Considering ivermectin for treatment of schistosomiasis.

Because of recent reports of praziquantel resistance in schistosome infections, there have been suggestions to employ ivermectin as a possible alternative, especially as its chemical composition is different from that of praziquantel, so cross-resistance is not expected. In order to ascertain possible damage and elimination of worms, we used ivermectin by oral gavage in infected mice, at a high dose (30.1 mg/kg, bordering toxicity). We also tested the efficacy of the drug at various times postinfection (PI), to check on possible effect on young and mature stages of the parasites. Thus, we treated mice on days 21 and 22 or on days 41 and 42 and even on days 21, 22, 41, and 42 PI. None of the treatment regimens resulted in cure rates or signs of lessened pathology in the mice. We also compared the effect of ivermectin to that of artemisone, an artemisinin derivative which had served us in the past as an effective anti-schistosome drug, and there was a stark difference in the artemisone's efficacy compared to that of ivermectin; while ivermectin was not effective, artemisone eliminated most of the worms, prevented egg production and granulomatous inflammatory response. We assume that the reported lack of activity of ivermectin, in comparison with praziquantel and artemisinins, originates from the difference in their mode of action. In wake of our results, we suggest that ivermectin is not a suitable drug for treatment of schistosomiasis.

Efficient Treatment of Experimental Cerebral Malaria by an Artemisone-SMEDDS System: Impact of Application Route and Dosing Frequency.

Artemisone (ART) has been successfully tested in vitro and in animal models against several diseases. However, its poor aqueous solubility and limited chemical stability are serious challenges. We developed a self-microemulsifying drug delivery system (SMEDDS) that overcomes these limitations. Here, we demonstrate the efficacy of this formulation against experimental cerebral malaria in mice and the impact of its administration using different routes (gavage, intranasal delivery, and parenteral injections) and frequency on the efficacy of the treatment. The minimal effective daily oral dose was 20 mg/kg. We found that splitting a dose of 20 mg/kg ART given every 24 h, by administering two doses of 10 mg/kg each every 12 h, was highly effective and gave far superior results compared to 20 mg/kg once daily. We obtained the best results with nasal treatment; oral treatment was ranked second, and the least effective route of administration was intraperitoneal injection. A complete cure of experimental cerebral malaria could be achieved through choosing the optimal route of application, dose, and dosing interval. Altogether, the developed formulation combines easy manufacturing with high stability and could be a successful and very versatile carrier for the delivery of ART in the treatment of human severe malaria.

Asymmetric trisalkylamine cyclopropenium derivatives with antimicrobial activity.

Cationic molecules are found in abundance as antimicrobial agents with a well-defined mechanism of action and significant therapeutic benefits. Quaternary ammonium-containing compounds are frequently employed due to their facile synthesis and tunable properties. Over time, however, bacterial resistance to these compounds has become a significant obstacle. We report here a series of asymmetric trisalkylamine cyclopropenium cationic derivatives as chemical isosteres of quaternary ammonium compounds, capable of strong antimicrobial activity and overcoming microbial resistance. These small molecules were prepared by one-pot reaction of tetrachlorocyclopropene (TCC) with unhindered secondary amines in the presence of Hünig's base. In this work we describe the synthesis, purification, and characterization of five trisamino-cyclopropenium derivatives and confirm their structures by spectral analysis and mass-spectrometry. Three of the compounds displayed considerable antimalarial activity (IC50 < 0.1 µM) without demonstrating significant toxic effects in vitro (TC50 > 1 µM). This class of cyclopropenium-based compounds provides an opening for the discovery of potent and non-toxic antimicrobial agents.

Exploration of Macroporous Polymeric Sponges As Drug Carriers.

