Simultaneous determination of tandospirone and its active metabolite, 1-[2-pyrimidyl]-piperazine in rat plasma by LC-MS/MS and its application to a pharmacokinetic study.

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometry method for the detection of tandospirone (TDS) and its active metabolite 1-[2-pyrimidyl]-piperazine (1-PP) in Sprague-Dawley rat plasma is described. It was employed in a pharmacokinetic study. These analytes and the internal standards were extracted from plasma using protein precipitation with acetonitrile, then separated on a CAPCELL PAK ADME C18 column using a mobile phase of acetonitrile and 5 mm ammonium formate acidified with formic acid (0.1%, v/v) at a total flow rate of 0.4 mL/min. The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. The method was validated to quantify the concentration ranges of 1.000-500.0 ng/mL for TDS and 10.00-500.0 ng/mL for 1-PP. Total time for each chromatograph was 3.0 min. The intra-day precision was between 1.42 and 6.69% and the accuracy ranged from 95.74 to 110.18% for all analytes. Inter-day precision and accuracy ranged from 2.47 to 6.02% and from 98.37 to 105.62%, respectively. The lower limits of quantification were 1.000 ng/mL for TDS and 10.00 ng/mL for 1-PP. This method provided a fast, sensitive and selective analytical tool for quantification of tandospirone and its metabolite 1-PP in plasma necessary for the pharmacokinetic investigation.

Buspirone Hydrochloride Loaded In Situ Nanovesicular Gel as an Anxiolytic Nasal Drug Delivery System: In Vitro and Animal Studies.

Nasal nanovesicular gels of buspirone hydrochloride (BH) were prepared and characterized aiming for sustained delivery and enhancing bioavailability. Buspirone hydrochloride has low bioavailability of about 4% after oral administration due to first pass metabolism. Buspirone hydrochloride nanovesicles were formulated by thin film hydration method (TFH). The selected nanovesicular formulation was incorporated into two types of in situ gels (pH-induced and thermoreversible) using carbopol 974P and poloxamer 407 (P407), respectively, together with different mucoadhesive polymers. The in situ gels were examined for pH, gelling capability, viscosity, content uniformity, mucoadhesiveness, and in vitro drug release. The ex vivo permeation performance of the in situ gel formulations that showed the most sustained release was also assessed. The in vivo study was done by the determination of BH blood level in albino rabbits after nasal administration. Results revealed that nanovesicles prepared using Span 60 and cholesterol in a ratio of 80:20 showed the highest EE% (70.57 ± 1.00%). The ex vivo permeation data confirmed higher permeability figures for carbopol formulation in comparison to poloxamer formulations. The in vivo study data showed an increase of 3.26 times in BH bioavailability when formulated into the carbopol nanovesicular in situ gel relative to control (nasal drug solution).

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry.

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h. Graphical Abstract.

Formulation and Evaluation of Niosomal in situ Nasal Gel of a Serotonin Receptor Agonist, Buspirone Hydrochloride for the Brain Delivery via Intranasal Route.

BACKGROUND: Buspirone Hydrochloride is an anxiolytic agent and serotonin receptor agonist belonging to azaspirodecanedione class of compounds used in the treatment of anxiety disorders. It has short half-life (2-3h) and low oral bioavailability (4%) due to extensive first pass metabolism. OBJECTIVE: The nasal mucosa has several advantages viz., large surface area, porous endothelial membrane, high blood flow, avoidance of first-pass metabolism and ready accessibility that lead to faster and higher drug absorption. Keeping these facts in mind, the objective of the present study was to develop Buspirone hydrochloride loaded niosomal in-situ nasal gel. METHODS: Buspirone hydrochloride niosomal in situ nasal gel was formulated, optimized and evaluated with the objective to deliver drug to the brain via intranasal route. Niosomes were prepared by thin film evaporation method and optimized using32 factorial design. Niosomes were characterized for particle size, zeta potential, entrapment efficiency and in vitro drug release. Buspirone hydrochloride loaded niosomes were further incorporated into Carbopol 934P and HPMC K4M liquid gelling system for the formation of in situ gel. The resultant solution was assessed for various parameters, viz., gelling time, gelling capacity, viscosity at pH 5 and pH 6. RESULTS: The vesicle size of all niosomal suspension batches ranges between 168.3 -310.5 nm. The vesicle size of optimized niosomal suspension F5 batch is 181.9±0.36nm. For F5 batch, the value of zeta potential was found to be -15.4 mV; this specifies that prepared niosomes have sufficient surface charge to prevent aggregation of the vesicles. % entrapment efficiency for all batches was found in the range 72.44±0.18% to 87.7±0.66%. The cumulative percent release of niosomal suspension ranges from 66.34±0.39 to 84.26±0.26%. Ex vivo permeation of Buspirone hydrochloride through the sheep nasal mucosa showed that 83.49% w/w drug permeated after 8 h. The SEM and Zeta potential studies showed the formation of stable vesicles. CONCLUSION: Thus, the application of niosomes proved the potential for intranasal delivery of Buspirone hydrochloride over the conventional gel formulations. Overall intranasal drug delivery for Buspirone hydrochloride has been successfully developed.

