The stereo inversion of 2-arylpropionic acid non-steroidal anti-inflammatory drugs and structurally related compounds by Verticillium lecanii

The fungus Verticillium lecanii has previously been shown to be capable of inverting the chirality of ibuprofen and 2-phenylpropionic acid from the (R)-enantiomer to the corresponding (S)-antipode, a phenomenon also observed in mammalian systems including man. An investigation is reported here into the substrate specificity of the enzyme system present in V. lecanii using the following 2-arylpropionic acids : ibuprofen, ketoprofen, indoprofen, suprofen, flurbiprofen and fenoprofen, together with the structurally related compounds 2-phenylbutyric acid, 2-phenoxypropionic acid, mandelic acid, atrolactic acid, etodolac and α-methoxyphenylpropionic acid. The results demonstrated that V. lecanii is capable of inverting the chirality of all the 2-arylpropionic acids investigated. All were inverted in the (R) to (S) direction with the exception of ketoprofen, where inversion was observed in the reverse direction. Using the structurally related compounds as substrates, the size of the alkyl substituent at the α-carbon at the methyl group, and the presence of the methyl group at the chiral centre, were found to be critical. These results suggest that V. lecanii could be used as a basis for the production of pure enantiomers of the 2-arylpropionic acids in commercial biotransformations.     

Microbial metabolism of 2-arylpropionic acids: effect of environment on the metabolism of ibuprofen by Verticillium lecanii

The effect of environment on the growth of Verticillium lecanii and its metabolic transformation of racemic ibuprofen are reported. The growth of V. lecanii exhibited a lag phase of up to 12 h followed by a period of rapid growth for up to 4 d. The optimal conditions for growth of the micro-organism were determined to be 24°C at pH 7.0 with a culture volume of up to one-tenth of the culture flask volume.
The metabolic oxidation of (R,S)-ibuprofen occurred in both growing cultures and washed cell suspensions of V. lecanii. Examination of the stereochemical composition of the remaining substrate indicated that under both conditions the oxidation was substrate stereoselective for the R-enantiomer of the drug. Using growing cultures of the micro-organism, quantitative conversion of the substrate to the metabolite was achieved following incubation for 14 d. Examination of the enantiomeric composition of the metabolic product indicated an excess of the S-isomer (ratio S/R = 2.1). The possible mechanisms for the apparent anomaly in the stereoselectivity of (R,S)-ibuprofen metabolism and the enantiomeric composition of the metabolite are discussed.     

Improvement of the efficacy of Verticillium lecanii used in biocontrol of Sphaerotheca fuliginea by addition of oil formulations

Biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea, by Verticillium lecanii is seriously hampered at low humidities. The effect is especially marked at low humidity (60% RH) during the three hours following the application of V. lecanii spores suspended in water. Formulations of V. lecanii spores in oil might improve the situation. Arachid oil (peanut oil) and two invert emulsions using either Sunspray 6N or paraffin oil were tested in formulations of V. lecanii spores. Arachid oil gave the best development of V. lecanii on mildewed cucumber leaves. V. lecanii formulated with arachid oil showed significantly better control of mildew than without. A concentration of 0.5% arachid oil was somewhat toxic to mildew but 0.05% was not. Arachid oil did not show toxicity to V. lecanii. The humidity requirements of V. lecanii formulated with and without 0.05% arachid oil were compared at 95, 90 and 85% RH. Arachid oil significantly reduced the humidity dependence of V. lecanii. Since arachid oil is safe for human consumption and not phytotoxic to cucumber leaves, low concentrations of arachid oil are recommended as an additive to increase the effectiveness of V. lecanii as a biocontrol agent of S. fuliginea.     

Preliminary evaluation of the effectiveness of a Verticillium lecanii isolate in the control of Thrips tabaci (Thysanoptera: Thripidae)

Thrips tabaci is a polyphagous pest that attacks many different crop and ornamental plant species. Recently, the need to reduce the use of insecticides in the control of thrips is more and more increasing. On that account, the possibility to make use of the entomopathogenic fungus Verticillium lecanii (DAOM198499) was evaluated in laboratory conditions. Suspension of 10(3) to 10(7) conidia/ml grown in liquid culture medium with a control (distilled water) treated on second larval stage of T. tabaci in controlled condition (16L: 8D photoperiod, 25 +/- 1 degrees C temperature, 98% RH in first 48h and 70% RH in next days). Preliminary results showed that mortality in lethal suspension (10(7) conidia/ml) of V. lecanii after 24 hours and 2-7 days in lower suspension appears. This suggests that strine DAOM 198499 of V. lecanii has the ability to control T. tabaci in greenhouse conditions.     

