ANDA申请中关于标签等包装标识物问题的探讨

目的：为我国药品制剂出口企业ANDA申请过程中有关标签等包装标识物资料申报和规范管理提出建议。方法：分析美国有关标签等包装标识物监管的法律、法规和指南的特征乖意图。结果和结论：我国药品制剂企业在ANDA申请工作中,标签等包装标识物部分存在问题,应对照美国的法律、法规进行修正。     

Serofendic Acid Promotes Stellation Induced by cAMP and cGMP Analogs in Cultured Cortical Astrocytes

We investigated the effect of serofendic acid, a neuroprotective substance derived from fetal calf serum, on the morphological changes in cultured cortical astrocytes. Cultured astrocytes developed a stellate morphology with several processes following exposure to dibutylyl cAMP (dbcAMP), a membrane-permeable cAMP analog; 8-Br-cGMP, a membrane-permeable cGMP analog; or phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator. Serofendic acid significantly accelerated the stellation induced by dbcAMP- and 8-Br-cGMP. In contrast, the PMA-induced stellation was not affected by serofendic acid. Next, we attempted to elucidate the mechanism underlying the dbcAMP-induced stellation and explore the site of action of serofendic acid. Both the stellation induced by dbcAMP and the promotional effect of serofendic acid were partially inhibited by KT5720, a specific protein kinase A (PKA) inhibitor. Furthermore, serofendic acid failed to facilitate the stellation induced by Y-27632, an inhibitor of Rho-associated kinase (ROCK). These results indicate that serofendic acid promotes dbcAMP- and 8-Br-cGMP-induced stellation and the promotional effect on dbcAMP-induced stellation is mediated at least partly by the regulation of PKA activity and not by controlling ROCK activity.     

Gonadal hormones decrease temporomandibular joint κ-mediated antinociception through a down-regulation in the expression of kappa opioid receptors in the trigeminal ganglia

We have previously demonstrated that activation of κ-opioid receptor located in the temporomandibular joint (TMJ) of rats induces a significantly greater TMJ antinociception in diestrus females than in proestrus females (higher estradiol serum levels than diestrus) and males. These findings indicate that gonadal hormones decrease TMJ kappa-mediated antinociception. The aim of this study was to investigate some of the mechanisms by which gonadal hormones decrease TMJ kappa-mediated antinociception. Western blot analysis demonstrated a significantly lower κ-opioid receptor expression in the trigeminal ganglia of intact males than in intact and ovariechtomized (OVX) females and orchidectomized (ORX) males. In females, κ-opioid receptor expression in the trigeminal ganglia was significantly lower in proestrus than in diestrus and OVX females. Taken together these findings suggest that gonadal hormones, especially male gonadal hormones, down-regulate κ-opioid receptor expression. Co-application of the NOS inhibitor l-NMMA or the NO-sensitive guanylyl cyclase inhibitor ODQ with the κ-opioid receptor agonist U50,488 blocked TMJ kappa-mediated antinociception in males and females. These findings suggest that antinociception induced by activation of kappa opioid receptors in the TMJ region is mediated by the l-arginine/NO/cGMP pathway in both sexes. Despite the involvement of the l-arginine/NO/cGMP pathway in TMJ kappa-mediated antinociception in both sexes, gonadal hormones do not diminish the activity of this pathway to decrease TMJ kappa-mediated antinociception. Alternatively, they significantly reduce κ-opioid receptor expression in the trigeminal ganglia.     