Achieving high drug loading capacity and controlling drug delivery are two main challenges related to drug carriers. In this study, polymeric macroporous sponges with very high pore volume and large porosity are introduced as a new-type of drug carrier. Due to the high pore volume (285 and 166 cm3/g for the sponges with densities of 3.5 and 6.0 mg/cm3, respectively), the sponges exhibit very high drug loading capacities with average values of 1870 ± 114 and 2697 ± 73 mg/g in the present study, which is much higher than the meso and microporous drug carriers (<1500 mg/g). In order to control the release profiles, an additional poly(p-xylylene) (PPX) coating was deposited by chemical vapor deposition on the drug loaded sponge. Consequently, Artemisone (ART) release in the aqueous medium could be retarded, depending on the density of the sponge and the thickness of the coating. In future, the new 3D polymeric sponges would be highly beneficial as drug carriers for the programmed release of drugs for treatment of chronic diseases.

PEG-Biscyanoacrylate Crosslinker for Octyl Cyanoacrylate Bioadhesive.

PEG400 (polyethylene glycol, MW 400) biscyanoacrylate is synthesized and copolymerized with 2-octyl cyanoacrylate for potential use as bioadhesive. PEG400 biscyanoacrylate is synthesized from the esterification of anthracenyl cyanoacrylic acid where the anthracene unit serves as vinyl-protecting group. Copolymerization increases the plasticity, mechanical strength, and resilience of the resulted polymer as determined by dynamic mechanical analysis. Peeling test confirms its superior bioadhesive properties. Surface morphology is characterized by SEM imaging. The formulations are cytocompatible and safe. This cyanoacrylate composition may provide improved bioadhesive cyanoacrylates.

Imaging the urinary pathways in mice by liposomal indocyanine green.

Intraoperative ureter identification can assist in the prevention of ureteral injury and consequently improve surgery outcomes. Our aim was to take advantage of the altered pharmacokinetics of liposomal indocyanine green (ICG), the only FDA-approved near-infrared (NIR) dye, for imaging of ureters during surgeries. ICG was passively adsorbed to liposomes. NIR whole mice body and isolated tissue imaging were used to study liposomal ICG properties vs. free ICG. In vivo, the urinary bladder could be clearly observed in most of the liposome-treated mice. Liposomal encapsulation of ICG enhanced ureteral emission up to 1.9 fold compared to free ICG (P<0.01). Increase in liposomal micropolarity and microviscosity and differential scanning calorimetry supported ICG localization within the liposomal bilayer. Our findings suggest that liposomal ICG could be utilized for ureteral imaging intra-operatively, thus potentially improving surgical outcomes. FROM THE CLINICAL EDITOR: Iatrogenic ureteral injury is a serious complication of abdominal surgery and intra-operative recognition of the ureters is usually the best method of injury prevention. In this article, the authors developed liposomal indocyanine green, which could be excreted via the urinary system and investigated its in-vivo use in mice.

Treatment of murine cerebral malaria by artemisone in combination with conventional antimalarial drugs: antiplasmodial effects and immune responses.

The decreasing effectiveness of antimalarial therapy due to drug resistance necessitates constant efforts to develop new drugs. Artemisinin derivatives are the most recent drugs that have been introduced and are considered the first line of treatment, but there are already indications of Plasmodium falciparum resistance to artemisinins. Consequently, drug combinations are recommended for prevention of the induction of resistance. The research here demonstrates the effects of novel combinations of the new artemisinin derivative, artemisone, a recently described 10-alkylamino artemisinin derivative with improved antimalarial activity and reduced neurotoxicity. We here investigate its ability to kill P. falciparum in a high-throughput in vitro assay and to protect mice against lethal cerebral malaria caused by Plasmodium berghei ANKA when used alone or in combination with established antimalarial drugs. Artemisone effects against P. falciparum in vitro were synergistic with halofantrine and mefloquine, and additive with 25 other drugs, including chloroquine and doxycycline. The concentrations of artemisone combinations that were toxic against THP-1 cells in vitro were much higher than their effective antimalarial concentration. Artemisone, mefloquine, chloroquine, or piperaquine given individually mostly protected mice against cerebral malaria caused by P. berghei ANKA but did not prevent parasite recrudescence. Combinations of artemisone with any of the other three drugs did completely cure most mice of malaria. The combination of artemisone and chloroquine decreased the ratio of proinflammatory (gamma interferon, tumor necrosis factor) to anti-inflammatory (interleukin 10 [IL-10], IL-4) cytokines in the plasma of P. berghei-infected mice. Thus, artemisone in combinations with other antimalarial drugs might have a dual action, both killing parasites and limiting the potentially deleterious host inflammatory response.