Chiral inversion of 2-phenylpropionic acid by Cordyceps militaris

Y.-F. HUNG, M.J. THOMASON, W. RHYS-WILLIAMS, A.W. LLOYD AND G.W. HANLON. 1996. Fifty-nine microbial cultures were screened for their ability to carry out the chiral inversion of (R,S) and (R)-2-phenylpropionic acid under both growing and non-growing conditions. While a number of these micro-organisms metabolized the substrate, only Verticillium lecanii IMI 68689 and Cordyceps militaris CBS 128.25 were able to invert the (R)-enantiomer to its (S)-antipode with no other biotransformation occurring. The rate of inversion was faster for C. militaris but in both cases the reaction was independent of growth and reached an equilibrium after 5 d incubation. Evidence suggests that the (R) to (S) inversion is inhibited in the presence of excess (S)-enantiomer which may account for the equilibrium enantiomeric ratios (S:R) of 90:10 and 70:30 for C. militaris and V. lecanii , respectively. Optimization studies revealed that the fastest rates of reaction for C. militaris were found at lower substrate concentrations (100 μg ml^-1) whilst the greatest yield of (S)-2-PPA was obtained at 250 μg ml^-1. Optimum inversion was found to occur using Sørensen's phosphate buffer at pH 5.5. These results may have implications for the use of micro-organisms in the production of pure enantiomers from racemic mixtures and, in addition, provide an in vitro model system for the study of a unique and interesting biotransformation.     

Topical administration of racemic ibuprofen

In vitro experiments to investigate possible stereoselective aspects of the topical administration of ibuprofen have been conducted. Incubation of ibuprofen with rat skin homogenates in the presence of coenzyme A, ATP, and magnesium provided no evidence for the formation of ibuprofenyl coenzyme A (the initial intermediate in the metabolic inversion of [R]- to [S]-ibuprofen). Similar incubation studies gave no indication of a change in the enantiomeric ratios of ibuprofen over the time course of the experiments. Percutaneous penetration studies of ibuprofen gel through porcine skin indicated that the ibuprofen enantiomer levels in the reservoir solutions were consistent with racemic ibuprofen having traversed the skin with no metabolic inversion. These results suggest that, in the models studied, skin metabolism does not result in the chiral inversion of (R)- to (S)-ibuprofen and that the topical administration of ibuprofen will result in the delivery of 50% “isomeric ballast.” Chirality 9:313–316, 1997. © 1997 Wiley-Liss, Inc.     

Pathogenicity of the Fungus Verticillium lecanii to Aphids and Powdery Mildew

Aspects of the host specificity and pathogenicity of the hyphomycete, Verticillium lecanii , were investigated under laboratory conditions. DiVerences were observed in the pathogenicities of three strains of V. lecanii (DAOM 198499, DAOM 216596 and Vertalec) to the potato aphid Macrosiphum euphorbiae and Sphaerotheca fuliginea , the causal agent of cucumber powdery mildew. The estimated median lethal concentration required to achieve 50% mortality (LC ), 50 median lethal time leading to 50% mortality (LT ) and aphid net reproductive rate ( R ) 50 0 indicated that Vertalec and strain 198499 were more virulent to aphids than strain 216596. The estimated median colonization time for 50% of fungal colonies (CT ) showed that strain 50 198499 was the best antagonist of cucumber powdery mildew. Further comparison suggested that the mean pathogenicity of V. lecanii strain 198499 to cucumber powdery mildew was almost equivalent to that of Sporothrix flocculosa , a biological control agent of greenhouse fungal pathogens. These observations provide convincing experimental evidence that V. lecanii is biologically active against both arthropods and fungi. The potential of using V. lecanii strain 198499 in biological control is discussed.     

