The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel.

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

Safety evaluation of the interchangeable use of robenacoxib in commercially-available tablets and solution for injection in cats.

BACKGROUND: Robenacoxib (Onsior™) is a non-steroidal anti-inflammatory drug developed for canine and feline use for the control of pain and inflammation. It is available as both tablets and solution for injection. The objective of this study was to evaluate the safety of the interchangeable use of commercially available robenacoxib formulations when administered to cats orally using 6 mg tablets and subcutaneously using a solution for injection containing 20 mg/mL. Thirty-four naïve healthy 4-month old cats were enrolled in this 37-day study and were randomized to four groups (three robenacoxib and one control). One robenacoxib group received the maximum recommended dose (MRD) rate of each formulation, while the other two received two and three times this dose rate. The cats underwent three 10-day treatment cycles comprised of seven days of once daily oral administration followed by three days of subcutaneous administration. The third cycle was followed by an additional seven days of oral treatment. The control group received oral empty gelatin capsules or subcutaneous saline injections. Assessment of safety was based on general health observations, clinical observations, physical, ophthalmic, electrocardiographic and neurological examinations, clinical pathology evaluations, food consumption, body weight, and macroscopic and microscopic examinations. Blood samples were collected for toxicokinetic evaluation. RESULTS: Blood concentrations of robenacoxib confirmed systemic exposure of all treated cats. All cats were in good health through study termination and there were no serious adverse events during the study. There were no changes in body weight, food consumption, ophthalmic, physical or neurological examinations during the study. Treatment-related abnormalities were of low occurrence at all doses and included injection site changes (transient edema with minimal or mild, subacute/chronic inflammation histologically) and prolongation of the QT interval. These findings were consistent with previously observed findings in studies with robenacoxib administered separately orally or subcutaneously in cats. Thus, there were no adverse effects that could be attributed specifically to the interchangeable use of oral and injectable robenacoxib. CONCLUSIONS: This 37-day laboratory study supports the safety of interchanging robenacoxib injection at a daily dose of 2 mg/kg with robenacoxib tablets at a daily dose of 1 mg/kg, or vice versa.

Testosterone Prevents Cutaneous Ischemia and Necrosis in Males Through Complementary Estrogenic and Androgenic Actions.

OBJECTIVE: Chronic nonhealing wounds are a substantial medical concern and are associated with morbidity and mortality; thus, new treatment strategies are required. The first step toward personalized/precision medicine in this field is probably in taking sex differences into account. Impaired wound healing is augmented by ischemia, and we previously demonstrated that 17ß-estradiol exerts a major preventive effect against ischemia-induced skin flap necrosis in female mice. However, the equivalent effects of testosterone in male mice have not yet been reported. We then investigated the role of steroid hormones in male mice using a skin flap ischemia model. APPROACH AND RESULTS: Castrated male mice developed skin necrosis after ischemia, whereas intact or castrated males treated with testosterone were equally protected. Testosterone can (1) activate the estrogen receptor after its aromatization into 17ß-estradiol or (2) be reduced into dihydrotestosterone, a nonaromatizable androgen that activates the androgen receptor. We found that dihydrotestosterone protected castrated wild-type mice by promoting skin revascularization, probably through a direct action on resistance arteries, as evidenced using a complementary model of flow-mediated outward remodeling. 17ß-estradiol treatment of castrated male mice also strongly protected them from ischemic necrosis through the activation of estrogen receptor-α by increasing skin revascularization and skin survival. Remarkably, 17ß-estradiol improved skin survival with a greater efficiency than dihydrotestosterone. CONCLUSIONS: Testosterone provides males with a strong protection against cutaneous necrosis and acts through both its estrogenic and androgenic derivatives, which have complementary effects on skin survival and revascularization.

Six-month safety evaluation of robenacoxib tablets (Onsior™) in dogs after daily oral administrations.

