Pharmacodynamics of mefloquine and praziquantel combination therapy in mice harbouring juvenile and adult Schistosoma mansoni

Praziquantel (PZQ) is currently the only drug widely used for the treatment of schistosomiasis, but the antimalarial drug mefloquine (Mef) possesses interesting antischistosomal properties. Combination therapy with these two drugs has been suggested as a strategy for transmission control, as PZQ is active against adult worms and Mef is active against schistosomula. To examine the efficacy of combination therapy, Schistosoma mansoni-reinfected mice were separated into seven groups: untreated (I), treated with PZQ in doses of 200 mg/kg (II) or 1,000 mg/kg (III), treated with Mef in doses of 200 mg/kg (IV) or 400 mg/kg (V); each dose was divided equally and given on two consecutive days. Group VI was treated with doses of PZQ + Mef as in groups II and IV, respectively, while group VII was treated with PZQ + Mef as in groups III and V, respectively. PZQ + Mef at the reduced doses of 200 mg/kg each enhanced the therapeutic efficacy over the reduced PZQ dose alone as shown by a very high reduction in the total numbers of mature worms (95% vs. 49%), immature worms (96% vs. 29%) and the complete eradication of immature females, mature females and immature eggs. The reduction in worm burden was associated with the healing of hepatic granulomatous lesions and the normalisation of all liver enzymes. Therefore, the use of Mef with PZQ is more effective than PZQ alone and should be considered for clinical trials in humans as a potential treatment regimen to prevent treatment failures in areas with high rates of schistosomiasis.     

Effect of Ketoconazole,a Cytochrome P450 Inhibitor,on the Efficacy of Quinine and Halofantrine against Schistosoma mansoni in Mice

The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. The antimalarials quinine (QN) and halofantrine (HF) possess moderate antischistosomal properties. The major metabolic pathway of QN and HF is through cytochrome P450 (CYP) 3A4. Accordingly, this study investigates the effects of CYP3A4 inhibitor, ketoconazole (KTZ), on the antischistosomal potential of these quinolines against Schistosoma mansoni infection by evaluating parasitological, histopathological, and biochemical parameters. Mice were classified into 7 groups: uninfected untreated (I), infected untreated (II), infected treated orally with PZQ (1,000 mg/kg) (III), QN (400 mg/kg) (IV), KTZ (10 mg/kg)+QN as group IV (V), HF (400 mg/kg) (VI), and KTZ (as group V)+HF (as group VI) (VII). KTZ plus QN or HF produced more inhibition (P<0.05) in hepatic CYP450 (85.7% and 83.8%) and CYT b5 (75.5% and 73.5%) activities, respectively, than in groups treated with QN or HF alone. This was accompanied with more reduction in female (89.0% and 79.3%), total worms (81.4% and 70.3%), and eggs burden (hepatic; 83.8%, 66.0% and intestinal; 68%, 64.5%), respectively, and encountering the granulomatous reaction to parasite eggs trapped in the liver. QN and HF significantly (P<0.05) elevated malondialdehyde levels when used alone or with KTZ. Meanwhile, KTZ plus QN or HF restored serum levels of ALT, albumin, and reduced hepatic glutathione (KTZ+HF) to their control values. KTZ enhanced the therapeutic antischistosomal potential of QN and HF over each drug alone. Moreover, the effect of KTZ+QN was more evident than KTZ+HF.     

Miltefosine, a promising novel agent for schistosomiasis mansoni

This research aims towards developing an alternative antischistosomal drug using miltefosine, which is primarily used in the treatment of leishmaniasis. The treatment and control of schistosomiasis, a notable neglected tropical disease (NTD), rely on a single drug, praziquantel (PZQ). The dependency on PZQ exclusively is quite alarming, given the spread of the disease (over 200 million people infected and close to 800 million people at risk in three continents) and the threat of drug resistance. This study shows that the oral administration of miltefosine in a daily dose of 20 mg/kg for five successive days to mice infected with either invasive, juvenile or adult stages of Schistosoma mansoni resulted in significant reduction of worm burden, hepatic granulomata size and amelioration of hepatic pathology. Scanning Electron Microscopy revealed that miltefosine induced severe tegumental damage in adult schistosomes. In conclusion, we believe this is the first study highlighting miltefosine as a promising novel agent for schistosomiasis mansoni.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.     

