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Milbemycin oximes are macrocyclic lactones that have a broad spectrum of activity against nematode infection in animals. They are known to block drug efflux, which increases the susceptibility of fungi to azoles. We investigated the effects of milbemycin on the azole susceptibility of fungi (Aspergillus fumigatus, Candida albicans, C. auris, Cryptococcus neoformans , and Trichophyton rubrum). To screen for changes in azole susceptibility, fungal growth was tested on a culture medium containing 1 μg/ml milbemycin. The results showed that milbemycin increased the azole susceptibility of azole-resistant strains of C. albicans, C. auris, C. neoformans , and T. rubrum. Thus, milbemycin might be useful against antifungal drug-resistant strains. [ABSTRACT FROM AUTHOR]

A possible synergistic effect of macrocyclic lactones' (MLs) combination has been previously described against resistant gastrointestinal nematodes of cattle. In addition to synergism, drug-drug interactions between MLs can also result in additive or antagonistic effect, considering the different MLs pharmacokinetics, pharmacodynamics, and interactions with molecular mechanisms of resistance. Therefore, the aim of the current work was evaluated the effect of different MLs combinations against Haemonchus contortus. Infecting larvae of two isolates (one susceptible and one resistant to ivermectin) were used in the larval migration inhibition test. After estimating the half maximal effective concentration of abamectin (ABA), eprinomectin, (EPR), ivermectin (IVM), and moxidectin (MOX) for both isolates, combinations were delineated by a simplex-centroid mixture experiment, and the mixture regression analysis was applied to the special cubic model. A synergistic effect was found for the EPR + MOX against the susceptible isolate as well as the EPR + MOX, IVM + MOX, and ABA + EPR + IVM against the resistant isolate. An antagonistic effect of ABA + IVM + MOX was found against the susceptible isolate. For the susceptible isolate, a higher inhibition was found with greater proportions of EPR and lower proportions of the other drugs compared to the reference mixture. For the resistant isolate, inhibition greater than that of the reference mixture was found with higher proportions of IVM as well as lower proportions of the other drugs. The synergistic and antagonistic effects were dependent on the following: (a) parasite drug resistance profile, (b) the composition of the combination, and (c) the proportions used, with EPR and IVM exerting a greater impact on these effects. [ABSTRACT FROM AUTHOR]

Energy-coupling factor transporters (ECFTs) are membrane-bound ATP-binding cassette (ABC) transporters in prokaryotes that are found in pathogens against which novel antibiotics are urgently needed. To date, just 54 inhibitors of three molecular-structural classes with mostly weak inhibitory activity are known. Target repurposing is a strategy that transfers knowledge gained from a well-studied protein family to under-studied targets of phylogenetic relation. Forty-eight human ABC transporters are known that may harbor structural motifs similar to ECFTs to which particularly multitarget compounds may bind. We assessed 31 multitarget compounds which together target the entire druggable human ABC transporter proteome against ECFTs, of which nine showed inhibitory activity (hit rate 29.0%) and four demonstrated moderate to strong inhibition of an ECFT (IC 50 values between 4.28 and 50.2 µM) as well as antibacterial activity against ECFT-expressing Streptococcus pneumoniae. Here, ivermectin was the most potent candidate (MIC 95 : 22.8 µM), and analysis of five ivermectin derivatives revealed moxidectin as one of the most potent ECFT-targeting antibacterial agents (IC 50 : 2.23 µM; MIC 95 : 2.91 µM). Distinct molecular-structural features of avermectins and derivatives as well as the differential biological response of the hit compounds in general provided first indications with respect to the structure-activity relationships and mode of action, respectively., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Milbemycin, a group of 16-membered macrolide antibiotics produced by Streptomyces milbemycinicus, has been widely used as an insecticide and an anthelmintic. To enhance the production of milbemycin, ribosomal engineering with heterologous expression of a regulatory gene was used to screen for a high milbemycin-producing mutant. The mutant S. milbemycinicus R2-6-5 isolated after the treatment of S. milbemycinicus A2079 with 6 μg/mL streptomycin produced 172.5 and 163.1% of milbemycins A3 and A4, respectively, compared with original strain. Analysis of the gene rsmG revealed a frameshift mutation, one cytidine unit being inserted into the 21 position (21C → 21CC). The heterologous regulatory gene aveR, which belongs to the LAL-family was integrated into the genome of S. milbemycinicus R2-6-5 denoted S. milbemycinicus J37 to enhance production of milbemycin. The production of milbemycins A3 and A4 in S. milbemycinicus J37 reached 758.9 and 279.0 μg/g respectively, representing 142 and 61% higher yields over S. milbemycinicus R2-6-5. The combination of ribosomal engineering and heterologous regulatory gene expression in S. milbemycinicus J37 resulted in an increase by 12.4-fold for milbemycin A3 and 11.7-fold for milbemycin A4, respectively, when compared to the original strain. Overall, these results demonstrate that combining available technologies for strain modification such as ribosome engineering technology and heterologous regulatory gene expression is an effective approach for development of high milbemycin-producing strains. [ABSTRACT FROM AUTHOR]

Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 μM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 μM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 μM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.

Seaweeds have increasing importance because of their bioactive compounds and their potential application in different industries. Liquid seaweed extract are commercially used as an agricultural biofertilizer. The current work investigated the potential of a commercial liquid seaweed extract, named TrueAlgaeMax (TAM), as a diet feed additive for Nile tilapia Oreochromis niloticus. Five diets (D0, D0.5, D1, D1.5, and D2) were supplemented with different TAM concentrations (0, 0.5, 1, 1.5, and 2%, respectively) to investigate the growth performance, feed utilization, and non-specific immunity of O. niloticus challenged with Aeromonas hydrophila. Healthy mono-sex O. niloticus fingerlings were randomly stocked for 70 days in net enclosures (3 m × 6 m × 1 m) at a density of 20 fish m−3 with an average initial body weight of 50 g fingerling−1 (three groups for each treatment with a total of 1080 fish per treatment). Diet D2 represented the highest weight gain (WG) value (161.29 g), average daily weight gain (ADWG) value (2.30 g day−1), and specific growth rate (SGR) value (2.40% day−1) and achieved the best feed conversion ratio (FCR) value (1.31). In addition, D2 achieved the highest serum protein (5.47 g dL−1), lysozyme (3.75 μg mL−1), and respiratory burst activity (1.89 mg mL−1). Fish mortality decreased by increasing TAM levels. The GC-Mass data of TAM showed nine main phytochemical compounds belonging to seven different important bioactive groups reported as immunity enhancer and growth promoting and showed antioxidant and antimicrobial properties. The results recommended that increasing the levels of TAM up to 2% as feed additive for O. niloticus diet strongly enhances growth performance, feed utilization, and non-specific immunity of Nile tilapia O. niloticus challenged with A. hydrophila. Considering seaweed additions (dry weight or extract) as aquaculture feed additive is of importance. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate the effects of heat on macrocyclic lactone residues in cows' milk. Ivermectin, abamectin, doramectin, eprinomectin and moxidectin were added to raw milk in three concentrations. The milk was then pasteurised (40 seconds at 74°C or 1 minute at 80°C) and boiled (10 minutes at 100°C). The analyses were performed with a validated method: LC-MS/MS. Thermal treatment resulted in a statistically significant decrease in the abamectin, eprinomectin, and moxidectin concentrations in the milk; however, the residues did not completely degrade. Boiling resulted in a greater decrease in the moxidectin concentrations than was observed with pasteurisation. The high pasteurisation and boiling processes had a greater effect on the eprinomectin residues than did the low pasteurisation process. The pasteurisation and boiling processes did not have an effect on the doramectin and ivermectin. The study concluded that the macrocyclic lactones are generally resistant to such processes. [ABSTRACT FROM AUTHOR]