Metabolic syndrome and urinary cGMP excretion in general population

To examine the relationship between metabolic syndrome and endothelial dysfunction, we investigated cross-sectionally the correlation between metabolic risk factors and urinary excretion of cyclic guanosine 3′,5′-monophosphate (cGMP), a second messenger of nitric oxide (NO), in 1541 Japanese men and women aged 40–79 years. The 24-h urinary excretion of cGMP was measured using a ¹²⁵I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The components of metabolic syndrome were defined based on the following criteria: body mass index (BMI) ≥ 25.0 kg/m², fasting plasma glucose ≥ 6.11 mmol/l or non-fasting plasma glucose level ≥ 11.1 mmol/l, systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg, high-density lipoprotein (HDL)-cholesterol < 1.03 mmol/l for men and <1.29 mmol/l for women, and triglyceride ≥ 1.69 mmol/l. The number of components of metabolic syndrome correlated inversely with urinary cGMP excretion; means of cGMP excretion for the whole group adjusted for age, sex, and cardiovascular risk factors were 53.6, 48.6, 47.9, 44.4 and 42.3 nmol/mmol for 0, 1, 2, 3, and 4–5 components of metabolic syndrome, respectively (p = 0.002). Our data suggest that a reduction of NO bioactivity concur with clustered features of the metabolic syndrome.     

Validated liquid chromatographic method and analysis of content of tilianin on several extracts obtained from Agastache mexicana and its correlation with vasorelaxant effect

Ethnopharmacological relevance

To optimize the obtention of tilianin, an antihypertensive flavonoid isolated from Agastache mexicana (Lamiaceae), a medicinal plant used in Mexico for the treatment of hypertension. Also, a validated HPLC method to quantify tilianin from different extracts, obtained by several extraction methods, was developed.

Materials and methods

The aerial parts of Agastache mexicana were dried at different temperatures (22, 40, 50, 90, 100 and 180 °C) and the dry material was extracted with methanol by maceration to compare the content of the active constituent tilianin in the samples. Furthermore, EtOH:H₂O (7:3), infusion and decoction extracts were prepared from air-dried samples at room temperature to compare the content and composition of the different extraction methods. Moreover, an ex vivo vasorelaxant test on endothelium-intact aortic rat rings was conducted, in order to correlate the presence of tilianin with the activity of each extract.

Results

Higher concentration and amounts of tilianin were determined from chromatograms in the obtained methanolic extracts from plant material dried at 90, 50, 40 and 22 °C, followed by 100 °C; however, lower concentrations were observed in dried at 180 °C and EtOH:H₂O (7:3). It is worth to notice that methanolic extracts with higher amount of tilianin were the most potent vasorelaxant extracts, even though these extracts were less potent than carbachol, a positive control used. Finally, decoction, infusion and EtOH:H₂O (7:3) extracts did not show any vasorelaxant effect.

Conclusion

Results suggest that extracts with higher concentration of tilianin possess the best vasorelaxant activity, which allowed us to have a HPLC method for future quality control for this medicinal plant.     

PACAP stimulation of maturational gonadotropin secretion in goldfish involves extracellular signal-regulated kinase, but not nitric oxide or guanylate cyclase, signaling

In goldfish, nitric oxide synthase (NOS) immunoreactivity is present in gonadotropes and extracellular signal-regulated protein kinase (ERK) mediates GnRH stimulation of gonadotropin release and synthesis. In this study, we tested the possible involvement of nitric oxide (NO) and ERK in mediating PACAP-stimulated maturational gonadotropin (GTH-II) release from primary cultures of dispersed goldfish pituitary cells. In static incubation experiments, PACAP-induced GTH-II release was unaffected by two inhibitors of NOS synthase, AGH and 1400W; whereas addition of a NO donor, SNAP, elevated GTH-II secretion. In perifusion experiments, neither NOS inhibitors (AGH, 1400W and 7-Ni) nor NO scavengers (PTIO and rutin hydrate) attenuated the GTH-II response to pulse applications of PACAP. In addition, the GTH-II responses to PACAP and the NO donor SNP were additive while PTIO blocked SNP action. Although dibutyryl cGMP increased GTH-II secretion in static incubation, inhibition of guanylate cyclase (GC), a known down-stream target for NO signaling, did not reduce the GTH-II response to pulse application of PACAP. On the other hand, GTH-II responses to PACAP in perifusion were attenuated in the presence of two inhibitors of ERK kinase (MEK), U 0126 and PD 98059. These results suggest that although increased availability of NO and cGMP can lead to increased GTH-II secretion, MEK/ERK signaling, rather than NOS/NO/GC activation, mediates PACAP action on GTH-II release in goldfish.     

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca²⁺ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca²⁺ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.     

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:

Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Experimental approach:

Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC).

Key results:

Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis.

Conclusion and implications:

Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.