TiO2 Photocatalysis-DESI-MS Rotating Array Platform for High-Throughput Investigation of Oxidation Reactions.

We present a new high-throughput platform for studying titanium dioxide (TiO2) photocatalytic oxidation reactions by performing reactions on a TiO2-coated surface, followed by direct analysis of oxidation products from the surface by desorption electrospray ionization mass spectrometry (DESI-MS). For this purpose, we coated a round glass wafer with photocatalytically active anatase-phase TiO2 using atomic layer deposition. Approximately 70 aqueous 1 µL samples can be injected onto the rim of the TiO2-coated glass wafer, before the entire wafer is exposed to UV irradiation. After evaporation of water, the oxidation products can be directly analyzed from the sample spots by DESI-MS, using a commercial rotating sample platform. The method was shown to provide fast photocatalytic oxidation reactions and analysis with throughput of about four samples per minute. The feasibility of the method was examined for mimicking phase I metabolism reactions of amodiaquine, buspirone and verapamil. Their main photocatalytic reaction products were mostly similar to the products observed earlier in TiO2 photocatalysis and in in vitro phase I metabolism assays performed using human liver microsomes.

Development of HPLC Method for the Determination of Buspirone in Rat Plasma Using Fluorescence Detection and Its Application to a Pharmacokinetic Study.

A simple and sensitive analytical method for the quantitative determination of buspirone in rat plasma by HPLC with fluorescence detection was developed and validated using naproxen as an internal standard. A relatively small-volume (150 µL) aliquot of rat plasma sample was prepared by a simple deproteinization procedure using acetonitrile as a precipitating organic solvent. Chromatographic separation was performed using Kinetex® C8 column with an isocratic mobile phase consisting of acetonitrile and 10-mM potassium phosphate buffer (pH 6.0) at a flow rate of 1.0 mL/min. The eluent was monitored by fluorescence detector at a wavelength pair of 237/380 nm (excitation/emission). The linearity was established at 20.0-5000 ng/mL, and the limit of detection was 6.51 ng/mL. The precision (≤14.6%), accuracy (89.2-108%), and stability (89.1-101%) were within acceptable ranges. The newly developed method was successfully applied to intravenous and oral pharmacokinetic studies of buspirone in rats.

Quantification of peptides using N-terminal isotope coding and C-terminal derivatization for sensitive analysis by micro liquid chromatography-tandem mass spectrometry.

Stable isotope-coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional-group derivatization of bioactive peptides followed by micro liquid chromatography-tandem mass spectrometry (LC-MS/MS). By separating the sensitivity-enhancement and isotope-coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde-d4 to afford N-alkylated products with different masses. This process is simple, quick and high-yield, and accurate comparative analysis can be achieved for the mass-differentiated peptides. Then, the carboxyl groups were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I-IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC-MS/MS. In all instances, a greater than 17-fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp-Arg-(Val-d8 )-Tyr-Ile-His-Pro-(Phe-d8 )-His-Leu]. Copyright © 2016 John Wiley & Sons, Ltd.