Macrocyclic lactone (ML) anthelmintics are the most important class of anthelmintics because of our high dependence on them for the control of nematode parasites and some ectoparasites in livestock, companion animals and in humans. However, resistance to MLs is of increasing concern. Resistance is commonplace throughout the world in nematode parasites of small ruminants and is of increasing concern in horses, cattle, dogs and other animals. It is suspected in Onchocerca volvulus in humans. In most animals, resistance first arose to the avermectins, such as ivermectin (IVM), and subsequently to moxidectin (MOX). Usually when parasite populations are ML-resistant, MOX is more effective than avermectins. MOX may have higher intrinsic potency against some parasites, especially filarial nematodes, than the avermectins. However, it clearly has a significantly different pharmacokinetic profile. It is highly distributed to lipid tissues, less likely to be removed by ABC efflux transporters, is poorly metabolized and has a long half-life. This results in effective concentrations persisting for longer in target hosts. It also has a high safety index. Limited data suggest that anthelmintic resistance may be overcome, at least temporarily, if a high concentration can be maintained at the site of the parasites for a prolonged period of time. Because of the properties of MOX, there are reasonable prospects that strains of parasites that are resistant to avermectins at currently recommended doses will be controlled by MOX if it can be administered at sufficiently high doses and in formulations that enhance its persistence in the host. This review examines the properties of MOX that support this contention and compares them with the properties of other MLs. The case for using MOX to better control ML-resistant parasites is summarised and some outstanding research questions are presented. Image 1 • Moxidectin (MOX) exhibits a long half-life and high potency. • MOX shows low toxicity in the host and low ecotoxicity. • MOX has properties suitable for high dose rate, long acting formulation. • MOX can be effective against macrocyclic lactone (ML)-resistant parasites. • Properties of MOX can be exploited to better control ML-resistant nematodes. [ABSTRACT FROM AUTHOR]

Fish lice, ectoparasites of the genus Argulus, are branchurian crustaceans that can significantly impact fish health by causing mechanical damage to cutaneous barriers and increasing susceptibility to other infections. While many treatments have been reported in teleosts and invertebrates, there are no published treatments for elasmobranchs. This study evaluated the safety and efficacy of a commercial formulation of milbemycin oxime and lufenuron in freshwater stingrays for treatment of Argulus spp. Seven juvenile Magdalena river stingrays (Potamotrygon magdalenae) and 10 juvenile smooth back river stingrays (Potamotrygon orbignyi) had severe infestations of Argulus spp. that were identified visually and microscopically. Animals were treated with milbemycin oxime and lufenuron (at 0.015 mg/L and 0.30 mg/L, respectively) in a 6-hr immersion once weekly for two treatments. They were visually examined for skin lesions as well as behavior and appetite daily by animal care staff. A subset of animals was euthanized and necropsied on days 8, 9, 43, and 78 after treatment initiation. There were no Argulus spp. detected at the time of the second treatment. Complete gross and histologic evaluations were completed for all animals. At all time points, no gross abnormalities were detected with the exception of thin body condition in some animals; no Argulus spp. were noted. Histologic lesions were all attributed to poor nutritional state at the time of acquisition. No histologic evidence of acute or chronic toxicosis was detected. The commercial formulation of milbemycin oxime and lufenuron, applied at the dose and for the exposure time used in this study, effectively eradicated Argulus spp. in a population of juvenile P. magdalenae and P. orbignyi, and did not cause mortality or clinical gross or histologic evidence of acute or chronic toxicity. [ABSTRACT FROM AUTHOR]

