Platensimycin-Encapsulated Liposomes or Micelles as Biosafe Nanoantibiotics Exhibited Strong Antibacterial Activities against Methicillin-Resistant Staphylococcus aureus Infection in Mice.

Platensimycin (PTM) is a promising natural product drug lead against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), while the clinical development was hampered by problems related to its poor solubility and pharmacokinetic properties. In this study, we used liposomes and micelles as carriers of PTM to prepare PTM nanoformulations for the treatment of MRSA infection in mice. PTM-loaded nanoparticles could effectively reduce residual bacteria in the MRSA-infected macrophage cell model, comparing to free PTM. More importantly, in vivo studies showed that encapsulation of PTM by liposomes or micelles effectively improved the pharmacokinetic properties of PTM in Sprague-Dawley rats and the survival rate of MRSA-infected C57BL/6J mice. Our study has thus suggested that the clinically used nanocarriers, such as liposome and micelle, might also be useful to improve the efficacy of other natural product drug leads to accelerate their in vivo evaluation and preclinical development.

2-PHENOXYETHANOL (2-PE) AND TRICAINE METHANESULFONATE (MS-222) IMMERSION ANESTHESIA OF AMERICAN HORSESHOE CRABS (LIMULUS POLYPHEMUS).

Despite extensive literature examining American horseshoe crab physiology, there are comparatively few publications addressing their medical care. Establishing anesthesia protocols for horseshoe crabs is integral to limiting the potential stress and pain associated with invasive procedures and for advancing euthanasia techniques. The objective of this study was to compare the effects of two immersion anesthetics, tricaine methanesulfonate (MS-222) at 1 g/L (buffered with sodium carbonate) and 2-phenoxyethanol (2-PE) at 2 mL/L, on horseshoe crabs. Twenty horseshoe crabs were assigned to one of two anesthetic treatment groups and individually anesthetized in natural seawater. Water quality, cardiac contractility, and hemolymph gas analytes were measured prior to anesthesia and at 30 min Animals were monitored via heart rate, gilling rate, and sedation score every 5 min until recovered. Transcarapacial ultrasonography was used to obtain heart rate, gilling rate, and percent fractional shortening. Light or surgical anesthesia was produced in 10/10 animals in the 2-PE group and 8/10 animals in the MS-222 group. There was no significant difference in sedation scores, induction time (median 15 min), or recovery time (median 20.5 min). Gilling rate and cardiac contractility decreased during anesthesia, whereas heart rate did not. Hemolymph pH and pO2 were not different among treatment groups or time points. Baseline pCO2 was higher than pCO2 at 30 min for both groups but significantly elevated only in the MS-222 group. This is attributed to increased activity during the handling of awake animals. Invasive blood pressure obtained via cardiac catheterization in two animals was markedly decreased during surgical anesthesia. In conclusion, 2-PE and MS-222 provided effective anesthesia with clinically useful induction and recovery times. 2-PE provided a subjectively more reliable and smoother anesthesia compared to MS-222.

Effect of repeated exposure to AQUI-S® on the viability and growth of Neoparamoeba perurans.

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28-day period to AQUI-S® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post-smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non-attached amoeba in the culture was assessed with a vital stain. AQUI-S® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

International survey on the use and welfare of zebrafish Danio rerio in research.

A survey was conducted regarding zebrafish Danio rerio use for scientific research with a focus on: anaesthesia and euthanasia; housing and husbandry; breeding and production; refinement opportunities. A total of 98 survey responses were received from laboratories in 22 countries in Europe, North America, South America, Asia and Australia. There appears a clear and urgent need to identify the most humane methods of anaesthesia and euthanasia. Aversive responses to MS-222 were widely observed raising concerns about the use of this anaesthetic for D. rerio. The use of anaesthesia in fin clipping for genetic identification is widely practised and there appears to be an opportunity to further develop less invasive methods and refine this process. Optimization (and potentially standardization) of feeding is an area for further investigation. Given that diet and body condition can have such profound effects on results of experiments, differences in practice could have significant scientific implications. Further research into transition between dark and light phases in the laboratory appears to represent an opportunity to establish best practice. Plants and gravel were not considered practical by many laboratories. The true value and benefits need to be established and communicated. Overproduction is a concern both from ethical and financial viewpoints. There is an opportunity to further reduce wastage of D. rerio. There are clear concerns and opportunities for the scientific community to work together to further improve the welfare of these important laboratory models.