Pulmonary inversion of 2-arylpropionic acids: influence of protein binding.

The possible contribution of pulmonary metabolism to the putative first-pass metabolism of 2-arylpropionic acid nonsteroidal antiinflammatory drugs has not been documented. Isolated perfused rabbit lungs, perfused with 4.5% bovine serum albumin or 5% dextran, were used to study the pulmonary elimination of (R)- and rac-ibuprofen, fenoprofen, and flurbiprofen. In the absence of protein binding, ibuprofen was metabolized via inversion and other pathways, whereas fenoprofen metabolism was essentially restricted to inversion of the (R)-enantiomer; fraction inverted (+/- SE) was 0.37 +/- 0.05 for (R)-ibuprofen and 0.85 +/- 0.03 for (R)-fenoprofen. In the presence of protein, neither ibuprofen nor fenoprofen was metabolized. Flurbiprofen did not undergo pulmonary elimination under any condition studied. This study illustrates that even though a tissue is capable of metabolism, particularly inversion of 2-arylpropionics, the quantitative importance of such elimination pathways may be minimal in the presence of the high degree of protein binding that is characteristic of these drugs.     

Acidic Nonsteroidal Anti-inflammatory Drugs Inhibit Rat Brain Fatty Acid Amide Hydrolase in a pH-dependent Manner

Previous studies have demonstrated that fatty acid amide hydrolase, the enzyme responsible for the metabolism of anandamide, is inhibited by the acidic non-steroidal anti-inflammatory drug (NSAID) ibuprofen with a potency that increases as the assay pH is reduced. Here we show that (R) -, (S) - and (R, S) -flurbiprofen, indomethacin and niflumic acid show similar pH-dependent shifts in potency to that seen with ibuprofen. Thus, (S) -flurbiprofen inhibited 2 μM [3 H]anandamide metabolism with IC 50 values of 13 and 50 μM at assay pH values of 6 and 8, respectively. In contrast, the neutral compound celecoxib was a weak fatty acid amide hydrolase inhibitor and showed no pH dependency (IC 50 values ~300 μM at both assay pH). The cyclooxygenase-2-selective inhibitors nimesulide and SC-58125 did not inhibit fatty acid amide hydrolase activity at either pH. The data are consistent with the conclusion that the non-ionised forms of the acidic NSAIDs are responsible for the inhibition of fatty acid amide hydrolase.     

Selection of Verticillium lecanii Isolates with High Potential for Biocontrol of Cucumber Powdery Mildew by Means of Components Analysis at Different Humidity Regimes

To identify characteristics for the selection of Verticillium lecanii isolates with high potential for biocontrol of Sphaerotheca fuliginea under glasshouse conditions, an exploratory study was performed on the effect of water limitation on the development of 14 isolates. The conidial germination, growth and sporulation of isolates of V. lecanii were studied in a tritrophic system on cucumber leaves and in a ditrophic system in Petri dishes. Their mycoparasitic ability was studied in S. fuliginea and Cladosporium cladosporioides . All characteristics were clearly affected by humidity. Four isolates showed good biocontrol potential. The performance of isolates on agar had less predictive value than on powdery mildew. The germination of isolates of V. lecanii was lower and the mycelial growth faster on agar than on mildewed leaves under corresponding humidity conditions. The results suggest that conditions in the phyllosphere differed from the set humidity in the surrounding air. A correlation was found between the lysis of C. cladosporioides growing in dual culture on agar with isolates of V. lecanii and the parasitism of powdery mildew on detached, rooted leaves. C. cladosporioides might offer a suitable substrate for testing isolates of V. lecanii for mycoparasitic potential under various environmental conditions. Conidial germination, growth and sporulation had limited predictive value.     

