Inhibition of Eph/ephrin interaction with the small molecule UniPR500 improves glucose tolerance in healthy and insulin-resistant mice.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations.

BACKGROUND AND PURPOSE: The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance. EXPERIMENTAL APPROACH: UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs. KEY RESULTS: UniPR129 disrupted EphA2-ephrin-A1 interaction with Ki = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects. CONCLUSIONS AND IMPLICATIONS: The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.

2-Amino/azido/hydrazino-5-alkoxy-5H-[1]benzopyrano[4,3-d]pyrimidines: synthesis and pharmacological evaluation.

New series of 5-alkoxy-benzopyranopyrimidine derivatives were developed from the chemical modulation of the substituent in position 2 of the scaffold, with the aim to produce analgesic/antiphlogistic agents more potent than analogues previously reported. The 2-hydrazino derivatives exhibited a good analgesic activity in writhing test; the analgesic doses of the compounds did not affect mice spontaneous locomotor activity thus any confounding sedative effect could be excluded. These derivatives revealed an aspirin-like profile with a strong inhibition of AA-induced platelet aggregation, probably due to a strong, non selective, inhibition of cyclooxygenases. In spite of the inhibition of COX activity displayed in vitro, the compounds did not cause gastric damage in rats after acute oral administration. A different pharmacological profile was observed for the 2-azido derivatives, particularly in vivo.

Comparative screening of plant essential oils: phenylpropanoid moiety as basic core for antiplatelet activity.

Essential oils extracted from different plants (Anthemis nobilis L., Artemisia dracunculus L., Cannabis sativa L., Cupressus sempervirens L., Cymbopogon citratus (DC.) Stapf., Curcuma longa L., Foeniculum vulgare L., Hypericum perforatum L., Hyssopus officinalis L., Mentha spicata L., Monarda didyma L., Ocimum basilicum L., Ocotea quixos Kosterm., Origanum vulgare L., Pinus nigra J.F. Arnold, Pinus silvestris L., Piper crassinervium Kunth., Rosmarinus officinalis L., Salvia officinalis L., Salvia sclarea L., Santolina chamaecyparissus L., Thymus vulgaris L., Zingiber officinaie L.) were screened in guinea pig and rat plasma in order to assess antiplatelet activity and inhibition of clot retraction. The oils were chemically analysed and a relationship between components and ability to affect hemostasis was evidenced. O. quixos, F. vulgaris, and A. dracunculus showed the highest antiplatelet activity against ADP, Arachidonic Acid and the Thromboxane A2 agonist U46619 (IC50, 4-132 microg ml(-1)), and a good ability to destabilize clot retraction (IC50, 19-180 microg ml(-1)). For these oils a significant correlation between antiplatelet potency and phenylpropanoids content (54-86%) was evidenced thus suggesting a key role for this moiety in the prevention of clot formation. These findings provide the rationale to take in account the antiplatelet activity in the pharmacological screening of natural products containing phenylpropanoids.

Novel antiplatelet and antithrombotic activities of essential oil from Lavandula hybrida Reverchon "grosso".

Lavender extracts are known to produce several mild effects at central and peripheral level. However, no studies are so far available about the potential effects of lavender essential oil on the hemostatic system. In this work, we demonstrated antiplatelet properties of lavender oil towards platelet aggregation induced by arachidonic acid, U46619, collagen and ADP (IC50 = 51, 84, 191 and 640 microg/ml, respectively) on guinea-pig platelet rich plasma (PRP) and its ability to destabilize clot retraction (IC50 = 149 microg/ml) induced by thrombin on rat PRP. Furthermore, antithrombotic properties were studied in an in vivo model of pulmonary thromboembolism induced by intravenous injection of a collagen-epinephrine mixture in mice subacutely treated with lavender oil. In this model, lavender oil (100 mg/kg/day os for 5 days) significantly reduced thrombotic events without inducing prohemorrhagic complications at variance with acetylsalicylic acid used as reference drug. Finally, main components of the oil were studied in vitro in order to assess their antiplatelet effects, but none of them possessed an activity comparable to the oil itself. These results provide the first experimental evidence of lavender oil's antiplatelet/antithrombotic properties which could be due to a synergistic effect of its components.