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites.

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.

Applicability of Redirecting Artemisinins for New Targets.

Employing new therapeutic indications for drugs that are already approved for human use has obvious advantages, including reduced costs and timelines, because some routine steps of drug development and regulation are not required. This work concentrates on the redirection of artemisinins (ARTS) that already are approved for clinical use, or investigated, for malaria treatment. Several mechanisms of action are suggested for ARTS, among which only a few have been successfully examined in vivo, mainly the induction of oxidant stress and anti-inflammatory effects. Despite these seemingly contradictory effects, ARTS are proposed for repurposing in treatment of inflammatory disorders and diverse types of diseases caused by viral, bacterial, fungal, and parasitic infections. When pathogens are treated the expected outcome is diminution of the causative agents and/or their inflammatory damage. In general, repurposing ARTS is successful in only a very few cases, specifically when a valid mechanism can be targeted using an additional therapeutic agent and appropriate drug delivery. Investigation of repurposing should include optimization of drug combinations followed by examination in relevant cell lines, organoids, and animal models, before moving to clinical trials.

Synthesis of artemiside and its effects in combination with conventional drugs against severe murine malaria.

This research describes the use of novel antimalarial combinations of the new artemisinin derivative artemiside, a 10-alkylamino artemisinin. It is a stable, highly crystalline compound that is economically prepared from dihydroartemisinin in a one-step process. Artemiside activity was more pronounced than that of any antimalarial drug in use, both in Plasmodium falciparum culture and in vivo in a murine malaria model depicting cerebral malaria (CM). In vitro high-throughput testing of artemiside combinations revealed a large number of conventional antimalarial drugs with which it was additive. Following monotherapy in mice, individual drugs reduced parasitemias to nondetectable levels. However, after a period of latency, parasites again were seen and eventually all mice became terminally ill. Treatment with individual drugs did not prevent CM in mice with recrudescent malaria, except for piperaquine at high concentrations. Even when CM was prevented, the mice developed later of severe anemia. In contrast, most of the mice treated with drug combinations survived. A combination of artemiside and mefloquine or piperaquine may confer an optimal result because of the longer half life of both conventional drugs. The use of artemiside combinations revealed a significant safety margin of the effective artemiside doses. Likewise, a combination of 1.3 mg/kg of body weight artemiside and 10 mg/kg piperaquine administered for 3 days from the seventh day postinfection was completely curative. It appears possible to increase drug concentrations in the combination therapy without reaching toxic levels. Using the drug combinations as little as 1 day before the expected death of control animals, we could prevent further parasite development and death due to CM or anemic malaria. Earlier treatment may prevent cognitive dysfunctions which might occur after recovery from CM.

Transdermal delivery of artemisinins for treatment of pre-clinical cerebral malaria.

Transdermal drug delivery avoids complications related to oral or parenteral delivery - the need for sterility, contamination, gastrointestinal side effects, patient unconsciousness or nausea and compliance. For malaria treatment, we demonstrate successful novel transdermal delivery of artemisone (ART) and artesunate. The incorporation of ART into a microemulsion (ME) overcomes the limitations of the lipophilic drug and provides high transcutaneous bioavailability. ART delivery to the blood (above 500 ng/ml) was proved by examining the sera from treated mice, using a bioassay in cultured Plasmodium falciparum. Skin spraying of ART-ME eliminated P. berghei ANKA in an infected mouse model of cerebral malaria (CM) and prevented CM, even after a late treatment with a relatively small amount of ART (13.3 mg/kg). For comparison, the artesunate (the most used commercial artemisinin) formulation was prepared as ART. However, ART-ME was about three times more efficient than artesunate-ME. The solubility and stability of ART in the ME, taken together with the successful transdermal delivery leading to animal recovery, suggest this formulation as a potential candidate for transdermal treatment of malaria.