BACKGROUND: Robenacoxib is a non-steroidal anti-inflammatory drug available for canine and feline use for the control of pain and inflammation marketed as Onsior™. The aim of this target animal safety study was to evaluate the 6-month safety profile of oral robenacoxib administration. It was a randomized, negative-controlled, parallel group study. Thirty-two healthy, young, experimentally naïve, purebred Beagle dogs were administered 0 (sham control, Group 1), 2, 6, and 10 mg/kg robenacoxib (corresponding to the upper end of the dosage range [1X, Group 2] and multiples thereof [3X and 5X, Group 3 and 4]), orally once daily for 6 months. Assessment of safety included general health and clinical observations, physical, neurological, ophthalmological and electrocardiographic examinations, gross and histopathological examinations and clinical pathology evaluations. Blood samples were collected for toxicokinetic assessment of robenacoxib. RESULTS: No serious adverse events were reported. When compared with control, no treatment effect was observed for body weight, feed or water consumption, clinical pathology, urinalysis and fecal examination parameters. There were no treatment-related changes in stifle joint tissues and microscopic/histopathology examinations of all tissues/organs were normal. Salivation and soft feces were noted in all groups but observed more frequently in the treated groups as compared with control. On Day 178, increased buccal mucosal bleeding times were observed in two treated animals (Group 3 and 4) and one dog in Group 4 displayed a retinal change. Decreased hopping and conscious proprioception was noted in four treated dogs. One dog in Group 2 had ventricular premature complexes. Post-mortem changes included mild, red foci on the cecum in one dog (Group 3) and minimal duodenal discoloration in one dog (Group 4), with no corresponding histological findings in either dog. Ovarian weights were decreased in females from Group 3 and 4 with no gross or histological changes in the ovaries. Blood concentrations of robenacoxib confirmed systemic exposure of treated dogs. Exposure increased with increasing doses and there were no accumulation of robenacoxib in blood. CONCLUSIONS: Robenacoxib was well tolerated at doses from 2 to 10 mg/kg/day and this 6-month study supports the safe use of Onsior™ (robenacoxib) tablets in dogs for the intended dosing regimen.

Safety evaluation of the interchangeable use of robenacoxib (Onsior™) tablets and solution for injection in dogs.

BACKGROUND: Robenacoxib (Onsior™) is a non-steroidal anti-inflammatory drug developed for canine and feline use for the control of pain and inflammation. It is available as both tablets and solution for injection. The objective of this safety study was to investigate the interchangeable use of two robenacoxib formulations in dogs using a novel study design alternating between oral tablets and subcutaneous injections. Thirty-two naïve healthy 4-month dogs were enrolled in this 88-day study and were randomized among four groups to be untreated or to receive robenacoxib at the highest recommended or elevated dose rates. The dogs were administered three 20-day treatment cycles each separated by a 14-day washout period. Each 20-day cycle was comprised of 10 days of once daily oral administration, 3 days of subcutaneous administration, followed by further 7 days of oral administration (Groups 2 to 4). The control group (Group 1) received oral empty gelatin capsules or subcutaneous saline injections. Assessment of safety was based on general health observations, clinical observations, physical and neurological examinations including ophthalmological examinations, electrocardiographic examinations and clinical pathology evaluations, food and water consumption, body weight, and macroscopic and microscopic examinations. Blood samples were collected for pharmacokinetic evaluation. RESULTS: Blood concentrations of robenacoxib confirmed systemic exposure of all treated dogs. All dogs were in good health through study termination and there were no serious adverse events during the course of the study. No changes in body weight, food consumption, ophthalmic, neurological examinations, electrocardiograms, buccal mucosal blood times, clinical pathology or organ weight were attributable to robenacoxib formulation administration. Primary treatment-related abnormalities were of low incidence at all doses. They were confined to macroscopic and microscopic changes observed locally at the subcutaneous injection sites and microscopic findings within the gastrointestinal tract. These findings were as expected based on previous studies with robenacoxib solution for injection alone and the known properties of this class of compound and mode of administration. There were no adverse effects which could be attributed specifically to the interchangeable use of oral and injectable robenacoxib. CONCLUSIONS: Alternating regimens of robenacoxib tablets and solution for injection were well tolerated in healthy young dogs.

Chronic estradiol treatment reduces platelet responses and protects mice from thromboembolism through the hematopoietic estrogen receptor α.

Although estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or ß were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including ß1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies.

Activation function 2 (AF2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing.

17ß-Estradiol (E2) regulates estrogen receptor-α (ERα) target gene transcription through the two independent activation functions (AFs), AF1 and AF2, located in the N-terminal and ligand binding domain of ERα, respectively. We previously reported that ERα is required for the E2 atheroprotective action as well as for its accelerative action on endothelial healing, but its AF1 function is dispensable. Here, we investigated the role of ERαAF2 in these two major beneficial actions of E2 by electively targeting ERαAF2 (named ERαAF2(0)). Our results prove four points. (i) Compared with WT ERα, the ability of ERαAF2(0) to stimulate the C3 complement or the estrogen response element-thymidine kinase promoter in two cell lines was dramatically decreased, confirming the importance of AF2 in the E2-induced transcriptional activity of ERα. (ii) The uterotrophic action of E2 was totally absent in ERαAF2(0) mice, showing the crucial role of ERαAF2 in E2-induced uterus hyperplasia. (iii) ERαAF2 was dispensable for the accelerative action of E2 on endothelial healing, underlining the functionality of ERαAF2(0) in vivo. (iv) Finally, the atheroprotective effect of E2 was abrogated in ERαAF2(0) LDL-r(-/-) mice. Thus, whereas ERαAF1 and ERαAF2 are both required for the uterotrophic action of E2, we show that only ERαAF2 is necessary for its atheroprotective effect.