Activity Profile of an FDA-Approved Compound Library against Schistosoma mansoni

Background

As plans to expand mass drug treatment campaigns to fight schistosomiasis form, worries about reliance on praziquantel as the sole available treatment motivate the investigation for novel antischistosomal compounds. Drug repurposing might be an inexpensive and effective source of novel antischistosomal leads.

Methodology

1600 FDA approved compounds were first assayed against Schistosoma mansoni schistosomula at a concentration of 10 µM. Active compounds identified from this screen were advanced to the adult worm screen at 33.33 µM, followed by hit characterization. Leads with complementary pharmacokinetic and toxicity profiles were then selected for in vivo studies.

Principal Findings

The in vitro screen identified 121 and 36 compounds active against the schistosomula and adult stage, respectively. Further, in vitro characterization and comparison with already available pharmacokinetic and toxicity data identified 11 in vivo candidates. Doramectin (10 mg/kg) and clofazimine (400 mg/kg) were found to be active in vivo with worm burden reductions of 60.1% and 82.7%, respectively.

Conclusions/Significance

The work presented here expands the knowledge of antischistosomal properties of already approved compounds and underscores variations observed between target-based and phenotypic approaches and among laboratories. The two in vivo-active drugs identified in this study, doramectin and clofazimine are widely available and present as novel drug classes as starting points for further investigation.     

Blocking of human T lymphocyte activation by channel antagonists

It has been established that early events in lymphocyte activation involve a rise in intracellular Ca++ as well as changes in the flux of other ions. Although a Ca++ channel has been postulated to participate in the early Ca++ rise, its presence in lymphocytes remains controversial. Also although yet undetected, electrophysiological data suggest the presence of a Ca++ activated K+ channel on human peripheral blood lymphocytes (HPBL). Here we report on the effect of specific channel blockers as an approach to the identification of these channels on HPBL. At 40 nM nifedipine, an inhibitor of voltage-gated Ca++ channels, fully inhibits the PHA-promoted activation of HPBL. This effect is concentration dependent with a half maximum effect at approximately 10 nM and is demonstrable whether the drug is added at the same time as or up to 18 h after the addition of the mitogen. This inhibition of activation is not seen if the lymphocytes are activated using IL-2 instead of PHA. Charybdotoxin a toxin which blocks a Ca++ activated K+ channel of muscle cells also blocks to almost 100 per cent the PHA-induced activation of HPBL. This inhibition can be demonstrated regardless of whether the blocker is added together with or up to 4 h after PHA. As opposed to nifedipine charybdotoxin shows no effect if added 18 h after the initiation of the activation process. When nifedipine and charybdotoxin were tested on mice splenocytes we found that nifedipine fully inhibits the LPS-promoted activation of these cells while charybdotoxin has no effect on their activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-gated calcium channel subunits from platyhelminths: potential role in praziquantel action

Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+ -dependent responses in muscles, nerves and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming alpha(1) subunit and other modulatory subunits, including the beta subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel beta subunits (Ca(v)betavar) found in these parasites. In particular, we examine the role these beta subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Ca(v)betavar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant beta subunits may thus represent a platyhelminth-specific gene family.     

Schistosome calcium channel beta subunits. Unusual modulatory effects and potential role in the action of the antischistosomal drug praziquantel

Schistosomes are parasitic flatworms that cause schistosomiasis, a major tropical disease. The current drug of choice against schistosomiasis is praziquantel (PZQ), which has minimal side effects and is potent against all schistosome species. The mode of action of PZQ is unknown, though the drug clearly affects Ca(2+) homeostasis in worms, and there is indirect evidence for interaction of PZQ with schistosome voltage-gated Ca(2+) channels. We have cloned and expressed two Ca(2+) channel beta subunits, one from Schistosoma mansoni and one from Schistosoma japonicum. These two subunits (SmCa(v)beta A and SjCa(v)beta) have structural motifs that differ from those found in other known beta subunits. Surprisingly, coexpression of either SmCa(v)beta A or SjCa(v)beta with a cnidarian (CyCa(v)1) or mammalian (Ca(v)2.3) Ca(2+) channel alpha(1) subunit results in a striking reduction in current amplitude. In the case of Ca(v)2.3, this current reduction can be partially reversed by addition of 100 nm PZQ, which results in a significant increase in current amplitude. Thus, these unusual schistosome beta subunits can confer PZQ sensitivity to an otherwise PZQ-insensitive mammalian Ca(2+) channel, indicating that a possible target for PZQ action is the interaction between beta subunits and pore-forming alpha(1) subunits in schistosomes.     

