Proof-of-concept study for a long-acting formulation of ivermectin injected in cattle as a complementary malaria vector control tool.

BACKGROUND: Domesticated animals play a role in maintaining residual transmission of Plasmodium parasites of humans, by offering alternative blood meal sources for malaria vectors to survive on. However, the blood of animals treated with veterinary formulations of the anti-helminthic drug ivermectin can have an insecticidal effect on adult malaria vector mosquitoes. This study therefore assessed the effects of treating cattle with long-acting injectable formulations of ivermectin on the survival of an important malaria vector species, to determine whether it has potential as a complementary vector control measure. METHODS: Eight head of a local breed of cattle were randomly assigned to either one of two treatment arms (2 × 2 cattle injected with one of two long-acting formulations of ivermectin with the BEPO® technology at the therapeutic dose of 1.2 mg/kg), or one of two control arms (2 × 2 cattle injected with the vehicles of the formulations). The lethality of the formulations was evaluated on 3-5-day-old Anopheles coluzzii mosquitoes through direct skin-feeding assays, from 1 to 210 days after treatment. The efficacy of each formulation was evaluated and compared using Cox proportional hazards survival models, Kaplan-Meier survival estimates, and log-logistic regression on cumulative mortality. RESULTS: Both formulations released mosquitocidal concentrations of ivermectin until 210 days post-treatment (hazard ratio > 1). The treatments significantly reduced mosquito survival, with average median survival time of 4-5 days post-feeding. The lethal concentrations to kill 50% of the Anopheles (LC50) before they became infectious (10 days after an infectious blood meal) were maintained for 210 days post-injection for both formulations. CONCLUSIONS: This long-lasting formulation of ivermectin injected in cattle could complement insecticide-treated nets by suppressing field populations of zoophagic mosquitoes that are responsible, at least in part, for residual malaria transmission. The impact of this approach will of course depend on the field epidemiological context. Complementary studies will be necessary to characterize ivermectin withdrawal times and potential environmental toxicity.

Effects of an injectable long-acting formulation of ivermectin on Onchocerca ochengi in zebu cattle.

The availability of a safe macrofilaricidal drug would help to accelerate onchocerciasis elimination. A trial was conducted in Cameroon to evaluate the effects of a subcutaneous injectable long-acting formulation of ivermectin (LAFI) on the microfilariae (mf) and adult stages of Onchocerca ochengi. Ten zebu cattle naturally infected with the parasite were injected subcutaneously with either 500 mg (group A, N = 4), or 1000 mg long-acting ivermectin (group B, N = 4) or the vehicle (group C, N = 2). Skin samples were collected from each animal before, and 6, 12, and 24 months after treatment to measure microfilarial densities (MFDs). Nodules excised before, and 6 and 12 months after treatment were examined histologically to assess the adult worms' viability and reproductive status. Blood samples were collected at pre-determined time-points to obtain pharmacokinetic data. Before treatment, the average O. ochengi MFDs were similar in the three groups. Six months after treatment, all animals in groups A and B were free of skin mf, whereas those in group C still showed high MFDs (mean = 324.5 mf/g). Only one ivermectin-treated animal (belonging to group A) had skin mf 12 months after treatment (0.9 mf/g). At 24 months, another animal in group A showed skin mf (10.0 mf/g). The histologic examination of nodules at 6 and 12 months showed that LAFI was not macrofilaricidal but had a strong effect on embryogenesis. The new LAFI regimen might be an additional tool to accelerate the elimination of human onchocerciasis in specific settings.