Highlights • Five macrocyclic lactones were studied in cheese that is a very complex food matrix. • An analytical method was developed and validated, following international guidelines. • Excellent figures of merit were obtained with very low limits of quantification. • Twenty-one of the twenty-two cheeses studied contained macrocyclic lactones. • This is the first simultaneous evaluation of five macrocyclic lactones in cheeses. Abstract A highly sensitive analytical method was developed and validated, following international guidelines, for the determination of the residues of five macrocyclic lactones (MLs) (abamectin, doramectin, eprinomectin, ivermectin and moxidectin) in cheese. The extracts were concentrated by rotary-evaporation and derivatized; no clean-up was necessary. Despite matrix complexity, no significant matrix-effect was verified, and standards were prepared in solvents. Linear working ranges varied from 0.25 to 5.0 μg L−1. Excellent limits of quantification (0.58–0.87 μg kg−1), mean recoveries (91–103%), and repeatability and intermediate precision (<5.8%) were obtained. Twenty-two samples of bovine and non-bovine cheeses were analyzed. Twenty-one samples showed residues of at least one ML (between 0.59 and 15.3 μg kg−1), but moxidectin was never detected; a sample of mozzarella was free of MLs. To the best of our knowledge, this is the first method describing the simultaneous evaluation of these MLs in cheese using HPLC and fluorescence detection. [ABSTRACT FROM AUTHOR]

γ-BHC and dieldrin are legacy insecticides that were extensively used after the second World War. When they were banned, their modes of action and metabolism were not known. This article aims at providing a picture of the metabolism of γ-BHC and the modes of action of γ-BHC and dieldrin. γ-BHC is metabolized via two independent metabolic pathways. One is a glutathione conjugation pathway resulting in the formation of dichlorophenyl mercapturic acid and the other is an oxidative metabolism catalyzed by microsomes to mainly 2,4,6-trichlorophenol (TCP) and (36/45)-1,2,3,4,5,6-hexachlorocyclohex-1-ene (HCCHE). Other metabolites of this pathway are 2,4,5-TCP, 2,3,4,6-tetrachlorophenol (TeCP), (36/45)- and (346/5)-1,3,4,5,6-pentachlorocyclohex-1-enes (PCCHE). Nowadays, γ-BHC and dieldrin are very important reagents which are used to study the GABA receptor in insects and mammals. They were found to be noncompetitive GABA antagonists blocking the chloride ion selective pores in the GABA-gated chloride channels and leading to inhibition of chloride ion conductance. [3H]EBOB binding data showed that γ-BHC, its analogs, dieldrin, and other cyclodiene insecticides interact with the same site on GABA receptor as picrotoxinin. Only γ-BHC, among other BHC isomers, exhibits this binding characteristic. Milbemycin, currently widely used as an insecticide, acaricide and nematicide, has been found to open the GABA-gated chloride channel. © Pesticide Science Society of Japan. [ABSTRACT FROM AUTHOR]

Macrocyclic lactones (MLs) are chemical compounds administered to livestock for parasite control. These compounds are poorly metabolized and pass relatively unchanged into the dung of treated animals. When coprophagous insects such as dung beetles (Coleoptera: Scarabaeoidea) are exposed to ML residues while feeding on dung, lethal and sublethal effects are often observed. This can lead to ML residues impairing ecosystem functions that underpin agricultural production. A strategy to reduce these negative effects involves the use of compounds that offer lower risk to non‐target invertebrates, such as the ML moxidectin. Considering two dung beetle species with differing sensitivities to agricultural intensification, we asked whether exposure to moxidectin residues influenced survival, reproductive output, and functioning (short‐ and long‐term estimates of dung removal). When exposed to moxidectin, adults of the sensitive species – Geotrupes spiniger Marsham (Scarabaeidae) – experienced a 43% reduction in survival. In contrast, survival of the non‐sensitive species – Aphodius rufipes L. (Scarabaeidae) – was unaffected. We were unable to determine whether exposure affected reproductive output of either species. We found little evidence to suggest moxidectin impaired dung removal. However, high densities of a species with relatively low individual functional importance (A. rufipes) can compensate for the loss of a functionally dominant species (G. spiniger). Over a longer time frame, earthworms decomposed dung fully, irrespective of moxidectin residues. This functional redundancy reinforces that wider justification for the conservation of biodiversity should remain integrated into agricultural policy and practice. Dung from cattle treated with moxidectin causes high mortality in the dung beetle Geotrupes spiniger, but does not affect the survival of Aphodius rufipes (both Coleoptera: Scarabaeidae). We demonstrate a high density of a small‐bodied dwelling dung beetle can have the same functional efficiency as a large‐bodied tunneling dung beetle. Over longer periods, earthworms may compensate for impaired dung removal caused by exposing coprophagous insects to anthelmintic residues. [ABSTRACT FROM AUTHOR]