Protein kinase C θ deficiency increases resistance of C57BL/6J mice to Plasmodium berghei infection-induced cerebral malaria.

Protein kinase C θ (PKCθ) functions as a core component of the immunological synapse and serves as a key protein in the integrated T-cell antigen receptor (TCR)/CD28-induced signaling cascade leading to T-cell activation. However, the involvement of PKCθ in host-mediated immune responses to pathogens has not been thoroughly investigated. We tested the consequences of PKCθ ablation on the host response to infection by Plasmodium berghei ANKA (PbA). We found that both PKCθ(+/+) and PKCθ(-/-) C57BL/6J mice are susceptible to infection with PbA. However, despite a similar parasite burden, PKCθ(+/+) mice had an earlier onset of neurological signs, characteristics of experimental cerebral malaria (ECM), resulting in an earlier death. These mice suffered from an early and pronounced splenomegaly with a concomitant increase in the total number of CD4(+) splenic T cells. In contrast, a large proportion of PbA-infected PKCθ(-/-) mice overcame the acute phase characterized by neurological symptoms and survived longer than PKCθ(+/+) mice. The partial resistance of PKCθ(-/-) mice to ECM was associated with an impaired production of Th1-type cytokines, including gamma interferon and tumor necrosis factor alpha/lymphotoxin-α, which are known to exacerbate symptoms leading to ECM. In addition, PbA infection-induced LFA-1 expression in CD8(+) T cells was suppressed in PKCθ-deficient T cells, suggesting a diminished ability to adhere to endothelial cells and sequester in brain microvasculature, which may explain the decrease in neurological symptoms. These data implicate PKCθ in CD4(+) Th1(+) and CD8(+) T-cell-mediated immune responses during PbA infection that contribute to the development of ECM.

Schistosoma mansoni infection reduces the incidence of murine cerebral malaria.

BACKGROUND: Plasmodium and Schistosoma are two of the most common parasites in tropical areas. Deregulation of the immune response to Plasmodium falciparum, characterized by a Th1 response, leads to cerebral malaria (CM), while a Th2 response accompanies chronic schistosomiasis. METHODS: The development of CM was examined in mice with concomitant Schistosoma mansoni and Plasmodium berghei ANKA infections. The effect of S. mansoni egg antigen injection on disease development and survival was also determined. Cytokine serum levels were estimated using ELISA. Statistical analysis was performed using t-test. RESULTS: The results demonstrate that concomitant S. mansoni and P. berghei ANKA infection leads to a reduction in CM. This effect is dependent on infection schedule and infecting cercariae number, and is correlated with a Th2 response. Schistosomal egg antigen injection delays the death of Plasmodium-infected mice, indicating immune involvement. CONCLUSIONS: This research supports previous claims of a protective effect of helminth infection on CM development. The presence of multiple parasitic infections in patients from endemic areas should therefore be carefully noted in clinical trials, and in the development of standard treatment protocols for malaria. Defined helminth antigens may be considered for alleviation of immunopathological symptoms.

Artemisone effective against murine cerebral malaria.

BACKGROUND: Artemisinins are the newest class of drug approved for malaria treatment. Due to their unique mechanism of action, rapid effect on Plasmodium, and high efficacy in vivo, artemisinins have become essential components of malaria treatment. Administration of artemisinin derivatives in combination with other anti-plasmodials has become the first-line treatment for uncomplicated falciparum malaria. However, their efficiency in cases of cerebral malaria (CM) remains to be determined. METHODS: The efficacy of several artemisinin derivatives for treatment of experimental CM was evaluated in ICR or C57BL/6 mice infected by Plasmodium berghei ANKA. Both mouse strains serve as murine models for CM. RESULTS: Artemisone was the most efficient drug tested, and could prevent death even when administered at relatively late stages of cerebral pathogenesis. No parasite resistance to artemisone was detected in recrudescence. Co-administration of artemisone together with chloroquine was more effective than monotherapy with either drug, and led to complete cure. Artemiside was even more effective than artemisone, but this substance has yet to be submitted to preclinical toxicological evaluation. CONCLUSIONS: Altogether, the results support the use of artemisone for combined therapy of CM.