Phase 1, open-label study of MEDI-547 in patients with relapsed or refractory solid tumors.

BACKGROUND: Targeting the cell-surface receptor EphA2, which is highly expressed in some solid tumors, is a novel approach for cancer therapy. We aimed to evaluate the safety profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of MEDI-547, an antibody drug conjugate composed of the cytotoxic drug auristatin (toxin) linked to a human anti-EphA2 monoclonal antibody (1C1), in patients with solid tumors relapsed/refractory to standard therapy. METHODS: In this phase 1, open-label study with planned dose-escalation and dose-expansion cohorts, patients received a 1-h intravenous infusion of MEDI-547 (0.08 mg/kg) every 3 weeks. RESULTS: Six patients received 0.08 mg/kg; all discontinued treatment. Dose escalation was not pursued. The study was stopped before cohort 2 enrollment due to treatment-related bleeding and coagulation events (hemorrhage-related, n = 3; epistaxis, n = 2). Therefore, lower doses were not explored and an MTD could not be selected. The most frequently reported treatment-related adverse events (AEs) were increased liver enzymes, decreased hemoglobin, decreased appetite, and epistaxis. Three patients (50%) experienced treatment-related serious AEs, including conjunctival hemorrhage, pain (led to study drug discontinuation), liver disorder, and hemorrhage. Best response included progressive disease (n = 5; 83.3%) and stable disease (n = 1; 16.7%). Minimal or no dissociation of toxin from 1C1 conjugate occurred in the blood. Serum MEDI-547 concentrations decreased rapidly, ~70% by 3 days post-dose. No accumulation of MEDI-547 was observed at 0.08 mg/kg upon administration of a second dose 3 weeks following dose 1. CONCLUSIONS: The safety profile of MEDI-547 does not support further clinical investigation in patients with advanced solid tumors.

Repeated exposure of goldfish (Carassius auratus) to tricaine methanesulfonate (MS-222).

Goldfish that have been repeatedly exposed to tricaine methanesulfonate (MS-222) require greater concentration of the drug to attain equivalent planes of anesthesia, but the mechanism for this increased anesthetic need is unknown. Minimum anesthetic concentration (MAC) is a commonly used method with which to compare anesthetics. It was hypothesized that fish exposed to MS-222 daily would have an increased MAC. It was also hypothesized that fish exposed daily to MS-222 would develop histomorphologic changes to their gills to explain the increasing demand. Forty-nine Serasa comet goldfish were enrolled and were divided into three populations (n = 15, n = 15, and n = 19). In trial 1, using an up-down method, MAC was determined daily after 4 min of exposure to MS-222 for which the starting concentration was 160 mg/L. In trial 2, MAC was determined following 2 min of exposure to MS-222 for which the starting concentration was 260 mg/L. In trial 3, four naive fish were euthanatized and gills collected for histology and electron microscopy (EM). The remaining fish were exposed to MS-222 daily for 4 wk. Four fish were euthanatized and their gills submitted for similar examination at 2 wk and 4 wk. MAC for fish exposed to MS-222 for 4 min increased from 120 to 160 mg/L. The regression line had a slope of 1.51 +/- 0.26 (R2 = 0.65; P < 0.0001). MAC for fish exposed to MS-222 for 2 min increased from 210 pmm to 220 mg/L; the regression line had a slope of 0.52 +/- 0.38 (R2 = 0.12; P = 0.2). Histologic and EM examination of gills did not show morphologic changes indicative of a reaction to MS-222. Goldfish in this study had an increased requirement for MS-222 following daily exposure for 4 min but not following daily exposure for 2 min at a higher concentration. The cause of this increased anesthetic need is not related to morphologic changes to the gills.

A Comparison of Buffered Tricaine Methanesulfonate (MS-222) and Isoeugenol Anesthesia for Caudal Fin Clipping in Zebrafish (Danio rerio).