Macrocyclic lactones (MLs), specifically the avermectins and milbemycins, are known for their effectiveness against a broad spectrum of disease-causing nematodes and arthropods in humans and animals. In most nematodes, drugs in this class induce paralysis, resulting in starvation, impaired ability to remain associated with their anatomical environment and death of all life stages. Initially, this was also thought to be the ML mode of action against filarial nematodes, but researchers have not been able to validate these characteristic effects of immobilization/starvation of MLs in vitro, even at higher doses than are possible in vivo. Relatively recently, ML receptor sites exclusively located proximate to the excretory-secretory (ES) apparatus were identified in Brugia malayi microfilaria and an ML-induced suppression of secretory protein release by B. malayi microfilariae was demonstrated in vitro. It is hypothesized here that suppression of these ES proteins prevents the filarial worm from interfering with the host's complement cascade, reducing the ability of the parasite to evade the immune system. Live microfilariae and/or larvae, thus exposed, are attacked and presented to the host's innate immune mechanisms and are ultimately killed by the immune response, not the ML drug. These live, exposed filarial worms stimulate development of innate, cellular and humoral immune responses that when properly stimulated are capable of clearing all larvae or microfilariae present in the host, regardless of their individual sensitivity to MLs. Additional research in this area can be expected to improve our understanding of the relationships among filarial worms, MLs and the host immune system, which likely would have implications in filarial disease management in humans and animals. [ABSTRACT FROM AUTHOR]

Avermectins and milbemycins are commonly used in agro-ecosystems for the control of parasites in domestic livestock. As integral members of agro-ecosystems with importance in maintaining pasture health through dung burial behaviour, dung beetles are an excellent nontarget bio-indicator taxon for examining potential detrimental effects of pesticide application. The current review focuses on the relative toxicity of four different anthelmintics (ivermectin, eprinomectin, doramectin and moxidectin) in dung residues using dung beetles as a bioindicator species. One of the implications of this review is that there could be an effect that extends to the entire natural assemblage of insects inhabiting and feeding on the dung of cattle treated with avermectin or milbemycin products. Over time, reduced reproductive rate would result in decreased dung beetle populations and ultimately, a decrease in the rate of dung degradation and dung burial.

Avermectins and milbemycin are widely used as veterinary drugs and as agricultural pesticides, and their residues have been detected in soil. This study reports a simple and high-throughput method for determining ivermectin (IVER), abamectin (ABA), doramectin (DORA), eprinomectin (EPRI), and moxidectin (MOXI) residues in soils, employing an on-line solid-phase extraction technique coupled with ultra-high performance liquid chromatography and tandem mass spectrometry (SPE-UHPLC–MS/MS). The method was validated and applied for the determination of ABA in soils from an orange plantation treated with this pesticide. The sample preparation procedure consisted of extraction of the compounds from soil using methanol (with recoveries of 73–85%), and subsequent on-line SPE cleanup and concentration using a C8 sorbent coupled to the UHPLC–MS/MS system. The optimal conditions were: water:methanol (40:60, v/v) sample solvent; water:methanol (96:4, v/v) loading solvent; 2 × 250 μL sample volume; and elution of the analytes retained on the SPE column in back flush with 5 mmol L −1 ammonium acetate:acetonitrile (10:90, v/v) chromatographic mobile phase. The method produced linear results in the ranges 0.1–10 ng g −1 (IVER, ABA, DORA, and MOXI) and 0.5–10 ng g −1 (EPRI), with linearity greater than 0.99. The precision of the method was better than 19% and accuracy was in the range 74–89%. The limits of quantitation were 0.2 ng g −1 for EPRI and 0.1 ng g −1 for the other compounds. The SPE column could be reused in more than 2000 analyses without loss of efficiency. The ABA concentration in the soil varied between 1.7 and 18 ng g −1 , and no dissipation was observed during five consecutive days after application of the pesticide to the orange plantation. [ABSTRACT FROM AUTHOR]