Development and Validation of a Luminescence-based,Medium-Throughput Assay for Drug Screening in Schistosoma mansoni

Background

Schistosomiasis, one of the world’s greatest neglected tropical diseases, is responsible for over 280,000 human deaths per annum. Praziquantel, developed in the 1970s, has high efficacy, excellent tolerability, few and transient side effects, simple administration procedures and competitive cost and it is currently the only recommended drug for treatment of human schistosomiasis. The use of a single drug to treat a population of over 200 million infected people appears particularly alarming when considering the threat of drug resistance. Quantitative, objective and validated methods for the screening of compound collections are needed for the discovery of novel anti-schistosomal drugs.

Methodology/Principal Findings

The present work describes the development and validation of a luminescence-based, medium-throughput assay for the detection of schistosomula viability through quantitation of ATP, a good indicator of metabolically active cells in culture. This validated method is demonstrated to be fast, highly reliable, sensitive and automation-friendly. The optimized assay was used for the screening of a small compound library on S. mansoni schistosomula, showing that the proposed method is suitable for a medium-throughput semi-automated screening. Interestingly, the pilot screening identified hits previously reported to have some anti-parasitic activity, further supporting the validity of this assay for anthelminthic drug discovery.

Conclusions

The developed and validated schistosomula viability luminescence-based assay was shown to be successful and suitable for the identification of novel compounds potentially exploitable in future schistosomiasis therapies.     

A Novel Biological Activity of Praziquantel Requiring Voltage-Operated Ca2+ Channel β Subunits: Subversion of Flatworm Regenerative Polarity

Background

Approximately 200 million people worldwide harbour parasitic flatworm infections that cause schistosomiasis. A single drug—praziquantel (PZQ)—has served as the mainstay pharmacotherapy for schistosome infections since the 1980s. However, the relevant in vivo target(s) of praziquantel remain undefined.

Methods and Findings

Here, we provide fresh perspective on the molecular basis of praziquantel efficacy in vivo consequent to the discovery of a remarkable action of PZQ on regeneration in a species of free-living flatworm (Dugesia japonica). Specifically, PZQ caused a robust (100% penetrance) and complete duplication of the entire anterior-posterior axis during flatworm regeneration to yield two-headed organisms with duplicated, integrated central nervous and organ systems. Exploiting this phenotype as a readout for proteins impacting praziquantel efficacy, we demonstrate that PZQ-evoked bipolarity was selectively ablated by in vivo RNAi of voltage-operated calcium channel (VOCC) β subunits, but not by knockdown of a VOCC α subunit. At higher doses of PZQ, knockdown of VOCC β subunits also conferred resistance to PZQ in lethality assays.

Conclusions

This study identifies a new biological activity of the antischistosomal drug praziquantel on regenerative polarity in a species of free-living flatworm. Ablation of the bipolar regenerative phenotype evoked by PZQ via in vivo RNAi of VOCC β subunits provides the first genetic evidence implicating a molecular target crucial for in vivo PZQ activity and supports the ‘VOCC hypothesis’ of PZQ efficacy. Further, in terms of regenerative biology and Ca²⁺ signaling, these data highlight a novel role for voltage-operated Ca²⁺ entry in regulating in vivo stem cell differentiation and regenerative patterning.     

Reduction of K+-Stimulated 45Ca2+ Influx in Synaptosomes with Age Involves Inactivating and Noninactivating Calcium Channels and Is Correlated with Temporal Modifications in Protein Dephosphorylation