Modulation of cerebral malaria by fasudil and other immune-modifying compounds.

Malaria continues to cause millions of deaths annually. No specific effective treatment has yet been found for cerebral malaria, one of the most severe complications of the disease. The pathology of cerebral malaria is considered to be primarily immunological. We examined a number of compounds with known effects on the immune system, in a murine model of cerebral malaria. Of the compounds tested, only fasudil and curcumin had significant effects on the progression of the disease. Although neither drug caused a reduction in parasitemia, survival of the treated mice was significantly increased, and the development of cerebral malaria was either delayed or prevented. Our results support the hypothesis that an immunomodulator efficient in preventing CM should be administered together with anti-plasmodial drugs to prevent severe malaria disease; curcumin and fasudil should be further investigated to determine efficiency and feasibility of treatment.

Treatment of Experimental Cerebral Malaria by Slow Release of Artemisone From Injectable Pasty Formulation.

Malaria caused by Plasmodium falciparum causes numerous cases of morbidity with about 400,000 deaths yearly owing, mainly, to inflammation leading to cerebral malaria (CM). CM conventionally is treated by repetitive administration of anti-plasmodial drugs and supportive non-specific drugs, for about a week. A mouse model of CM caused by Plasmodium berghei ANKA, in which brain and systemic clinical pathologies occur followed by sudden death within about a week, was used to study the effect of artemisone, a relatively new artemisinin, within an injectable pasty polymer formulated for its controlled release. The parasites were exposed to the drug over several days at a non-toxic concentrations for the mice but high enough to affect the parasites. Artemisone was also tested in cultures of bacteria, cancer cells and P. falciparum to evaluate the specificity and suitability of these cells for examining the release of artemisone from its carrier. Cultures of P. falciparum were the most suitable. Artemisone released from subcutaneous injected poly(sebacic acid-ricinoleic acid) (PSARA) pasty polymer, reduced parasitemias in infected mice, prolonged survival and prevented death in most of the infected mice. Successful prophylactic treatment before infection proved that there was a slow release of the drug for about a week, which contrasts with the three hour half-life that occurs after injection of just the drug. Treatment with artemisone within the polymer, even at a late stage of the disease, helped to prevent or, at least, delay accompanying severe symptoms. In some cases, treatment prevented death of CM and the mice died later of anemia. Postponing the severe clinical symptoms is also beneficial in cases of human malaria, giving more time for an appropriate diagnosis and treatment before severe symptoms appear. The method presented here may also be useful for combination therapy of anti-plasmodial and immunomodulatory drugs.

Oral Administration of Artemisone for the Treatment of Schistosomiasis: Formulation Challenges and In Vivo Efficacy.

Artemisone is an innovative artemisinin derivative with applications in the treatment of malaria, schistosomiasis and other diseases. However, its low aqueous solubility and tendency to degrade after solubilisation limits the translation of this drug into clinical practice. We developed a self-microemulsifying drug delivery system (SMEDDS), which is easy to produce (simple mixing) with a high drug load. In addition to known pharmaceutical excipients (Capmul MCM, Kolliphor HS15, propylene glycol), we identified Polysorb ID 46 as a beneficial new additional excipient. The physicochemical properties were characterized by dynamic light scattering, conductivity measurements, rheology and electron microscopy. High storage stability, even at 30 °C, was achieved. The orally administrated artemisone SMEDDS formulation was highly active in vivo in S. mansoni infected mice. Thorough elimination of the adult worms, their eggs and prevention of the deleterious granuloma formation in the livers of infected mice was observed even at a relatively low dose of the drug. The new formulation has a high potential to accelerate the clinical use of artemisone in schistosomiasis and malaria.