Azapirones for Attention Deficit Hyperactivity Disorder: A Systematic Review.

INTRODUCTION: No meta-analysis has evaluated azapirones (serotonin1A receptor partial agonists) as anxiolytics for attention deficit hyperactivity disorder (ADHD). METHODS: Randomized controlled trials (RCTs) and single-arm trials published before October 27, 2015 were retrieved from major healthcare databases and clinical trial registries. Relative risk and 95% confidence intervals were calculated. RESULTS: 5 RCTs (n=429) and 3 single-arm studies (n=70) were identified. 3 RCTs compared buspirone vs. methylphenidate in children/adolescents, one buspirone patches vs. placebo patches in children/adolescents, and one atomoxetine plus buspirone vs. atomoxetine vs. placebo in adults. The single-arm studies were buspirone trails in children/adolescents. All-cause discontinuation rates and adverse events did not differ between pooled buspirone and methylphenidate groups. No other meta-analyses of buspirone efficacy and safety vs. comparators were conducted due to insufficient data. 2 RCTs found no significant differences in parent and teacher ADHD-Rating Scale total scores between buspirone and methylphenidate, while one reported that methylphenidate improved parent and teacher ADHD-RS total scores vs. buspirone. DISCUSSION: It remains unclear whether buspirone use has benefit for ADHD patients and therefore further evidence is needed for better clinical use of buspirone in patients with ADHD.

Controlled release effervescent buccal discs of buspirone hydrochloride: in vitro and in vivo evaluation studies.

In the present study controlled release effervescent buccal discs of buspirone hydrochloride (BS) were designed using HPMC as rate controlling and bioadhesive polymer by direct compression method. Sodium bicarbonate and citric acid were used in varying amounts as effervescence forming agents. Carbon dioxide evolved due to reaction of sodium bicarbonate and citric acid was explored for its potential as buccal permeation enhancer. The designed buccal discs were evaluated for physical characteristics and in vitro drug release studies. Bioadhesive behavior of designed buccal discs was assessed using texture analyzer. In vivo animal studies were performed in rabbits to study bioavailability of BS in the designed buccal discs and to establish permeation enhancement ability of carbon dioxide. It was observed that effervescent buccal discs have faster drug release compared to non-effervescent buccal discs in vitro and effervescent buccal discs demonstrated significant increase in bioavailability of drug when compared to non-effervescent formulation. Hence, effervescent buccal discs can be used as an alternative to improve the drug permeation resulting in better bioavailability. However, the amount of acid and base used for generation of carbon dioxide should be selected with care as this may damage the integrity of bioadhesive dosage form.

Comparison of TiO2 photocatalysis, electrochemically assisted Fenton reaction and direct electrochemistry for simulation of phase I metabolism reactions of drugs.

The feasibility of titanium dioxide (TiO2) photocatalysis, electrochemically assisted Fenton reaction (EC-Fenton) and direct electrochemical oxidation (EC) for simulation of phase I metabolism of drugs was studied by comparing the reaction products of buspirone, promazine, testosterone and 7-ethoxycoumarin with phase I metabolites of the same compounds produced in vitro by human liver microsomes (HLM). Reaction products were analysed by UHPLC-MS. TiO2 photocatalysis simulated the in vitro phase I metabolism in HLM more comprehensively than did EC-Fenton or EC. Even though TiO2 photocatalysis, EC-Fenton and EC do not allow comprehensive prediction of phase I metabolism, all three methods produce several important metabolites without the need for demanding purification steps to remove the biological matrix. Importantly, TiO2 photocatalysis produces aliphatic and aromatic hydroxylation products where direct EC fails. Furthermore, TiO2 photocatalysis is an extremely rapid, simple and inexpensive way to generate oxidation products in a clean matrix and the reaction can be simply initiated and quenched by switching the UV lamp on/off.

Lyophilized sustained release mucoadhesive chitosan sponges for buccal buspirone hydrochloride delivery: formulation and in vitro evaluation.

