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.

Evaluation of Loco-Regional Skin Toxicity Induced by an In Situ Forming Depot after a Single Subcutaneous Injection at Different Volumes and Flow Rates in Göttingen Minipigs.

The present study aims to investigate the loco-regional tolerability and injection parameters (i.e., flow rate and administration volume) of an in situ forming depot (ISFD) in Göttingen minipigs, to secure both the therapeutic procedure and compliance in chronic medical prescriptions. The ISFD BEPO® technology (MedinCell S.A.) is investigated over 10 days, after a single subcutaneous injection of test item based on a DMSO solution of diblock and triblock polyethylene glycol-polylactic acid copolymers. Injection sites are systematically observed for macroscopic loco-regional skin reactions as well as ultrasound scanning, enabling longitudinal in vivo imaging of the depot. Observations are complemented by histopathological examinations at 72 h and 240 h post-injection. Overall, no treatment-emergent adverse effects are macroscopically or microscopically observed at the subcutaneous injection sites, for the tested injection flow rates of 1 and 8 mL/min and volumes of 0.2 and 1 mL. The histopathology examination confirms an expected foreign body reaction, with an intensity depending on the injected volume. The depot morphology is similar irrespective of the administration flow rates. These results indicate that the ISFD BEPO® technology can be considered safe when administered subcutaneously in Göttingen minipigs, a human-relevant animal model for subcutaneous administrations, in the tested ranges.

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.

BEPO®: Bioresorbable diblock mPEG-PDLLA and triblock PDLLA-PEG-PDLLA based in situ forming depots with flexible drug delivery kinetics modulation.

This article presents BEPO®, an in situ forming depot (ISFD) technology mediated by a solvent-exchange mechanism. The matrix of the in situ formed drug delivery depot is composed of the combination of a diblock (DB) and a triblock (TB) polyethylene glycol-polyester copolymer. This combination offers a broad capability to tune the release of a wide variety of drugs to the desired pharmacokinetics. The work described in the present article demonstrates that the delivery rate and profile can be adjusted by changing the composition of either TB or DB or the relative ratio between them, among other parameters. It has been shown that the polymeric composition of the formulation has a substantial impact on the solvent exchange rate between the organic solvent and the surrounding aqueous medium which subsequently determines the internal structure of the resulting depot and the delivery of the therapeutic cargo. This has been demonstrated studying the in vitro release of two model molecules: bupivacaine and ivermectin. Formulations releasing these drugs have been administered to animal models to show the possibility of delivering therapeutics from weeks to months by using BEPO® technology.

Solid emulsion gel as a novel construct for topical applications: synthesis, morphology and mechanical properties.

A series of the solid emulsion gels with the oil volume fraction in the range of 0-50% were synthesized through a polycondensation reaction between activated p-nitrophenyl carbonate poly(ethylene glycol) and protein-stabilized oil-in-water emulsions. The resultant structures were investigated in terms of swelling behavior, composition, morphology, mechanical and skin hydration properties. Solid emulsions gels share the properties of both hydrogel and emulsion. Similar to the classical hydrogel, the SEG swells in water up to equilibrium swelling degree, which decreases as the oil volume fraction increases, and comprises immobilized drops of protein-stabilized oil. The impregnation of the oil phase is found to reduce tensile stiffness of the material, but improves material's extensibility. The mechanical properties of the constructs (Young moduli in the range of 9-15 kPa and the elongation at break of 120-220%) are interpreted according to the "rule of elasticity mixture" that considers the elasticity of the composite material to be a sum of the contributions from individual components, i.e. hydrogel and dispersed oil drops. An idealized model that takes into account the history of the material preparation has been proposed to explain the improved extensibility of the constructs. The results of the mechanical tests, equilibrium swelling, and the skin hydration effect of the solid emulsion gels in vivo are discussed from the perspective of the biomedical applications of the solid emulsion gels, in particular, for the transdermal delivery of hydrophilic and lipophilic drugs.

Solid emulsion gel as a vehicle for delivery of polyunsaturated fatty acids: implications for tissue repair, dermal angiogenesis and wound healing.

The paper describes preparation and biological characterization of the solid hybrid biomaterial that was designed for cell-targeted lipid delivery in healing tissues. The material referred to as 'solid emulsion gel' combines a protein-stabilized lipid emulsion and a hydrogel structure in a single compartment. The potential of the omega-3 (n-3)-fatty acids rich solid emulsion gel for tissue repair applications was investigated at the macro-, micro-, molecular and gene expression levels, using human fibroblasts and endothelial cells and a porcine model of full-thickness wounds. Being non-cytotoxic in vitro and in vivo, the biomaterial was found to affect cell metabolism, modulate expression of certain genes, stimulate early angiogenesis and promote wound repair in vivo. The neovascular response in vivo was correlated with upregulated expression of the genes involved in lipid transport (e.g. adipophilin), anti-apoptosis (e.g. heat shock proteins, haem oxygenase 1) and angiogenesis (vascular endothelial growth factor, placental growth factor). Collectively, the results of this study provide first evidence that the angiogenic response provided by solid emulsion gel-mediated delivery of n-3 fatty acids is an alternative to the topical administration of exogenous growth factors or gene therapy, and can be advantageously used for the stimulation of tissue repair in complex wounds.

Mechanical and microstructural properties of hybrid poly(ethylene glycol)-soy protein hydrogels for wound dressing applications.