The purpose of this study was to compare the safety and efficacy of buffered tricaine methanesulfonate (MS-222) and isoeugenol for the anesthesia of zebrafish undergoing caudal fin clipping. Eighty 9 mo Danio rerio (AB strain) zebrafish were allocated to one of 2 equal groups: buffered MS-222 (168 mg/L, n = 40) or isoeugenol (20 mg/L, n = 40). The time to induction of anesthesia was significantly shorter in the isoeugenol group (141 ± 70 s) than in the MS-222 group (207 ± 103 s). The time to recovery from anesthesia was also shorter in the MS-222 group (373 ± 125 s) than in the isoeugenol group (491 ± 176 s). No obvious displays of distress or aversion to anesthesia were observed in either group. No difference was detected in the proportion of zebrafish that became anesthetized with either drug. One male zebrafish in the buffered MS-222 group was found dead at the 1-h post-procedural monitoring time point, but there was no difference between groups in the proportion of fish that survived anesthesia to the end of experiment. In conclusion, the safety and efficacy of buffered MS-222 (168 mg/L) and isoeugenol (20 mg/L) was similar for zebrafish undergoing anesthesia for caudal fin clipping.

Effects of ambasilide in isolated perfused guinea pig heart: use dependence.

There has been increased interest in class III antiarrhythmic drugs for con-version of atrial fibrillation to sinus rhythm. Ambasilide, a class III antiarrhythmic, has been shown to block multiple cardiac channels in a variety of animals including humans. Although the electrophysiological effects of ambasilide are characterized on the cellular level, its effects on an organ level have yet to be investigated. We investigated escalating doses of ambasilide in isolated, per-fused guinea pig hearts. Ambasilide prolonged the RR, PQ, QRS, QT, and QTc (F) in a concentration-dependent manner in either normal sinus rhythm or with reduced heart rate (atriectomy). dP/dtmin was increased (became less negative) in the presence of increasing concentrations of ambasilide, whereas the vehicle produced less negative lusitropy. Ambasilide demonstrated use dependence by prolonging QTc (F) less at slower heart rates. Ambasilide also inhibited isoproterenol-induced tachycardia, suggesting it exerts beta-adrenergic blocking properties. In conclusion, this study suggests that ambasilide has multichannel blocking properties including beta-adrenergic antagonism.

Analysis of Short-Term Cortisol Stress Response in Channel Catfish by Anesthetization with Metomidate Hydrochloride and Tricaine Methanesulfonate.

The ability of the anesthetics metomidate hydrochloride and tricaine methanesulfonate (MS-222) to mitigate the cortisol stress response of Channel Catfish Ictalurus punctatus was evaluated during a 10-min confinement stress. The cortisol concentrations of Channel Catfish anesthetized in metomidate hydrochloride remained consistent throughout the 10-min exposure; however, for fish anesthetized with MS-222 and nonanesthetized fish, cortisol concentrations were approximately 7- and 22-fold higher, respectively, than the baseline concentrations. While both anesthetics reduced cortisol concentrations relative to those of nonanesthetized fish, these results suggest that MS-222 is an appropriate anesthetic to use during the initial 5 min of sedation and that metomidate hydrochloride is appropriate for longer periods of sedation.

Methemoglobinemia induced by a topical anesthetic spray (cetacaine).

In two seriously ill patients, cyanosis developed shortly after a topical anesthetic spray (Cetacaine) was used. In both cases the presence of methemoglobinemia was suggested by a discrepancy between the arterial oxygen tension and the oxygen saturation of hemoglobin, as measured spectrophotometrically. The characteristic responses of the spectrophotometric oximeter to blood containing different concentrations of methemoglobin are described. Physicians administering this topical anesthetic spray (Cetacaine) should be aware of the possible development of methemoglobinemia.

In vitro complement consumption by contrast materials and analogues: reactors vs. nonreactors.

Blood samples from previous contrast reactors and nonreactor controls were incubated with diatrizoate and several contrast analogues. Total complement levels were assayed. All the agents caused complement activation generally proportional to their concentration. Reactors' sera responded to lower concentrations of contrast or analogues than did control sera. Such studies might be of value in predicting contrast reactors.

Symptomatic methemoglobinemia with a commonly used topical anesthetic, cetacaine.

Severe cyanosis resulting from acquired methemoglobinemia after application of a topical anesthetic, Cetacaine spray, occurred in a 37-year-old patient following bronchoscopy for postoperative atelectasis. Response to methylene blue therapy was dramatic and complete. Attention is drawn to a dangerous adverse effect of this commonly used topical anesthetic agent.

Phototoxicity to a sunscreen ingredient. Padimate A.