The voltage-dependent calcium uptake in rat brain synaptosomes was measured under conditions in which [Ca2+]o/[Na+]i exchange was minimized to characterize the voltage-sensitive calcium channels from rats of different ages. In solutions of CaCl2 concentrations of less than 500 microM, the initial (5-s) calcium uptake declined by approximately 20-50% in 12- and 24-month-old rats relative to 3-month-old adults. Depolarization of synaptosomes from 3-month-old rats in a calcium-free medium or in the presence of 0.5 mM CaCl2 led to an exponential decline of the calcium uptake rate after 20 s (voltage- or voltage-and-calcium-dependent inactivation) to approximately 66 and 34% of the initial value with a t1/2 of 1.6 or 0.7 s, respectively. The presence of 1 microM nifedipine resulted in a 15-25% reduction of 45Ca2+ uptake rates, which appeared to affect noninactivating calcium channels, but addition of the calcium channel agonist Bay K 8644 was without effect. In 24-month-old rats, inactivation of 45Ca2+ uptake in calcium-free media was nondetectable, and in the presence of 0.5 mM CaCl2, the rate and extent of inactivation were also much lower than in 3-month-old animals (the t1/2 was 0.9 s, and the calcium uptake rate at 20 s was 55% of its initial value). Moreover, the presence of 1 microM nifedipine was without effect on initial calcium uptake or inactivation in synaptosomes from 24-month-old rats. These results indicate that the decrease in calcium channel-mediated 45Ca2+ uptake involves an inhibition or block of both dihydropyridine-resistant and -sensitive calcium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-type calcium channels are preferentially coupled to endocytosis in bovine chromaffin cells

Exocytosis and endocytosis are Ca(2+)-dependent processes. The contribution of high-voltage activated Ca(2+) channels subtypes to exocytosis has been thoroughly studied in chromaffin cells. However, similar reports concerning endocytosis are unavailable. Thus, we studied here the effects of blockers of L (nifedipine), N (omega-conotoxin GVIA) and P/Q (omega-agatoxin IVA) Ca(2+) channel on Ca(2+) currents (I(Ca)), Ca(2+) entry (Q(Ca)), as well as on the changes in membrane capacitance (C(m)) in perforated-patch voltage-clamped bovine adrenal chromaffin cells. Using 500-ms pulses to 0 or +10 mV, given from a holding potential of -80 mV and 2 mM Ca(2+) we found that omega-conotoxin GVIA affected little the exo-endocytotic responses while omega-agatoxin IVA markedly blocked those responses. However, nifedipine blocked little exocytosis but almost completely inhibited endocytosis. We conclude that L-type Ca(2+) channels seem to be selectively coupled to endocytosis.     

Ca2 + channels and praziquantel: A view from the free world

Targeting the cellular Ca^2 + channels and pumps that underpin parasite Ca^2 + homeostasis may realize novel antihelmintic agents. Indeed, the antischistosomal drug praziquantel (PZQ) is a key clinical agent that has been proposed to work in this manner. Heterologous expression data has implicated an action of PZQ on voltage-operated Ca^2 + channels, although the relevant in vivo target of this drug has remained undefined over three decades of clinical use. The purpose of this review is to bring new perspective to this issue by discussing the potential utility of free-living planarian flatworms for providing new insight into the mechanism of PZQ action. First, we discuss in vivo functional genetic data from the planarian system that broadly supports the molecular data collected in heterologous systems and the ‘Ca^2 + hypothesis’ of PZQ action. On the basis of these similarities we highlight our current knowledge of platyhelminth voltage operated Ca^2 + channels, their unique molecular pharmacology and the downstream functional PZQ interactome engaged by dysregulation of Ca^2 + influx that has potential to yield novel antischistosomal targets. Overall the broad dataset underscores a common theme of PZQ-evoked disruptions of Ca^2 + homeostasis in trematodes, cestodes and turbellarians, and showcases the utility of the planarian model for deriving insight into drug action and targets in parasitic flatworms.     

Inhibition or Knockdown of ABC Transporters Enhances Susceptibility of Adult and Juvenile Schistosomes to Praziquantel

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ). Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1) and other ATP binding cassette (ABC) transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR). Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3–4 weeks post infection), normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R)-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R)-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that parasite ABC multidrug transporters might serve as important targets for enhancing the action of PZQ. They also suggest a potentially novel and readily-available strategy for overcoming reduced PZQ susceptibility of schistosomes.     

Identification of hit compounds with anti-schistosomal activity on in vitro generated juvenile worms in cell-free medium

BackgroundAnthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay.Methodology/Principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3–6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug.ConclusionWith this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug).     

