased oxidative stress and inhibited the apoptosis of cardiomyocytes, as determined by a decrease in caspase activities, increased expression of B­cell lymphoma 2 (Bcl­2) and reduced expression of cleaved caspase­3, cleaved caspase­9 and Bcl­2­associated X. Furthermore, treatment with TIM upregulated the levels of apelin in the plasma and myocardium of ISO­treated rats. The results indicated that TIM protected cardiomyocytes against ISO­induced MI, potentially via the apelin/apelin receptor signaling pathway. The results of the present study suggested that TIM may be a potential novel therapy for the treatment of MI.

Enhanced Survival in Mice Exposed to Ionizing Radiation by Combination of Gamma-Tocotrienol and Simvastatin.

Ionizing radiation exposure is a major concern for active military service members, as well as civilian population. Considering that the exposure is not predictable, it is imperative that strategies to counteract radiation damage must be discovered. Recent in vitro studies performed in our laboratory demonstrated that the vitamin E analog gamma-tocotrienol (GT3) in combination with cholesterol-lowering drugs (Statins), synergistically induced endothelial thrombomodulin, an anticoagulant with radio-protective efficacy. It was hypothesized that the combination of treatment with both GT3 along with Statins would provide better radiation protection in vivo than each drug individually. CD2F1 mice were injected subcutaneously with either vehicle or single dose of GT3 (200 mg/kg body weight) 24 hours before irradiation followed by oral or subcutaneous administration of various doses of simvastatin (25, 50, and 100 mg/kg body weight) before exposure to lethal doses (11.5 and 12 Gy) of Cobalt-60 (60Co) gamma-irradiation. The combined treatment group exhibited enhanced radiation lethality protection substantially, accelerated white blood cell recovery, and augmented restoration of bone marrow cellularity when compared to the animals treated with either drug exclusively. This information clearly suggests that combined treatment could be used as a safeguard for military personnel from exposure to harmful ionizing radiation.

Design, synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKKε for the treatment of obesity.

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R7 and R8 A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC50 values as low as 210 nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R7 cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.

γ-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport.

BACKGROUND: Ceramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport. METHODS: The effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5. RESULTS: GT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis. CONCLUSIONS: Our results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

The 6-hydroxychromanol derivative SUL-109 ameliorates renal injury after deep hypothermia and rewarming in rats.

Background: Mitochondrial dysfunction plays an important role in kidney damage in various pathologies, including acute and chronic kidney injury and diabetic nephropathy. In addition to the well-studied ischaemia/reperfusion (I/R) injury, hypothermia/rewarming (H/R) also inflicts acute kidney injury. Substituted 6-hydroxychromanols are a novel class of mitochondrial medicines that ameliorate mitochondrial oxidative stress and protect the mitochondrial network. To identify a novel 6-hydroxychromanol that protects mitochondrial structure and function in the kidney during H/R, we screened multiple compounds in vitro and subsequently assessed the efficacy of the 6-hydroxychromanol derivatives SUL-109 and SUL-121 in vivo to protect against kidney injury after H/R in rats. Methods: Human proximal tubule cell viability was assessed following exposure to H/R for 48/4 h in the presence of various 6-hydroxychromanols. Selected compounds (SUL-109, SUL-121) or vehicle were administered to ketamine-anaesthetized male Wistar rats (IV 135 µg/kg/h) undergoing H/R at 15°C for 3 h followed by rewarming and normothermia for 1 h. Metabolic parameters and body temperature were measured throughout. In addition, renal function, renal injury, histopathology and mitochondrial fitness were assessed. Results: H/R injury in vitro lowered cell viability by 94 ± 1%, which was counteracted dose-dependently by multiple 6-hydroxychomanols derivatives. In vivo, H/R in rats showed kidney injury molecule 1 expression in the kidney and tubular dilation, accompanied by double-strand DNA breaks and protein nitrosylation. SUL-109 and SUL-121 ameliorated tubular kidney damage, preserved mitochondrial mass and maintained cortical adenosine 5'-triphosphate (ATP) levels, although SUL-121 did not reduce protein nitrosylation. Conclusions: The substituted 6-hydroxychromanols SUL-109 and SUL-121 ameliorate kidney injury during in vivo H/R by preserving mitochondrial mass, function and ATP levels. In addition, both 6-hydroxychromanols limit DNA damage, but only SUL-109 also prevented protein nitrosylation in tubular cells. Therefore SUL-109 offers a promising therapeutic strategy to preserve kidney mitochondrial function.

(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one reduces lipoteichoic acid-induced damage in rat cardiomyoblast cells.