This work aims to prepare sustained release buccal mucoadhesive lyophilized chitosan sponges of buspirone hydrochloride (BH) to improve its systemic bioavailability. Chitosan sponges were prepared using simple casting/freeze-drying technique according to 3(2) factorial design where chitosan grade was set at three levels (low, medium, and high molecular weight), and concentration of chitosan solution at three levels (0.5, 1, and 2%). Mucoadhesion force, ex vivo mucoadhesion time, percent BH released after 8 h (Q8h), and time for release of 50% BH (T50%) were chosen as dependent variables. Additional BH cup and core buccal chitosan sponge were prepared to achieve uni-directional BH release toward the buccal mucosa. Sponges were evaluated in terms of drug content, surface pH, scanning electron microscopy, swelling index, mucoadhesion strength, ex vivo mucoadhesion time, and in vitro drug release. Cup and core sponge (HCH 0.5E) were able to adhere to the buccal mucosa for 8 h. It showed Q8h of 68.89% and exhibited a uni-directional drug release profile following Higuchi diffusion model.

Conformational analysis and geometry optimization of buspirone-A 5-HT1A receptor agonist.

Buspirone, a partial 5-HT1A receptor agonist, is a clinically prescribed anxiolytic. In the present study, conformational analysis and geometry optimization of buspirone were done as per Hartree-Fock (HF) calculation method by Argus Lab 4.0.1 software. The minimum potential energy was calculated by geometry convergence function by Argus Lab software. The results indicate that the best conformation of molecule is present at minimum potential energy of-100679.5513 kcal/mol. At this point, buspirone will be more active.

Enhanced bioavailability of buspirone hydrochloride via cup and core buccal tablets: formulation and in vitro/in vivo evaluation.

This work aims to prepare sustained release buccal mucoadhesive tablets of buspirone hydrochloride (BH) to improve its systemic bioavailability. The tablets were prepared according to 5×3 factorial design where polymer type was set at five levels (carbopol, hydroxypropyl methylcellulose, sodium alginate, sodium carboxymethyl cellulose and guar gum), and polymer to drug ratio at three levels (1:1, 2:1 and 3:1). Mucoadhesion force, ex vivo mucoadhesion time, percent BH released after 8 h (Q8h) and time for release of 50% BH (T(50%)) were chosen as dependent variables. Additional BH cup and core buccal tablets were prepared to optimize BH release profile and make it uni-directional along with the tablets mucoadhesion. Tablets were evaluated in terms of content uniformity, weight variation, thickness, diameter, hardness, friability, swelling index, surface pH, mucoadhesion strength and time and in vitro release. Cup and core formula (CA10) was able to adhere to the buccal mucosa for 8h, showed the highest Q8h (97.91%) and exhibited a zero order drug release profile. Pharmacokinetic study of formula CA10 in human volunteers revealed a 5.6 fold increase in BH bioavailability compared to the oral commercial Buspar® tablets. Conducting level A in vitro/in vivo correlation showed good correlation (r²=0.9805) between fractions dissolved in vitro and fractions absorbed in vivo.

Interaction of calcium sulfate with xanthan gum: effect on in vitro bioadhesion and drug release behavior from xanthan gum based buccal discs of buspirone.

Bioadhesive polymers in buccal drug delivery systems play an important role in delivery of therapeutic drug molecules for local and systemic action. Xanthan gum, a GRAS listed natural polymer was used to design buccal discs of buspirone hydrochloride by direct compression method. Effect of calcium sulfate on bioadhesive and drug release behavior of xanthan gum buccal discs was studied. Varying amount of calcium sulfate (0%, 5%, 10%, 20%, 30%, 40% and 50%, w/w) in combination with xanthan gum was used to prepare buccal bioadhesive discs. Increase in calcium sulfate concentration resulted in faster drug release and decreased the bioadhesive strength of the designed discs. Further, in rheological evaluation it was observed that viscosity of xanthan gum gel reduces with increasing concentration of calcium sulfate. Compatibility of drug with various excipients was assessed using DSC and FTIR techniques.

Determination of diosbulbin B in rat plasma and urine by LC-MS/MS and its application in pharmacokinetic and urinary excretion studies.