Electrophysiological characterization of a schistosome transient receptor potential channel activated by praziquantel

Ion channels have proved to be productive targets for anthelmintic chemotherapy. One example is the recent discovery of a parasitic flatworm ion channel targeted by praziquantel (PZQ), the main clinical therapy used for treatment of schistosomiasis. The ion channel activated by PZQ – a transient receptor potential ion channel of the melastatin subfamily, named TRPM_PZQ – is a Ca²⁺-permeable ion channel expressed in all parasitic flatworms that are PZQ-sensitive. However, little is currently known about the electrophysiological properties of this target that mediates the deleterious action of PZQ on many trematodes and cestodes. Here, we provide a detailed biophysical characterization of the properties of Schistosoma mansoni TRPM_PZQ channel (Sm.TRPM_PZQ) in response to PZQ. Single channel electrophysiological analysis demonstrated that Sm.TRPM_PZQ when activated by PZQ is a non-selective, large conductance, voltage-insensitive cation channel that displays distinct properties from human TRPM paralogs. Sm.TRPM_PZQ is Ca²⁺-permeable but does not require Ca²⁺ for channel gating in response to PZQ. TRPM_PZQ from Schistosoma japonicum (Sj.TRPM_PZQ) and Schistosoma haematobium (Sh.TRPM_PZQ) displayed similar characteristics. Profiling Sm.TRPM_PZQ responsiveness to PZQ has established a biophysical signature for this channel that will aid future investigation of endogenous TRPM_PZQ activity, as well as analyses of endogenous and exogenous regulators of this novel, druggable antiparasitic target.     

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

BackgroundSchistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.Conclusions/SignificanceOur data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.     

Calcium, calcium channels, and calcium channel antagonists

Voltage-dependent Ca2+ channels are an important pathway for Ca2+ influx in excitable cells. They also represent an important site of action for a therapeutic group of agents, the Ca2+ channel antagonists. These drugs enjoy considerable use in the cardiovascular area including angina, some arrhythmias, hypertension, and peripheral vascular disorders. The voltage-dependent Ca2+ channels exist in a number of subclasses characterized by electrophysiologic, permeation, and pharmacologic criteria. The Ca2+ channel antagonists, including verapamil, nifedipine, and diltiazem, serve to characterize the L channel class. This channel class has been characterized as a pharmacologic receptor, since it possesses specific drug-binding sites for both antagonists and activators and it is regulated by homologous and heterologous influences. The Ca2+ channels of both voltage- and ligand-regulated classes are likely to continue to be major research targets for new drug design and action.     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

Miltefosine Lipid Nanocapsules for Single Dose Oral Treatment of Schistosomiasis Mansoni: A Preclinical Study

Miltefosine (MFS) is an alkylphosphocholine used for the local treatment of cutaneous metastases of breast cancer and oral therapy of visceral leishmaniasis. Recently, the drug was reported in in vitro and preclinical studies to exert significant activity against different developmental stages of schistosomiasis mansoni, a widespread chronic neglected tropical disease (NTD). This justified MFS repurposing as a potential antischistosomal drug. However, five consecutive daily 20 mg/kg doses were needed for the treatment of schistosomiasis mansoni in mice. The present study aims at enhancing MFS efficacy to allow for a single 20mg/kg oral dose therapy using a nanotechnological approach based on lipid nanocapsules (LNCs) as oral nanovectors. MFS was incorporated in LNCs both as membrane-active structural alkylphospholipid component and active antischistosomal agent. MFS-LNC formulations showed high entrapment efficiency (EE%), good colloidal properties, sustained release pattern and physical stability. Further, LNCs generally decreased MFS-induced erythrocyte hemolytic activity used as surrogate indicator of membrane activity. While MFS-free LNCs exerted no antischistosomal effect, statistically significant enhancement was observed with all MFS-LNC formulations. A maximum effect was achieved with MFS-LNCs incorporating CTAB as positive charge imparting agent or oleic acid as membrane permeabilizer. Reduction of worm load, ameliorated liver pathology and extensive damage of the worm tegument provided evidence for formulation-related efficacy enhancement. Non-compartmental analysis of pharmacokinetic data obtained in rats indicated independence of antischistosomal activity on systemic drug exposure, suggesting possible gut uptake of the stable LNCs and targeting of the fluke tegument which was verified by SEM. The study findings put forward MFS-LNCs as unique oral nanovectors combining the bioactivity of MFS and biopharmaceutical advantages of LNCs, allowing targeting via the oral route. From a clinical point of view, data suggest MFS-LNCs as a potential single dose oral nanomedicine for enhanced therapy of schistosomiasis mansoni and possibly other diseases.