Background: Milbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes. Results: milR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. β-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR’s function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04. Conclusions: A LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics. [ABSTRACT FROM AUTHOR]

Two single-site, laboratory, negatively controlled, masked, randomised dose confirmation studies were performed: one in dogs, the other in cats. After a period of acclimatisation, both the dogs and cats were orally infected with Echinococcus multilocularis protoscoleces. In the dog study, 10 dogs received a single dose of Milpro® tablets at a minimum dose of 0.5 mg/kg milbemycin oxime and 5 mg/kg praziquantel 18 days post-infection and 10 dogs received no treatment. In the cat study, 10 cats received a single dose of Milpro® tablets at a minimum dose of 2 mg/kg milbemycin oxime and 5 mg/kg praziquantel 7 days post-infection, 10 cats received a single dose of the treatment 18 days post-infection and 10 cats remained untreated. In both studies, intestinal worm counts were performed 23 days post-infection at necropsy. No worms were retrieved from any of the 30 treated animals. Nine of 10 control dogs had multiple worms (geometric mean 91, arithmetic mean 304) and all 10 control cats had multiple worms (geometric mean 216, arithmetic mean 481). The difference in worm counts between all three treated groups and their controls was highly significant (ANOVA p values of log transformed data <0.0001). Efficacy of 100 % was demonstrated for the elimination of adult E. multilocularis in dogs and cats as well as for elimination of immature E. multilocularis in cats as evidenced by the effectiveness of treatment 7 days post-infection. The treatments were well accepted and tolerated, and there were no adverse drug reactions observed. [ABSTRACT FROM AUTHOR]

Azole resistance in Candida glabrata, a pathogenic yeast, has prompted studies of compounds that have therapeutic potential by reversing azole resistance. Milbemycin A4 oxime blocked azole efflux and enhanced azole susceptibility fourfold in 28 clinical isolates of C. glabrata. Specificity of the milbemycin A4 oxime effect depended on the drug transporter and the substrate being effluxed. The major effect of milbemycin A4 oxime was inhibition of azole and rhodamine 6G efflux by the ATP-binding cassette ( ABC) transporters Cg CDR1 and PDH1. Milbemycin A4 oxime effect did not extend to oligomycin, transported by the ABC transporter YOR1 or to benomyl, transported by the major facilitator superfamily transporter, Cg FLR1. Milbemycin A4 oxime did not suppress transcription of Cg CDR1 but increased Cg CDR1 expression 126-fold. Selectivity of the effect is compatible with the concept that milbemycin A4 oxime may interact directly with one or more drug-binding sites of the major azole transporters. [ABSTRACT FROM AUTHOR]

Streptomyces bingchenggensis is a soil bacterium that produces milbemycins, a family of macrolide antibiotics that are commercially important in crop protection and veterinary medicine. In addition, S. bingchenggensis produces many other natural products including the polyether nanchangmycin and novel cyclic pentapeptides. To identify the gene clusters involved in the biosynthesis of these compounds, and better clarify the biochemical pathways of these gene clusters, the whole genome of S. bingchenggensis was sequenced, and the transcriptome profile was subsequently investigated by microarray. In comparison with other sequenced genomes in Streptomyces, S. bingchenggensis has the largest linear chromosome consisting of 11 936 683 base pairs (bp), with an average GC content of 70.8%. The 10 023 predicted protein-coding sequences include at least 47 gene clusters correlated with the biosynthesis of known or predicted secondary metabolites. Transcriptional analysis demonstrated an extremely high expression level of the milbemycin gene cluster during the entire growth period and a moderately high expression level of the nanchangmycin gene cluster during the initial hours that subsequently decreased. However, other gene clusters appear to be silent. The genome-wide analysis of the secondary metabolite gene clusters in S. bingchenggensis, coupled with transcriptional analysis, will facilitate the rational development of high milbemycins-producing strains as well as the discovery of new natural products. [ABSTRACT FROM AUTHOR]