A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for determination of diosbulbin B in rat plasma and urine after oral administration. The detector was a Q-trap™ mass spectrometer with an electro-spray ionization interface operating in the multiple reaction monitoring mode. After extracted with methyl tert-butyl ether, diosbulbin B and busprione (internal standard, IS) were separated on an Agilent Zorbax C18 column (4.6mm×50mm, 3.5µm) using a gradient mobile phase consisting of water and methanol. Linearity was obtained over the concentration range of 5-5000ng/ml for diosbulbin B in rat plasma and urine. The lower limit of quantitation was 5.0ng/ml. The accuracy (relative error, RE) and precision (relative standard deviation, RSD) of disobulblin B in two biological matrices ranged from -8.2% to 1.4% RE and 1.9 to 10.1% RSD, respectively. The fully validated method was applied to a pharmacokinetic and urine excretion study for the first time. The main pharmacokinetic parameters Tmax, Cmax, T1/2, and Ke were 1.88±0.22h, 18.0±3.1ng/ml, 6.89±1.0h and 0.103±0.01l/h, respectively. A cumulative excretion of disobulbin B in rat urine was 2.69±0.43µg at 60h after dosing, accounting for 0.89% of the total dose.

Synthesis, in vitro binding studies and docking of long-chain arylpiperazine nitroquipazine analogues, as potential serotonin transporter inhibitors.

It is well known that 6-nitroquipazine exhibits about 150-fold higher affinity for the serotonin transporter (SERT) than quipazine and recently we showed quipazine buspirone analogues with high to moderate SERT affinity. Now we have designed and synthesized several 6-nitroquipazine buspirone derivatives. Unexpectedly, their SERT binding affinities were moderate, and much lower than that of the previously studied quipazine buspirone analogues. To explain these findings, docking studies of both groups of compounds into two different homology models of human SERT was performed using a flexible target-ligand docking approach (4D docking). The crystal structures of leucine transporter from Aquifex aeolicus in complex with leucine and with tryptophan were used as templates for the SERT models in closed and outward-facing conformations, respectively. We found that the latter conformation represents the most reliable model for binding of buspirone analogues. Docking into that model showed that the nitrated compounds acquire a rod like shape in the binding pocket with polar groups (nitro- and imido-) at the ends of the rod. 6-Nitro substituents gave steric clashes with amino acids located at the extracellular loop 4, which may explain their lower affinity than corresponding quipazine buspirone analogues. The results from the present study may suggest chemical design strategies to improve the SERT modulators.

An efficient synthesis of aripiprazole, buspirone and NAN-190 by the reductive alkylation of amines procedure.

The reductive alkylation of amines procedure was applied for the synthesis of aripiprazole 1a, buspirone 1b, and NAN-190 1c.

Quantitative analysis of atractylenolide I in rat plasma by LC-MS/MS method and its application to pharmacokinetic study.

A new high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) was developed for quantitative analysis of atractylenolide I in rat plasma using buspirone as internal standard (I.S.). Rat plasma samples were deproteined with methanol and acetonitrile (1:1, v/v). Atractylenolide I and I.S. were separated on a Phenomenex Gemini C(18) column (50 mm × 2.0 mm, 5 µm) with gradient mobile phase at the flow rate of 0.4 ml/min. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The linear calibration curve of atractylenolide I in rat plasma ranged 2.0-5000 ng/ml (R>0.9979). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.6 ng/ml and 2.0 ng/ml, respectively. Both accuracy and precision of the assay were satisfactory. The recoveries of atractylenolide I and I.S. were 91.4% and 87.8%, respectively. This fully validated method was applied to a pharmacokinetic study of atractylenolide I in rats administered with 20 g/kg Atractylodis extract. The main pharmacokinetic parameters T(max) (the time to peak), C(max) (the concentration to peak), T(0.5) (the biological half time), and K(e) (the elimination rate constant) were 0.81 ± 0.11h, 7.99 ± 1.2 ng/ml, 1.94 ± 0.27 h, 0.365 ± 0.06/h, respectively.

Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction.

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Application of a fluorescence-based continuous-flow bioassay to screen for diversity of cytochrome P450 BM3 mutant libraries.

A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants.