Chronic Pharmacological and Safety Evaluation of Hematide™, a PEGylated Peptidic Erythropoiesis-Stimulating Agent, in Rodents

Hematide™ is a synthetic peptide-based, PEGylated erythropoiesis-stimulating agent, which is being developed for the chronic treatment of anaemia associated with chronic renal failure. To support the safety of long-term dosing of chronic renal failure patients, a comprehensive toxicology programme was implemented including rat subchronic and chronic studies. Rats were administered 0, 0.1, 1 and 10 mg/kg of Hematide every 3 weeks for 3 months via subcutaneous injection or for 6 months via intravenous injection. The dosing period was followed by a 6-week follow-up period. The primary pharmacology of Hematide resulted in erythroid polycythemia as measured by elevated haemoglobin levels that were time-and dose-dependent. The pharmacology profiles were similar regardless of administration route. For example, for male rats at Day 90, subcutaneous dosing resulted in haemoglobin increases of 2.7, 4.5 and 6.9 g/dl for 0.1, 1 and 10 mg Hematide/kg respectively, compared to 2.8, 5.7 and 7.4 g/dl increases for intravenous dosing. Histopathological changes were related to the prolonged severe polycythemia induced in normocythemic animals administered an erythropoiesis-stimulating agent. The findings included extramedullary haematopoiesis in the spleen and liver, bone marrow hypercellularity and organ congestion. Microscopic findings were reversible, demonstrating a return towards control findings within 6 weeks following cessation of dosing. Systemic exposures, based on both area under the curve (AUC) and maximum concentration (Cmax), were substantially greater for intravenous than subcutaneous administration. No Hematide-specific antibodies were detected. In conclusion, Hematide is a potent erythropoiesis-stimulating agent, and the studies provide support for the safety of clinical development, including chronic dosing, for the treatment of anaemia associated with chronic renal failure

Evaluation of Silver Nanoparticle Toxicity in Skin in Vivo and Keratinocytes in Vitro

IntroductionProducts using the antimicrobial properties of silver nanoparticles (Ag-nps) may be found in health and consumer products that routinely contact skin.ObjectivesThis study was designed to assess the potential cytotoxicity of Ag-nps in human epidermal keratinocytes (HEKs) and their inflammatory and penetrating potential into porcine skin in vivo.Materials and MethodsWe used eight different Ag-nps in this study [unwashed/uncoated (20, 50, and 80 nm particle diameter), washed/uncoated (20, 50, and 80 nm), and carbon-coated (25 and 35 nm)]. Skin was dosed topically for 14 consecutive days. HEK viability was assessed by MTT, alamarBlue (aB), and CellTiter 96 AQueous One (96AQ). Release of the proinflammatory mediators interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) were measured.ResultsThe effect of the unwashed Ag-nps on HEK viability after a 24-hr exposure indicated a significant dose-dependent decrease (p < 0.05) at 0.34 μg/mL with aB and 96AQ and at 1.7 μg/mL with MTT. However, both the washed Ag-nps and carbon-coated Ag-nps showed no significant decrease in viability at any concentration assessed by any of the three assays. For each of the unwashed Ag-nps, we noted a significant increase (p < 0.05) in IL-1β, IL-6, IL-8, and TNF-α concentrations. We observed localization of all Ag-nps in cytoplasmic vacuoles of HEKs. Macroscopic observations showed no gross irritation in porcine skin, whereas microscopic and ultrastructural observations showed areas of focal inflammation and localization of Ag-nps on the surface and in the upper stratum corneum layers of the skin.ConclusionThis study provides a better understanding Ag-nps safety in vitro as well as in vivo and a basis for occupational and risk assessment. Ag-nps are nontoxic when dosed in washed Ag-nps solutions or carbon coated

Thermoreversible Gel-Loaded Amphotericin B for the Treatment of Dermal and Vaginal Candidiasis

The present study was designed to develop a thermoreversible gel of Pluronic (P407) loaded amphotericin B (AmB-gel) for the dermal and vaginal treatment of candidiasis. P407 was used as a copolymer to exploit potential advantages related to increasing drug concentration in the tissue layer in order to provide a local effect. Parameters including internal structure, swelling, porosity, and short-term stability were determined. In addition, drug release profile and ex vivo skin and vaginal permeation studies were carried out. Antifungal efficacy was evaluated against strains of Candida spp. and atomic force microscopy (AFM) supported the results. The tolerance of AmB-gel was studied by evaluating biomechanical properties of skin and determining the irritation level in scarified rabbit skin supported by histological analysis. Results confirmed the development of a thermoreversible AmB-gel with high porosity exhibiting Newtonian behavior at 4 ºC and pseudoplasticity at 32 ºC as well as optimal stability for at least 90 days. The Amb-gel provided a sustained drug release following a Boltzmann sigmoidal model. Non permeation was observed in skin and vaginal mucosa, showing a high retained amount of AmB of 960.0 and 737.3 ug/g/cm2, respectively. In vitro antifungal efficacy showed that AmB-gel was more effective than Free-AmB in inhibiting strains of Candida spp. and these results were corroborated by AFM. Finally, tolerance studies showed that its application did not induce skin irritation nor alter its biophysical properties. Together, these results confirmed that AmB-gel could be proposed as a promising candidate for the clinical status in the treatment of skin and vaginal candidiasis.This research was funded by L’Agència de Gestiód’Ajuts Universitarisi de Recerca (AGAUR) grant number [SGR-2017 1744]. Lilian Sosa also expresses gratitude for the grant awarded by the Institute of Nanoscience and Nanotechnology IN2UB number [2017.3.IN2UB.2] in the completion of her doctoral thesis. Marcelle Silva-Abreu acknowledges the support from the Coordination for the Improvement Personnel (CAPES-Brazil)

In Vitro Cell Models for Ophthalmic Drug Development Applications

© Sara Shafaie et al. 2016; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.Tissue engineering is a rapidly expanding field that aims to establish feasible techniques to fabricate biologically equivalent replacements for diseased and damaged tissues/organs. Emerging from this prospect is the development of in vitro representations of organs for drug toxicity assessment. Due to the ever-increasing interest in ocular drug delivery as a route for administration as well as the rise of new ophthalmic therapeutics, there is a demand for physiologically accurate in vitro models of the eye to assess drug delivery and safety of new ocular medicines. This review summarizes current existing ocular models and highlights the important factors and limitations that need to be considered during their use.Peer reviewe

Enhanced hydrogen peroxide generation accompanies the beneficial bioenergetic effects of methylene blue in isolated brain mitochondria

The redox dye methylene blue (MB) is proven to have beneficial effects in various models of neurodegenerative diseases. Here we investigated the effects of MB (100 nM, 300 nM, and 1 μM) on key bioenergetic parameters and on H2O2 production/elimination in isolated guinea pig brain mitochondria under normal as well as respiration-impaired conditions. As measured by high-resolution Oxygraph the rate of resting oxygen consumption was increased, but the ADP-stimulated respiration was unaffected by MB with any of the substrates (glutamate malate, succinate, or α-glycerophosphate) used for supporting mitochondrial respiration. In mitochondria treated with inhibitors of complex I or complex III MB moderately but significantly increased the rate of ATP production, restored ΔΨm, and increased the rate of Ca2+ uptake. The effects of MB are consistent with transferring electrons from upstream components of the electron transport chain to cytochrome c, which is energetically favorable when the flow of electrons in the respiratory chain is compromised. On the other hand, MB significantly increased the production of H2O2 measured by Amplex UltraRed fluorimetry under all conditions, in resting, ATP-synthesizing, and respiration-impaired mitochondria, with each substrate combination supporting respiration. Furthermore, it also decreased the elimination of H2O2. Generation of H2O2 without superoxide formation, observed in the presence of MB, is interpreted as a result of reduction of molecular oxygen to H2O2 by the reduced MB. The elevated generation and impaired elimination of H2O2 should be considered for the overall oxidative state of mitochondria treated with MB

A Synthetic Adjuvant to Enhance and Expand Immune Responses to Influenza Vaccines

Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (TH1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines

Vaccination with Plasmodium knowlesi AMA1 Formulated in the Novel Adjuvant Co-Vaccine HT™ Protects against Blood-Stage Challenge in Rhesus Macaques

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC50 values correlated with estimated in vivo growth rates