Para-aminobenzoic acid and certain of its esters are widely used in sunscreens. We found that one of these, amyl paradimethylaminobenzoate (padimate A) was capable of provoking phototoxic reactions; With ultraviolet A (UV-A) dosages of about 30 joules/sq cm, a reaction grossly resembling sunburn developed in most subjects. The dominant histological feature was vacuolar degeneration of the epidermis without sunburn cells. Proprietary sunscreens containing padimate A were also shown to produce a similar reaction. Our explanation for the paradox of a sunscreen that promotes "sunburn" is that, as affected users have concluded, the product was ineffective. The similarity between sunburn and a phototoxic response has delayed recognition of this adverse effect until now.

Allergic contact photodermatitis to para-aminobenzoic acid.

Allergic photocontact dermatitis developed in a patient to a commercial sunscreen preparation containing para-aminobenzoic acid (PABA) in an alcohol base. Photopatch testing showed photosensitivity to PABA. Sensitization could only be detected when PABA was tested in an alcohol vehicle; a photopatch test to PABA in petrolatum was negative.

Allergic contact dermatitis from sunscreen preparations complicating photodermatitis.

Two men with known histories of photosensitivity had apparent exacerbations of their dermatoses. Patch-testing showed the presence of allergic contact sensitivity to the sunscreens that they were using. The clinical distribution patterns of adverse reactions to sunscreens naturally simulate sun-induced reactions, thus providing clinical problems that require evaluation by both patch and photo-patch tests. These findings emphasize that allergic contact or photo-contact dermatitis from sunscreens that are used for prophylaxis of photodermatitis can actually complicate or aggravate the preexisting state of photodermatitis for which the sunscreens are prescribed.

Retinal toxicity associated with occupational exposure to the fish anesthetic MS-222.

PURPOSE: To report a case of retinopathy associated with chronic occupational exposure to ethyl-m-aminobenzoic acid methanesulfonate (MS-222), a retinotoxic fish anesthetic. METHOD: Case report with electroretinograms to document changes in visual electrophysiology. RESULTS: An ichthyologist with a long history of skin exposure to MS-222 was initially examined for decreased vision, photophobia, and photopsia. His electroretinogram abnormalities were similar to those seen in animal models of acute MS-222 toxicity. After terminating MS-222 contact for 7 months, his vision returned to normal, and his electroretinogram improved. CONCLUSION: Individuals with occupational exposure to MS-222 should exercise caution to avoid systemic absorption of this retinotoxic compound.

Infantile methemoglobinemia induced by a topical anesthetic, Cetacaine.

A 2-month-old infant developed severe methemoglobinemia following topical pharyngeal application of a common benzocaine containing agent ( Cetacaine ). Although a number of reports of this complication have appeared in recent years, this is apparently the first case reported in the Otolaryngology literature. The pathophysiology, pharmacology, and treatment of this condition are reviewed.

Efficacy and safety of 5 anesthetics in adult zebrafish (Danio rerio).

Although the safety and efficacy of tricaine methanesulfonate (MS222) for anesthesia of fish are well established, other anesthetics used less commonly in fish have been less extensively evaluated. Therefore, we compared gradual cooling, lidocaine hydrochloride (300, 325, and 350 mg/L), metomidate hydrochloride (2, 4, 6, 8, and 10 mg/L), and isoflurane (0.5 mL/L) with MS222 (150 mg/L) for anesthesia of adult zebrafish. The efficacy and safety of each agent was evaluated by observing loss of equilibrium, slowing of opercular movement, response to tail-fin pinch, recovery time, and anesthesia-associated mortality rates. At 15 min after anesthetic recovery, we used a novel-tank test to evaluate whether anesthetic exposure influenced short-term anxiety-like behavior. Behavioral parameters measured included latency to enter and number of transitions to the upper half of the tank, number of erratic movements, and number of freezing bouts. Behavior after anesthesia was unaltered regardless of the anesthetic used. Efficacy and safety differed among the anesthetics evaluated. Gradual cooling was useful for short procedures requiring immobilization only, but all instrumentation and surfaces that come in contact with fish must be maintained at approximately 10 °C. MS222 and lidocaine hydrochloride at 325 mg/L were effective as anesthetic agents for surgical procedures in adult zebrafish, but isoflurane and high-dose lidocaine hydrochloride were unsuitable as sole anesthetic agents due to high (30%) mortality rates. Although MS222 remains the best choice for generating a surgical plane of anesthe- sia, metomidate hydrochloride and gradual cooling were useful for sedation and immobilization for nonpainful procedures.