OBJECTIVE: Infective endocarditis is usually caused by Streptococcus sanguinis and characterized by inflammatory responses in the endocardium. This study aimed to investigate if the new compound (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) isolated from Alpinia katsumadai Hayata could provide protection against lipoteichoic acid (LTA)-induced cell damage in embryonic rat heart cells (H9c2). METHODS: LTA-induced cell damage was established in H9c2, and the protective effects of TIM against the cell damage were examined at different concentrations (0.1-2.5 µM). The inflammatory response and oxidative stress in H9c2 cells were also measured. RESULTS: Treatment with TIM (0.1-2.5 µM) significantly decreased LTA-induced toxicity in H9c2 cells, which was indicated by increase in cell viability, elevation in the mitochondrial membrane potential, decrease in the release of cytochrome-c and DNA damage, inhibition of caspase-3/9 activities, and change in apoptosis-related protein expression in LTA-treated H9c2 cells. TIM treatment also significantly attenuated the redox imbalance in H9c2 cells by decreasing malondialdehyde and intracellular reactive oxygen species levels as well as by enhancing superoxide dismutase activities and glutathione levels by increasing nuclear factor (erythroid-derived 2)-like 2 protein expression. Moreover, TIM treatment decreased interleukin 1 ß, interleukin 12, and tumor necrosis factor α levels by inhibiting nuclear factor kappa B protein expression. CONCLUSION: Our data indicated that TIM protected H9c2 cells against LTA-induced toxicity, at least partially through inhibiting the inflammatory response and oxidative stress, providing scientific rational to develop TIM to treat infective endocarditis.

HDAC4 preserves skeletal muscle structure following long-term denervation by mediating distinct cellular responses.

BACKGROUND: Denervation triggers numerous molecular responses in skeletal muscle, including the activation of catabolic pathways and oxidative stress, leading to progressive muscle atrophy. Histone deacetylase 4 (HDAC4) mediates skeletal muscle response to denervation, suggesting the use of HDAC inhibitors as a therapeutic approach to neurogenic muscle atrophy. However, the effects of HDAC4 inhibition in skeletal muscle in response to long-term denervation have not been described yet. METHODS: To further study HDAC4 functions in response to denervation, we analyzed mutant mice in which HDAC4 is specifically deleted in skeletal muscle. RESULTS: After an initial phase of resistance to neurogenic muscle atrophy, skeletal muscle with a deletion of HDAC4 lost structural integrity after 4 weeks of denervation. Deletion of HDAC4 impaired the activation of the ubiquitin-proteasome system, delayed the autophagic response, and dampened the OS response in skeletal muscle. Inhibition of the ubiquitin-proteasome system or the autophagic response, if on the one hand, conferred resistance to neurogenic muscle atrophy; on the other hand, induced loss of muscle integrity and inflammation in mice lacking HDAC4 in skeletal muscle. Moreover, treatment with the antioxidant drug Trolox prevented loss of muscle integrity and inflammation in in mice lacking HDAC4 in skeletal muscle, despite the resistance to neurogenic muscle atrophy. CONCLUSIONS: These results reveal new functions of HDAC4 in mediating skeletal muscle response to denervation and lead us to propose the combined use of HDAC inhibitors and antioxidant drugs to treat neurogenic muscle atrophy.

(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one ameliorates retinal degeneration in Pde6brd10 mice.

As a severe photoreceptor-degenerative disease, retinitis pigmentosa (RP) is currently incurable and eventually leads to partial or complete blindness. (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) is a novel antioxidant isolated from the plant of Alpinia katsumadai Hayata, with protective effects on photoreceptor cells against lipoteichoic acid-induced damage through inhibiting oxidative stress. The present study was to further demonstrate whether TIM could ameliorate retinal degeneration of Pde6brd10 (rd10) mice, a mouse model of RP. rd10 mice were treated with TIM by intraperitoneal injection daily from postnatal Day 10 (P10) to P26. Retinal function was tested by electroretinography. Histology was evaluated by toluidine blue staining and TUNEL assay. Oxidative stress markers were measured by ELISA. Immunohistochemistry, real-time PCR, and western blotting were applied to explore the protective mechanism. Results showed TIM significantly improved the retinal function and decreased photoreceptor cell apoptosis in rd10 mice through reducing oxidative stress. For the first time, this study demonstrated the protective effects of TIM against retinal degeneration in rd10 mice, providing scientific rationale to use TIM treating the RP.

Preliminary results of a single-arm pilot study to assess the safety and efficacy of visnadine, prenylflavonoids and bovine colostrum in postmenopausal sexually active women affected by vulvovaginal atrophy.

This single-arm pilot study enrolled 47 post-menopausal women affected by vulvovaginal atrophy (VVA). The Vaginal Health Index Score (VHIS) was evaluated for all women and all completed the Female Sexual Function Index (FSFI) questionnaire at baseline (T0) and after 15 days of vaginal cream treatment with one application per day (T1). Following treatment there was a significant improvement in all VHIS parameters and total score (p < 0.0001). Similarly, there was a significant improvement on four FSFI domains (lubrication, orgasm, satisfaction and pain) and total score (p = 0.001). None of the patients reported any local or systemic side-effects during treatment.

Randomized crossover study investigating daily versus on-demand vulvar Visnadine spray in women affected by female sexual arousal disorder.