Acidic nonsteroidal anti-inflammatory drugs inhibit rat brain fatty acid amide hydrolase in a pH-dependent manner

Previous studies have demonstrated that fatty acid amide hydrolase, the enzyme responsible for the metabolism of anandamide, is inhibited by the acidic non-steroidal anti-inflammatory drug (NSAID) ibuprofen with a potency that increases as the assay pH is reduced. Here we show that (R)-, (S)- and (R,S)-flurbiprofen, indomethacin and niflumic acid show similar pH-dependent shifts in potency to that seen with ibuprofen. Thus, (S)-flurbiprofen inhibited 2 microM [3H]anandamide metabolism with IC50 values of 13 and 50 microM at assay pH values of 6 and 8, respectively. In contrast, the neutral compound celecoxib was a weak fatty acid amide hydrolase inhibitor and showed no pH dependency (IC50 values approximately 300 microM at both assay pH). The cyclooxygenase-2-selective inhibitors nimesulide and SC-58125 did not inhibit fatty acid amide hydrolase activity at either pH. The data are consistent with the conclusion that the non-ionised forms of the acidic NSAIDs are responsible for the inhibition of fatty acid amide hydrolase.     

Microbial metabolism of 2-arylpropionic acids: Chiral inversion of ibuprofen and 2-phenylpropionic acid

The metabolism of (R,S)-ibuprofen has been investigated in 24 microbial cultures. Of these Cunninghamella elegans, Mucor hiemalis, and Verticillium lecanii catalyzed the oxidation of the drug to 2-[4-(2-hydroxy-2-methylpropyl)phenyl]propionic acid, a known mammalian metabolite. The extent of metabolism was greatest with V. lecanii, with some 47% of the substrate being consumed over a 7-day incubation period. Enantiomeric analysis indicated stereoselective metabolism of (R)-ibuprofen, the enantiomeric composition of the residual substrate being R/S = 0.25. Following a preparative scale incubation of (R,S)-ibuprofen with V. lecanii, in which the reaction was allowed to go to completion, the metabolite was found to be predominantly of the S-configuration (S/R = 2.1), suggesting that chiral inversion of either the drug and/or the metabolite had taken place. Analysis of extracts following incubation of (R,S)-, (R)-, and (S)-2-phenylpropionic acid with V. lecanii, for 21 days, indicated that chiral inversion of the (R)-enantiomer to its optical antipode had taken place. The results of these investigations indicate that microorganisms, in addition to mammals, are able to mediate the chiral inversion of 2-arylpropionic acids. This observation may have implications for the preparation of optically pure 2-arylpropionic acids. © 1993 Wiley-Liss, Inc.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

Preventative and Curative Applications of Verticillium lecanii for Biological Control of Cucumber Powdery Mildew

The effect of timing of the application of the mycoparasite , Verticillium lecanii, on cucumber powdery mildew , Sphaerotheca fuliginea, was studied in a rooted cucumber leaf bioassay . The mycoparasite was applied at different times before and after mildew inoculation . At near - to maximum humidity ( 95% relative humidity) , early preventative (9 and 5 days before mildew inoculation) and early curative control treatments (2 days after mildew inoculation) gave considerable reduction in mildewed leaf areas , while late curative treatments resulted in greater mildewed leaf area but ultimately a reduced amount of healthy mildewed leaf area ( 20 % ) . Appropriate timing of biocontrol treatment application of V. lecanii is important to achieve good control .     

Determination of the epimeric composition of ibuprofenyl-CoA

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.     

Pheromone Traps for the Dissemination of an Entomopathogen by the Damson-hop Aphid Phorodon humuli

Two types of sex pheromone-emitting trap, a simple louvred trap and a commercially-produced wasp trap (Waspy®) designed or modified as delivery systems for entomopathogens, were tested using an isolate of the pathogen Verticillium lecanii . Their efficiencies for capturing winged autumn females (gynoparae) and males of Phorodon humuli in a hop garden and a plum orchard were compared with each other and with catches by yellow-painted water traps. Gynoparae rarely entered either type of pathogen dissemination trap. Males only entered traps that released (1 R , S ,4a R , 7 S , 7a S )-nepetalactol, the sex pheromone of P. humuli . In the hop garden, approximately five times as many males entered the modified Waspy® as entered the louvred traps, whereas in the plum orchard similar numbers of males were caught in both traps. On average, males visited the modified Waspy® trap for 34.1 ±7.9 (SE) min. Males spent about 50% of their time in the arena where the entomopathogen would normally be present and a further 20% of the time on the inside of the arena lid alongside the pheromone source. Out of a sample of 16 live aphids removed from a trap dispensing V. lecanii , 9 died from infection by the pathogen and 5 of the 16 initiated colonies of the fungus after they walked on a sterile agar plate for five minutes. None of the 15 aphids collected from traps without V. lecanii became infected or initiated colonies of the fungus on agar plates.     