A highly convergent and flexible synthetic approach to stereochemically defined spiroketals is reported. Substituents can be incorporated at various positions around the spiroketal framework without significant disruption to the synthetic scheme. The approach has been exploited to prepare the spiroketal fragment of milbemycin β14. [ABSTRACT FROM AUTHOR]

Abstract: Avermectins and milbemycins (AM) are potent compounds against all major nematode parasites, but their continuous usage has led to the development of widespread resistance in many of the important species of ruminant and equine parasites. The exception to this has been the cyathostomins, where AM resistance was recently first reported only after decades of drug exposure. Data from a Brazilian study suggests that AM resistance has developed in cyathostomins and reports of shortened egg reappearance periods after ivermectin treatment have been published recently from USA and Germany. Thus, AM resistance in cyathostomins is an emerging worldwide concern, but there is only limited amount data on the extent of this problem. To limit the development and spread of AM-resistant cyathostomins the equine industry must implement new strategies for worm control, and the veterinary parasitology community must develop and validate improved protocols for detecting anthelmintic resistance in the field. [Copyright &y& Elsevier]

Abstract: Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [3H]MLM A4 were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P316S mutant channels were 37- and 100-fold less sensitive to MLM A4 when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the β10 strand, the M2–M3 linker, and the M3 region influence MLM A4 binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A4. [Copyright &y& Elsevier]

Abstract: Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy, and the overexpression of P-glycoprotein (P-gp) is one of the major mechanisms of MDR. In this paper, we evaluated the effects of two new milbemycin compounds, milbemycin β 14 and secomilbemycin D, isolated from fermentation broth of S. bingchenggensis on reversing MDR of adriamycin-resistant human breast carcinoma (MCF-7/adr) cells. We observed that the both milbemycins (5μM) showed strong potency to increase adriamycin cytotoxicity toward MCF-7/adr cells with reversal fold (RF) of 13.5 and 10.59, respectively. In addition, the mechanisms of milbemycins on reversing P-gp-mediated MDR demonstrated that they significantly increased the accumulations of adriamycin and Rh123 via inhibiting P-gp efflux in MCF-7/adr cells. Furthermore, the results also revealed that milbemycin β 14 and secomilbemycin D could regulate down the expression of P-gp, but not affect the expression of MDR1 gene. In conclusion, our observations suggest that the two new milbemycin compounds probably represent the promising agents for reversing MDR in cancer therapy. [Copyright &y& Elsevier]

Abstract: The effects of milbemycin A4 (MB A4), milbemycin oxime A4 (MBO A4) and milbemycin β 1 (MB β 1) on reversing multidrug resistance (MDR) of tumor cells were firstly conducted according to the following research, including MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, the accumulation of adriamycin, the accumulation and efflux of rhodamine 123 (Rh123), the regulations of MDR1 gene, and expression of P-gp. The three milbemycins (5μM) showed strong potency to increase adriamycin cytotoxicity toward adriamycin-resistant human breast carcinoma cells MCF-7/adr with reversal fold (RF) of 21.42, 19.06 and 14.89, respectively. In addition, the mechanisms of milbemycins on P-glycoprotein (P-gp)-mediated MDR demonstrated that the milbemycins significantly increased the intracellular accumulations of adriamycin and Rh123 via inhibiting P-gp transport function. Based on the analysis of the P-gp and MDR1 gene expression using flow cytometry and RT-PCR, the results revealed that milbemycin compounds, particularly MB A4, could regulate down the expression of the P-gp and MDR1 gene. These findings suggest that the milbemycins probably represent promising agents for overcoming MDR in cancer therapy, and especially MB A4 is better modulator with the lowest toxicity. [ABSTRACT FROM AUTHOR]