The immunomodulatory effect of Sambucol on leishmanial and malarial infections.

A nontoxic dose of Sambucol, an immunomodulator commercially sold as an immune stimulator, was examined in murine models of leishmaniasis and malaria. Sambucol causes a shift in the immune response, as demonstrated in human monocyte cultures, to Th1 (inflammation-associated) responses. Treatment of leishmania-infected mice with Sambucol delayed the development of the disease. As there was no direct IN VITRO anti-leishmanial effect, the observed partial protection IN VIVO is most likely related to immune modulation. Although increased Th1 responses are associated with protection from leishmaniasis, they are considered to be the main immunopathological processes leading to cerebral malaria. Administration of Sambucol to mice prior to and following infection with Plasmodium berghei ANKA increased the incidence of cerebral malaria, while administration of Sambucol after infection had no effect on the disease. The results demonstrate how an inflammatory-like response may alleviate or exacerbate clinical symptoms of disease and hint at the importance of administration timing. The overall effect of immunomodulator administration depends on the ongoing immune response and the Th1/Th2 balance determined by both host and parasite defense mechanisms.

Surface characterization and biocompatibility of restorative resin containing nanoparticles.

Composite resins that are used to restore hard tissues have several drawbacks including the accumulation of biofilm on teeth and restorations. Recently, quaternary ammonium poly(ethylene imine) (QA-PEI) nanoparticles were developed for additional antibacterial activity of restorative composite resins. QA-PEI nanoparticles were synthesized from cross-linked poly(ethylene imine) that was N-alkylated with octyl halide, followed by quaternary methylation with methyl iodide. QA-PEI particles that were embedded in restorative composite resin at 1% w/w resulted in the complete growth inhibition of Streptococcus mutans. Moreover, the antibacterial activity was retained for at least 3 months. The active substances on the surface of the restorative composite resin that were incorporated with QA-PEI particles were detected by X-ray photoelectron spectroscopy (XPS) and confocal microscopy measurements. The in vitro cytotoxicity tests showed a similar effect on the viability of the cell line that was tested with composites including modified and unmodified dental composite resins. In vivo toxicity studies, which were assessed on Wistar rats by the implantation of modified composite specimens, revealed no inflammation response 1 week after the implantation of restorative composite resin that was embedded with up to 2% w/w QA-PEI.

Paclitaxel tumor biodistribution and efficacy after intratumoral injection of a biodegradable extended release implant.

PURPOSE: The aim of this study was to investigate the effectiveness of paclitaxel controlled release from intratumorally injected polymer. METHODS: The effectiveness of paclitaxel-polymer formulation injected intratumorally was tested in mouse bladder tumor model. To determine paclitaxel biodistribution in tumor at predetermined time periods the tumor was excised, frozen and sectioned, and the paclitaxel concentrations were determined in the tumor tissue and in plasma by HPLC. Histopathological evaluation of the necrosis and inflammation was performed on tumor sections. RESULTS: In the paclitaxel/polymer group mice were injected intratumorally with 0.2 ml of the 10% (w/w) paclitaxel formulation, the tumor disappeared completely 5 days after injection, and mice survived till the end of the study (50 days post-tumor cells inoculation). In biodistribution studies, the highest paclitaxel concentration in the tumor tissue was 40 microg/mg 1 day after the intratumoral injection and decreased gradually during 10 days to 5 microg/mg that is still high enough to induce cytotoxic effect, and the necrotic effect of paclitaxel on the tumors was confirmed by histopathology. CONCLUSIONS: Treatment with local injection of polymer-paclitaxel formulation inhibited the growth of solid tumors. Distribution studies of paclitaxel after intratumoral injection showed high and effective drug concentrations in tumor.