Differentiation of mitochondrial subgroups in the Verticillium lecanii species complex

Presumptive mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were obtained from 54 fungal isolates identified as Verticillium lecanii, V. psalliotae or ' V. lecanii -like'. Analysis of the mtDNA RFLPs showed 20 different patterns, indicating considerable genetic variation within the V. lecanii species complex. There was no direct correlation between host and mtDNA pattern, or between mtDNA patterns and previously described isoenzyme-defined specific groups. Isolates from tropical and subtropical areas showed considerable variation in genotypes, while isolates from temperate regions appeared less variable.     

Compatibility of Verticillium lecani (Zimm.) with several pesticides

The entomopathogenic fungus, Verticillium lecanii (Zimm.) Viégas is a widespread hyphomycete consisting of a heterogeneous species aggregate. Conidia survival may be effected by environmental factors or by bio-pesticides and chemical products used to protect crop plants. In this research compatibility of mentioned fungi with imidachlopride, flufenoxuron, teflubenzuron+phozalone, endosulfan, amitraz, and effect of these pesticides on vegetative growth of the fungus were studied. The formulations of pesticides were tested in three concentration (mean concentration-MC, half MC and twice the MC) and using Duncan multiple range test, treatment were grouped. Effects of the pesticides on V. lecanii vegetative growth in concerned results have shown that almost all formulations studies significantly inhibition fungal development. The results indicated that flufenoxuron (at three concentrations), teflubenzuron+phozalone (at MC and twice the MC), induced levels of vegetative growth inhibition higher than 68%. Only data on amitraz and imidachlopride (at half MC) were not significantly different from the control, which is indicated the compatible formulations with V. lecanii. These formulations could be used simultaneously with this entomopathogenic in integrated pest management.     

Comparison of laboratory infection of aphids by Metarrhizium anisopliae and Verticillium lecanii

The pathogenicity of the only documented ‘aphid’ strain of Metarrhizium anisopliae for the aphid, Macrosiphoniella sanborni, was established by experimental infection. Its aphicidal potential was assessed by conducting parallel experiments with Verticillium lecanii, a fungus of proven efficacy in controlling aphids. The spore germination, growth and sporulation rates of M. anisopliae were slower than V. lecanii and were most likely the principal factors determining the higher LC., values in bioassay and slower propagation of this fungus amongst aphid populations. It is concluded that M. anisopliae, except perhaps in the salt marsh habitat from which it was originally isolated, would not prove as useful an agent as V. lecanii in biological control of aphids.     

Efficient synthesis and chiral separation of 11C-labeled ibuprofen assisted by DMSO for imaging of in vivo behavior of the individual isomers by positron emission tomography

The pharmacological mechanisms focusing on chiral isomer of ibuprofen are not fully understood. Only the (S)-isomer of ibuprofen inhibits cyclooxygenases, which mediates the generation of prostanoids and thromboxanes. Consequently, (S)-isomers represent a major promoter of the anti-inflammatory effect, and the effects of the (R)-isomers have not been widely discussed. However, more recently, the cyclooxygenase-independent pharmacological effects of ibuprofen have been elucidated. Pharmacokinetic studies with individual isomers of ibuprofen by positron emission tomography should aid our understanding of the pharmacological mechanisms of ibuprofen. The efficient (11)C-labeling of ibuprofen for chiral separation via the TBAF-promoted α-[(11)C]methylation was achieved by using DMSO rather than THF as the reaction solvent. The robust production of the radiochemically labile (11)C-labeled ibuprofen ester was realized by the protective effect of DMSO on radiolysis. After intravenous injection of each enantiomer of [(11)C]ibuprofen, significantly high radioactivity was observed in the joints of arthritis mice when compared to the levels observed in normal mice. However, the high accumulation was equivalent between the enantiomers, indicating that ibuprofen is accumulated in the arthritic joints regardless of the expression of cyclooxygenases.