Prevention of skin flap necrosis by estradiol involves reperfusion of a protected vascular network.

Although 17beta-estradiol (E2) is protective in experimental models of myocardial and brain ischemia, its effect on skin ischemia remains unknown. Here, we assessed the protective effect of E2 in a mouse model of skin ischemia, mimicking the surgery of skin flaps. Whereas necrosis appeared in the half portion of the skin flap within 1 week after surgery in ovariectomized mice, it was reduced up to 10-fold when mice were pretreated with E2, at least 3 days before the surgery. The beneficial effect of E2 appeared to be attributable to an increase in skin survival, revealed by measuring viability of ex vivo explants and enhancement of the antiapoptotic Bcl-2 protein expression in vivo. This protective effect on the skin contributed to the protection of the vascular network and facilitated reperfusion, which was found to be accelerated in ovariectomized E2-treated mice, whereas hemorrhages were observed in untreated mice. E2 also increased expression of fibroblast growth factor-2 isoforms in the skin and circulating vascular endothelial growth factor in the serum. Finally, this protective effect of E2 was abolished in estrogen receptor-deficient mice (ERalpha(-/-)) but maintained in chimeric mice reconstituted with ERalpha-deficient bone marrow, indicating dispensable action of E2 in bone marrow-derived cells. This protective effect of E2 was mimicked by treatment with tamoxifen, a selective estrogen receptor modulator. In conclusion, we have demonstrated for the first time that E2 exerts a major preventive effect of skin flap necrosis through a prevention of ischemic-induced skin lesions, including those of the vascular network, which contributes to accelerate the reperfusion of the skin flap.

Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization.

OBJECTIVE: E2 accelerates reendothelialization through estrogen receptor alpha (ER alpha), and we now aimed at defining the precise local and systemic cellular actors of this process. METHODS AND RESULTS: The respective roles of endothelial and hematopoietic targets of E2 were investigated in a mouse carotid injury model, using confocal microscopy, to follow endothelium repair. Grafting ER alpha(-/-) mice with ER alpha(+/+) bone marrow (BM) was not sufficient to restore the accelerative effect of E2 on reendothelialization, demonstrating the necessary role of extrahematopoietic ER alpha. Using an endothelial-specific inactivation of ER alpha (Cre-Lox system), we showed that endothelial ER alpha plays a pivotal role in this E2 action. Conversely, in ER alpha(+/+) grafted with ER alpha(-/-) BM, the E2 regenerative effect was abolished, demonstrating that ER alpha-expressing hematopoietic cells are also needed. As eNOS expression in BM was required for this action, both endothelial progenitor cells and platelets could be the hematopoietic targets that participate to this beneficial E2 effect. CONCLUSIONS: We demonstrate that endothelial ER alpha plays a pivotal role in E2-mediated reendothelialization. However, endothelial targeting alone is not sufficient because the concomitant stimulation of a subpopulation of BM ER alpha is necessary. This cooperation should be taken into account in strategies aimed at optimizing in-stent reendothelialization.

Pharmacokinetics of lotilaner following a single oral or intravenous administration in cats.

BACKGROUND: CredelioTM (lotilaner) is an oral ectoparasiticide from the isoxazoline class developed for the treatment of flea and tick infestations in cats. It is formulated as a pure S-enantiomer in flavoured chewable tablets. The pharmacokinetics of lotilaner were investigated after intravenous or oral administration and under fed or fasted conditions in cats. Twenty-six adult cats were enrolled in a pharmacokinetic study evaluating either intravenous or oral administration of lotilaner. Following the oral administration at a dosage of 6 mg/kg, under fed or fasted conditions, or intravenous administration of 3 mg/kg, blood samples were collected up to 35 days after treatment. Lotilaner blood concentrations were measured using a validated liquid chromatography/tandem mass spectrometry method. Pharmacokinetic parameters were calculated by non-compartmental analysis. In addition, in vivo enantiomer stability of lotilaner was evaluated in a separate bioanalytical study. RESULTS: Following oral administration in fed cats, lotilaner was readily absorbed and peak blood concentrations reached within four hours. The terminal half-life was 33.6 days. Food enhanced the absorption, providing close to 100% oral bioavailability and reduced the inter-individual variability. Following intravenous administration, lotilaner had a low clearance of 0.13 l/kg/day, large volumes of distribution Vz and Vss of 5.34 and 5.37 l/kg, respectively and a terminal half-life of 28.7 days. In addition, there was no in vivo racemization of lotilaner. CONCLUSIONS: The pharmacokinetic properties of lotilaner administered orally as a flavoured chewable tablet (CredelioTM) were studied in detail. With a Tmax of 4 h and a terminal half-life of 33.6 days under fed conditions, lotilaner provides a rapid onset of flea and tick killing activity with consistent and sustained efficacy for at least one month in cats.