The aim of the study was to verify the efficacy of vulvar Visnadine spray in premenopausal women affected by female sexual arousal disorder (FSAD). Thirty-eight women aged 25-40 years affected by FSAD were enrolled in the randomized crossover study, by two possible sequences: on-demand, washout, daily (A sequence); and daily, washout, on-demand (B sequence). The Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale (FSDS) were used to assess sexual function and sexual distress, respectively. Color Doppler ultrasonography was used to measure clitoral blood flow. The study had two follow-ups at 30 (T1) and 60 days (T2). Thirty-one women completed the study. Mean (SD) sexual activity and vulvar Visnadine spray usage was 1 ± 0.9 weekly during on-demand administration for both the sequences (Vs T0, p = NS). The mean sexual activity during daily usage was 2 ± 0.9 (Vs T0, p < .004) and 2 ± 0.8 (Vs T0, p < .001) for A and B sequences, respectively. FSFI total score, particularly genital arousal, improved more during the daily than during on-demand phases of both sequences (p < .001). Finally, clitoral blood flow improved significantly during daily usage of both the sequences (p < .001). Our study suggests that vulvar Visnadine spray could improve sexual performance of women affected by FSAD, producing changes in subjective and objective sexual aspects.

Efficacy and tolerability of a spray formulation containing Visnadine in women self-reporting sexual symptoms: a randomized double-blind placebo-controlled pilot study.

PURPOSE: The aim of this pilot, double-blind, randomized, placebo-controlled study, was to evaluate both the efficacy and the tolerability of a formulation for vulvar application containing Visnadine, a natural extractive substance with vasoactive properties, (ReFeel® spray, IDI Integratori Dietetici Italiani S.r.l., Italy) in women self-reporting sexual symptoms. METHODS: Sixty women (age range 18-60 years) volunteered to test the product against placebo (PL): Two puffs in the vulvar area, 10 min before sexual stimulation, for 30 days and for a minimum of six times. The main outcome measure was the improvement of the Female Sexual Function Index (FSFI) score (cut-off ≤ 26.55 for female sexual dysfunction [FSD]). Secondary outcomes were sexual satisfaction and tolerability with the product. RESULTS: PL group (n = 28) and Visnadine group (n = 30) were comparable for age, sexual function and rate of FSD at baseline (T0). After 1 month (T1), women in Visnadine group scored from 25.0 ± 3.8 to 27.9 ± 2.4 (p < 0.001), whereas no changes were evident in PL group (from 25.4 ± 5.0 to 25.6 ± 4.7). Statistically significant differences at T1 were reported in women with a positive (p < 0.001) or a negative FSD diagnosis (p < 0.01) using active treatment. Women with FSD reported significantly more improvement in satisfaction with their sexual function when treated with Visnadine spray compared to PL (p < 0.001), as well as more excitation (p < 0.001), pleasure (p < 0.001) and less time to reach orgasm (p < 0.003). No significant side effects were evident in both groups. CONCLUSIONS: On demand, 1-month use of Visnadine spray displayed positive effects on sexual function in women with and without FSD and it was well tolerated. Topical Visnadine may not only be part of multimodal strategies to manage clinically relevant sexual symptoms but also simply to help women to enhance their subjective impaired perception of sexual response.

Therapeutic effects of the mitochondrial ROS-redox modulator KH176 in a mammalian model of Leigh Disease.

Leigh Disease is a progressive neurometabolic disorder for which a clinical effective treatment is currently still lacking. Here, we report on the therapeutic efficacy of KH176, a new chemical entity derivative of Trolox, in Ndufs4 -/- mice, a mammalian model for Leigh Disease. Using in vivo brain diffusion tensor imaging, we show a loss of brain microstructural coherence in Ndufs4 -/- mice in the cerebral cortex, external capsule and cerebral peduncle. These findings are in line with the white matter diffusivity changes described in mitochondrial disease patients. Long-term KH176 treatment retained brain microstructural coherence in the external capsule in Ndufs4 -/- mice and normalized the increased lipid peroxidation in this area and the cerebral cortex. Furthermore, KH176 treatment was able to significantly improve rotarod and gait performance and reduced the degeneration of retinal ganglion cells in Ndufs4 -/- mice. These in vivo findings show that further development of KH176 as a potential treatment for mitochondrial disorders is worthwhile to pursue. Clinical trial studies to explore the potency, safety and efficacy of KH176 are ongoing.

Rational Design and Multibiological Profiling of Novel Donepezil-Trolox Hybrids against Alzheimer's Disease, with Cholinergic, Antioxidant, Neuroprotective, and Cognition Enhancing Properties.

A novel series of donepezil-trolox hybrids were designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological assays showed that these derivatives possessed moderate to good inhibitory activities against acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B) as well as remarkable antioxidant effects. The optimal compound 6d exhibited balanced functions with good inhibition against hAChE (IC50 = 0.54 µM) and hMAO-B (IC50 = 4.3 µM), significant antioxidant activity (41.33 µM IC50 by DPPH method, 1.72 and 1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation, and Aß1-42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d has very low toxicity and is capable of combating oxidative toxin (H2O2, rotenone, and oligomycin-A) induced neurotoxicity. Most importantly, oral administration of 6d demonstrated notable improvements on cognition and spatial memory against scopolamine-induced acute memory deficit as well as d-galactose (d-gal) and AlCl3 induced chronic oxidative stress in a mouse model without acute toxicity and hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a valuable candidate for the development of a safe and effective anti-Alzheimer's drug.