TITLE: Effets d'une formulation injectable d'ivermectine à activité prolongée sur Onchocerca ochengi chez les bovins zébu. ABSTRACT: La disponibilité d'un médicament macrofilaricide et sans danger permettrait d'accélérer l'élimination de l'onchocercose. Un essai a été mené au Cameroun pour évaluer les effets d'une formulation injectable en sous-cutané d'ivermectine à activité prolongée (FIAP) sur les microfilaires (mf) et les stades adultes d'Onchocerca ochengi. Dix vaches zébu infectées naturellement par le parasite ont reçu une injection sous-cutanée de 500 mg (groupe A, N = 4) ou de 1000 mg d'ivermectine à activité prolongée (groupe B, N = 4) ou le véhicule (groupe C, N = 2). Des échantillons de peau ont été collectés de chaque animal avant, puis 6, 12 et 24 mois après traitement pour mesurer les densités microfilariennes (DMF). Des nodules prélevés avant et 6 et 12 mois après traitement ont été examinés histologiquement pour évaluer la viabilité et le statut reproductif des vers adultes. Des échantillons de sang ont été prélevés pour obtenir des données de pharmacocinétique. Avant traitement, les DMF à O. ochengi étaient similaires dans les 3 groupes. Six mois après traitement, aucun des animaux des groupes A et B ne présentait de mf dermiques, alors que ceux du groupe C présentaient encore des DMF élevées (moyenne : 324,5 mf/g). Parmi les animaux traités par ivermectine, un seul (du groupe A) avait des mf dermiques 12 mois après traitement (0,9 mf/g). A 24 mois, un autre animal du groupe A avait des mf (10,0 mf/g). L'examen histologique des nodules collectés à 6 et 12 mois montrait que la FIAP n'était pas macrofilaricide mais avait un effet marqué sur l'embryogénèse. La nouvelle FIAP pourrait représenter un outil pour accélérer l'élimination de l'onchocercose dans certaines circonstances spécifiques.

Retroinverso analogs of spadin display increased antidepressant effects.

RATIONALE: Although depression is the most common mood disorder, only one third of patients are treated with success. Finding new targets, new drugs, and also new drug intake way are the main challenges in the depression field. Several years ago, we identified a new target with the TWIK-related potassium channel-1 (TREK-1) potassium channel, and more recently, we have discovered a peptide of 17 amino acids with antidepressant properties. This peptide, that we called spadin, can be considered as a new concept in antidepressant drug design. Spadin derives from a larger peptide resulting to a posttranslational maturation of sortilin; consequently, spadin can be considered as a natural molecule. Moreover, spadin acts more rapidly than classical antidepressants and does not induce side effects. OBJECTIVES: In this work, we sought analogs of spadin displaying a better affinity on TREK-1 channels and an increased action duration. METHODS: Analogs were characterized by electrophysiology measurements, by behavioral tests, and by their ability to induce neurogenesis. RESULTS: We identified two retro-inverso peptides that have kept the antidepressant properties of spadin; particularly, they increased the hippocampal neurogenesis after a 4-day treatment. As spadin, these analogs did not induce side effects on either pain, epilepsy processes, or at the cardiac level. CONCLUSIONS: Together, our results indicated that spadin retro-inverso peptides could represent new potent antidepressant drugs. As exemplified by spadin in the field of depression, retro-inverso strategies could represent a useful technique for developing new classes of drugs in a number of pathologies.

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac.

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.

The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling.

RGS (regulator of G-protein signalling) proteins stimulate the intrinsic GTPase activity of the a subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 has been shown previously to stimulate the GTPase activities of Ga(o) and Ga(i) subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to G(i). It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Ga(i). In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G(i) signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on G(i) signalling via the bg pathway in a stable cell line ectopically expressing the G(i)-coupled M2 muscarinic acetylcholine receptor (HEK-m2). We show that the RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G(i). Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G(i) signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. We therefore propose that RGS14 combines the inhibition of G(i) activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Ga(i)-GTP via its RGS domain to negatively regulate signalling downstream of G(i).

Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells.

Corticotropin-releasing factor receptor type 2beta (CRF R2beta) is a member of the Class B heptahelical G protein-coupled receptors. This receptor is positively coupled to adenylate cyclase and is bound preferentially by the CRF-related peptides, urocortin (Ucn), Ucn II and Ucn III. In the rodent, CRF R2beta messenger RNA (mRNA) is expressed in the cardiovascular system, where its levels can be modulated by Ucn. In the present study, we investigated regulation of CRF R2beta levels by Ucn in A7r5 aortic smooth muscle cells. Ribonuclease protection assays show that A7r5 cells expressed the CRF R2beta subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4. Ucn induced accumulation of intracellular cAMP via CRF R2beta in this cell line. In addition to the treatment with Ucn, cAMP agonists or analogues themselves caused a significant decrease in CRF R2beta mRNA levels. Blockade of Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels by H7, a broad protein kinase inhibitor, suggested that a protein kinase pathway might be involved in this regulation. H89, a protein kinase A inhibitor, partially blocked Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels. Thus, Ucn induces intracellular cAMP to downregulate CRF R2beta mRNA expression in A7r5 cells.