The intravenous and oral pharmacokinetics of lotilaner in dogs.

BACKGROUND: Lotilaner is a new oral ectoparasiticide from the isoxazoline class developed for the treatment of flea and tick infestations in dogs. It is formulated as pure S-enantiomer in flavoured chewable tablets (Credelio™). The pharmacokinetics of lotilaner were thoroughly determined after intravenous and oral administration and under different feeding regimens in dogs. METHODS: Twenty-six adult beagle dogs were enrolled in a pharmacokinetic study evaluating either intravenous or oral administration of lotilaner. Following the oral administration of 20 mg/kg, under fed or fasted conditions, or intravenous administration of 3 mg/kg, blood samples were collected up to 35 days after treatment. The effects of timing of offering food and the amount of food consumed prior or after dosing on bioavailability were assessed in a separate study in 25 adult dogs. Lotilaner blood concentrations were measured using a validated liquid chromatography/tandem mass spectrometry (LC-MS/MS) method. Pharmacokinetic parameters were calculated by non-compartmental analysis. In addition, in vivo enantiomer stability was evaluated in an analytical study. RESULTS: Following oral administration in fed animals, lotilaner was readily absorbed and peak blood concentrations reached within 2 hours. The terminal half-life was 30.7 days. Food enhanced the absorption, providing an oral bioavailability above 80% and reduced the inter-individual variability. Moreover, the time of feeding with respect to dosing (fed 30 min prior, fed at dosing or fed 30 min post-dosing) or the reduction of the food ration to one-third of the normal daily ration did not impact bioavailability. Following intravenous administration, lotilaner had a low clearance of 0.18 l/kg/day, large volumes of distribution Vz and Vss of 6.35 and 6.45 l/kg, respectively and a terminal half-life of 24.6 days. In addition, there was no in vivo racemization of lotilaner. CONCLUSIONS: The pharmacokinetic properties of lotilaner administered orally as a flavoured chewable tablet (Credelio™) were studied in detail. With a Tmax of 2 h and a terminal half-life of 30.7 days under fed conditions, lotilaner provides a rapid onset of flea and tick killing activity with consistent and sustained efficacy for at least 1 month.

FGF2 translationally induced by hypoxia is involved in negative and positive feedback loops with HIF-1alpha.

BACKGROUND: Fibroblast growth factor 2 (FGF2) is a major angiogenic factor involved in angiogenesis and arteriogenesis, however the regulation of its expression during these processes is poorly documented. FGF2 mRNA contains an internal ribosome entry site (IRES), a translational regulator expected to allow mRNA expression during cellular stress. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have developed a skin ischemia model in transgenic mice expressing a reporter transgene under the control of the FGF2 IRES. The results reveal that FGF2 is induced at the protein level during ischemia, concomitant with HIF-1alpha induction and a decrease in FGF2 mRNA. In addition, the FGF2 IRES is strongly activated under these ischemic conditions associated with hypoxia, whereas cap-dependent translation is repressed by 4E-BP hypophosphorylation. We also show that up-regulation of FGF2 protein expression in response to hypoxia correlates with the increase of FGF2 IRES activity in vitro, in human retinoblasts 911. The use of siRNAs targeting HIF or FGF2 indicates that FGF2 and HIF-1alpha reciprocally regulate their expression/accumulation, by a negative feedback loop in early hypoxia, followed by a positive feedback loop in late hypoxia. CONCLUSION/SIGNIFICANCE: FGF2 expression is up-regulated in vivo and in vitro in response to hypoxia. Strikingly, this up-regulation is not transcriptional. It seems to occur by an IRES-dependent mechanism, revealing new mechanistic aspects of the hypoxic response. In addition, our data show that FGF2 interacts with HIF-1alpha in a unique crosstalk, with distinct stages in early and late hypoxia. These data reveal the physiological importance of IRES-dependent translation during hypoxic stress and underline the complexity of the cellular response to hypoxia, suggesting a novel role of FGF2 in the regulation of HIF-1alpha during the induction of angiogenesis.