Contact-dependent signaling during the late events of platelet activation.

Signaling events downstream from collagen receptors and G protein-coupled receptors are responsible for the initiation and extension of platelet plug formation. This creates the platelet plug and hopefully results in the cessation of bleeding. It is not, however, all that is required for hemostasis, and growing evidence is emerging that the perpetuation of a stable hemostatic plug requires additional intracellular signaling. At least part of this process is made possible by the persistent close contacts between platelets that can only occur after the onset of aggregation. This review discusses several examples of such signaling mechanisms that help to perpetuate the platelet plug in a contact-dependent manner, including outside-in signaling through integrins, signaling though Eph kinases and ephrins, and the role of CD40L.

Synthesis and pharmacological evaluation of 2,5-cycloamino-5H-[1]benzopyrano[4,3-d]pyrimidines endowed with in vitro antiplatelet activity.

A series of new 2,5-cycloamino-5H-[1]benzopyrano[4,3-d]pyrimidines 3a-i have been synthesized and tested in vivo for the anti-inflammatory/analgesic/antipyretic effects and in vitro to evaluate the antiplatelet activity on guinea-pig platelet-rich plasma aggregated by collagen, adenosine-5'-diphosphate (ADP) and arachidonic acid (AA). Title compounds were ineffective in vivo; however, the pyrrolidino derivatives 3a and 3c exhibited an antiplatelet activity against all the aggregants differing from that of acetylsalicylic acid (ASA) while the 5-morpholino derivatives 3g-i showed the most potent ASA-like antiplatelet activity.

New polycyclic pyrimidine derivatives with antiplatelet in vitro activity: synthesis and pharmacological screening.

The preparation and the pharmacological screening of novel anti-aggregatory/antiphlogistic polycyclic pyrimidine derivatives are described. The compounds were developed starting from bioactive 2-aminobenzopyranopyrimidine derivatives in order to assess the importance of the benzopyrano[4,3-d]pyrimidine structure and the role of an amino basic moiety in position 2. Antiplatelet activity was assessed in vitro against ADP and arachidonic acid-induced aggregation in guinea-pig plasma. Anti-inflammatory/analgesic/antipyretic activities were studied in rat paw oedema, mouse writhing test and E. coli-induced rat fever. Ulcerogenic and gastroprotective effects were also investigated in vivo on rat gastric mucosa. Among the tested compounds, the 5-substituted benzopyranopyrimidine derivatives 3d and 4d proved to be the most active antiplatelet agents as potent as acetylsalicylic acid against arachidonic acid-stimulated aggregation. Furthermore the 2-methylthio derivative 4d was endowed with greater efficacy against ADP aggregation suggesting that additional non-TXA2 dependent mechanisms are involved in its biological activity. Orally administered at 100 mg kg(-1) in rats this latter compound displayed antiphlogistic acitivity comparable to indomethacin (10 mg kg(-1)) coupled with an unusual gastroprotective effect on ethanol-induced ulcers. In conclusion, these findings indicate that the 5-pyrrolidino-2-methylthiobenzopyrano[4,3-d]pyrimidine 4d fulfils the chemical requirements to exhibit antiplatelet activity associated with gastroprotective effect.

2-Amino-Benzo[d]isothiazol-3-one derivatives: synthesis and assessment of their antiplatelet/spasmolytic effects.

We describe a series of 2-amino-benzo[d]isothiazol-3-one derivatives (2-8), which were synthesized and screened in vitro for inhibition of platelet aggregation and for their spasmolytic activity, with the awareness that the development of antiplatelet agents with additional vasodilation activity could be beneficial in the treatment of various vaso-occlusive disorders. The tested compounds show a powerful antiplatelet activity and various modifications resulted in molecules possessing antiaggregating effects as well as spasmolytic actions.