Biomimetic hydrogel made of poly(ethylene glycol) and soy protein with a water content of 96% has been developed for moist wound dressing applications. In this study, such hybrid hydrogels were investigated by both tensile and unconfined compression measurements in order to understand the relationships between structural parameters of the network, its mechanical properties and protein absorption in vitro. Elastic moduli were found to vary from 1 to 17 kPa depending on the composition, while the Poisson's ratio (approximately 0.18) and deformation at break (approximately 300%) showed no dependence on this parameter. Further calculations yielded the crosslinking concentration, the average molecular weight between crosslinks (M(C)) and the mesh size. The results show that reactions between PEG and protein create polymeric chains comprising molecules of PEG and protein fragments between crosslinks. M(C) is three times higher than that expected for a "theoretical network." On the basis of this data, we propose a model for the 3D network of the hydrogel, which is found to be useful for understanding drug release properties and biomedical potential of the studied material.

Electrically conductive biodegradable polymer composite for nerve regeneration: electricity-stimulated neurite outgrowth and axon regeneration.

Normal and electrically stimulated PC12 cell cultures and the implantation of nerve guidance channels were performed to evaluate newly developed electrically conductive biodegradable polymer composites. Polypyrrole (PPy) doped by butane sulfonic acid showed a significantly higher number of viable cells compared with PPy doped by polystyrenesulfonate after a 6-day culture. The PC12 cells were left to proliferate for 6 days, and the PPy-coated membranes, showing less initial cell adherence, recorded the same proliferation rate as did the noncoated membranes. Direct current electricity at various intensities was applied to the PC12 cell-cultured conductive membranes. After 7 days, the greatest number of neurites appeared on the membranes with a current intensity approximating 1.7-8.4 microA/cm. Nerve guidance channels made of conductive biodegradable composite were implanted into rats to replace 8 mm of sciatic nerve. The implants were harvested after 2 months and analyzed with immunohistochemistry and transmission electron microscopy. The regenerated nerve tissue displayed myelinated axons and Schwann cells that were similar to those in the native nerve. Electrical stimulation applied through the electrically conductive biodegradable polymers therefore enhanced neurite outgrowth in a current-dependent fashion. The conductive polymers also supported sciatic nerve regeneration in rats.

In vivo evaluation of a novel electrically conductive polypyrrole/poly(D,L-lactide) composite and polypyrrole-coated poly(D,L-lactide-co-glycolide) membranes.

This study evaluated the in vivo biocompatibility and biodegradation behavior of a novel polypyrrole (PPy)/poly(D,L-lactide) (PDLLA) composite and PPy-coated poly(D,L-lactide-co-glycolide) membranes. Test membranes were implanted subcutaneously in rats for 3-120 days. The biocompatibility was assessed by quantifying the alkaline and acid phosphatase secretion, the immunohistochemical staining of the ED-2-positive macrophages, and the histology at the tissue/material interface. The degradation was investigated using scanning electron microscopy. Pure PDLLA and poly(D,L-lactide-co-glycolide) membranes were used as references, whereas expanded polytetrafluoroethylene and a commercial styrene-butadiene rubber were used as controls. The enzyme activity of the PPy-containing specimens was shown to be similar to that of the references. The histological findings were consistent with the enzymatic results, showing a mild-to-moderate acute inflammation followed by a resolution of the inflammatory response with a decrease in inflammatory cells for each biodegradable membrane. The tissue reactions to the PPy, which was either in the form of nanoparticles or surface coating, were comparable to the response to the neighboring biodegradable materials. Elevated ED-2-positive macrophage populations appeared as early as day 3 in the loose connective tissue surrounding the implants. The density of these populations was related to the degree of inflammation. Scanning electron microscopy showed that the degradation of the PPy/PDLLA composite was not affected by the presence of PPy.

A biodegradable electrical bioconductor made of polypyrrole nanoparticle/poly(D,L-lactide) composite: A preliminary in vitro biostability study.

The electrical stability of a novel polypyrrole (PPy)/poly(D,L-lactide) (PDLLA) composite was studied in vitro and compared with that of PPy-coated polyester fabrics. Specimens were incubated in Ringer's solution at 37 degrees C for up to 8 weeks with or without the circulation of DC current under a constant 100 mV voltage. In situ current variation with incubation time was recorded. The AC volume electrical conductivity of the specimens before and after incubation in phosphate-buffered saline was recorded using a frequency analyzer. Water absorption and weight loss were monitored metrologically. Changes in the oxidation state of incubated PPy were analyzed with X-ray photoelectron spectroscopy. The morphological changes were observed with scanning electron microscopy, and the glass transition temperature of the PDLLA was investigated using differential scanning calorimetry. The PPy/PDLLA composite in Ringer's solution sustained a relatively stable conductivity up to 8 weeks after an initial period of "conditioning." The PPy-coated fabrics experienced a rapid loss of conductivity when subjected to electrical circulation and regained part of it when disconnected. The volume conductivity of the nonincubated PPy/PDLLA membrane behaved as a typical conductor in the low-frequency range. The mechanisms involved in the various electrical behaviours of the PPy/PDLLA composite and PPy-coated fabrics are discussed. In conclusion, the PPy/PDLLA composite was able to deliver a biologically significant electrical current in a simulated biological solution for up to 8 weeks and therefore may be considered as a first-generation synthetic biodegradable bioconductor.