Therapeutic drug monitoring (TDM) has been shown to be clinically beneficial for critically ill patients. However, this is a burden for neonates or children with small circulating blood volumes. Here, we aimed to develop and validate a microsampling TDM platform (including dried blood spots (DBS) and capillary microsamples (CMS)) for the simultaneous quantification of vancomycin, meropenem, and linezolid.Paired DBS and CMS samples were obtained from an intensive care unit (ICU) to evaluate its clinical application. Estimated plasma concentrations (EPC) were calculated from DBS concentrations. Agreement between methods was evaluated using Deming regression and Bland-Altman difference plots.r > 0.98). The drug concentration ratio in DBS samples to that in CMS was 1.39 for vancomycin, 1.34 for meropenem, and 0.94 for linezolid. The EPC calculated from the DBS using individual hematocrit ranges presented comparable absolute values for vancomycin (slope: 1.06) and meropenem (slope: 1.04), with a mean of 98% and 99% of the measured CMS concentrations, respectively.The microsampling methods validation showed excellent reliability and compatibility with the analysis of the sample matrix and hematocrit range of the studied population. The DBS and CMS accuracy and precision results were within accepted ranges and samples were stable at room temperature for at least 2 days and 8 h, respectively. Hematocrit had no impact on CMS, but sightly impacted DBS measurements. The CMS and DBS antibiotic concentrations correlated well (This study provides a microsampling TDM platform validated for clinical use for a rapid quantification of three antibiotics and is suitable for real-time TDM-guided personalization of antimicrobial treatment in critically ill children. Therapeutic drug monitoring (TDM) offers a significant opportunity to improve treatment efficacy while minimizing toxicity. However, the small circulating blood volume in critically ill children does not allow for frequent determination of drug concentrations . TherefoVancomycin (VAN), meropenem (MEM), and linezolid (LZN) are frequently used in neonatal and pediatric intensive care units (ICUs) . HoweverBarco et al. published an analytical method for the simultaneous determination of 14 antibiotics  in plasma  but did There are two main aims of this study: the first is to set up and apply a fully validated microsampling TDM for the simultaneous determination and quantification of three antibiotics in DBS and CMS samples; the second is to evaluate the reliability of two microsampling methods applied in routine TDM in critically ill children.The reference standard of vancomycin (Lot No.130360-202103), meropenem (Lot No.130506), and linezolid (Lot No.130640-201901) complied with ChP Reference Standards. The internal standards (IS) for norvancomycin (Lot No.130338-201704), meropenem-2H6 (Lot No.21B063-A5), and linezolid-2H3 (Lot No.21B035-A1) were purchased from ZZBIO . Ultrapure water was provided by a smart2Pure pro water purification system . Acetonitrile (ACN), methanol (MeOH), and formic acid (FA) were obtained from Sigma-Aldrich , and all solvents were of LC-MS grade. Whatman 903® paper was purchased from GE Healthcare, and capillary tubes (70 µl) were purchased from Hirschmann Co. Ltd .Stock solutions of VAN, MEM, and LZN were prepared in MeOH at concentrations of 4, 4, and 2 mg/ml, respectively. All stock solutions were stored at −80°C and were further diluted in 50% MeOH to obtain a working solution. All individual IS stock solutions were prepared in ACN at 1 mg/ml and also stored at −80°C. The IS working solution consisted of 2.5 µg/ml norvancomycin, 5.0 µg/ml meropenem-2H6, and 1.0 µg/ml linezolid-2H3.Calibrators were prepared at eight concentrations, based on therapeutic ranges, by spiking working solutions to blank human plasma and whole blood at concentrations of 1, 2, 4, 8, 15, 30, 50, and 100 µg/ml for VAN; 0.4, 0.8, 1.6, 3.2, 6, 12, 20, and 40 µg/ml for MEM; and 0.2, 0.4, 0.8, 1.6, 3, 6, 10, and 20 µg/ml for LZN. QC samples  were prepared by spiking working solutions to blank plasma and whole blood at 1, 3, 7.5, and 75 µg/ml for VAN; 0.4, 1.2, 3, and 30 µg/ml for MEM; 0.2, 0.6, 1.5, and 15 µg/ml for LZN. The amount of solvent added to the matrix, for both the calibrators and QCs, did not exceed 5%. Calibrators and QC samples were aliquoted and stored at −80°C until analysis.All samples were subjected to protein precipitation. To prepare the CMS samples, the whole blood samples of patients were filled in capillary tubes and sealed with wax on both ends. The CMS samples were sent to the lab and centrifuged for 5 min at 3,000 rpm to separate the plasma and blood cells. The volume of the plasma sample was estimated by the length of the plasma fraction and cut down by special capillary pliers. The cut plasma fraction was washed using blank plasma (for standardizing the plasma volume to 50 µl), 15 µl formic acid, and 50 µl ACN in turn. Similarly, the STD and QC samples were prepared using an aliquot of 50 µl calibrator and QC plasma samples, mixed with the same reagents. All samples were mixed with 150 µl IS working solution, shaken at 2,000 rpm for 5 min and centrifuged for 5 min at 12,500 rpm. 10 µl supernatant was transferred and diluted 20-fold in water. Finally, 5 µl diluent was injected into the LC-MS/MS for analysis.For DBS samples, an aliquot of 50 µl whole blood samples was used for the calibration curve. QC and unknown samples were spotted on Whatman 903® paper cards and dried in a fume hood at an ambient temperature for at least 30 min. DBS samples were cut off at 10 mm diameter and transferred into Eppendorf tubes. Subsequently, 200 µl of aqueous solution  and 200 µl of IS working solution was added into the tubes. Samples were shaken at 2,000 rpm for 20 min at room temperature and 5 min at 12,500 rpm. The supernatant was diluted and injected in the same manner as the CMS samples.Chromatographic analysis was performed using an ACQUITY UPLC system . Mobile phases consisting of 0.2% FA in water (A) and ACN (B) at a flow rate of 0.3 ml/min were used. Separation of the components was achieved by gradient elution: the mobile phase B gradient was initiated at 10% B at 0.5 min then linearly increased to 70% at 2.8 min and to 90% at 3 min. This was maintained at 90% at 3.3 min, continuously decreased to 10% at 3.5 min, and then maintained at 10% until the end of the run, resulting in a total run time of 4 min. The UPLC system was equipped with an Acquity UPLC® HSS T3 column  , fitted with a corresponding BEH C18 guard column, and maintained at 40°C. The autosampler temperature was set at 10°C.The LC system was coupled to a Xevo TQ-S Micro triple quadrupole mass spectrometer , equipped with an electrospray ionization (ESI) source operating in positive ion mode. Multiple reaction monitoring (MRM) was applied for the detection of the components. The desolvation temperature was 550°C, the cone gas flow was 150 L/hr, and the desolvation gas flow was 900 L/hr. MRM transitions with corresponding MS parameters for quantifiers, qualifiers, and ISs are listed in The method validation followed the Chinese Pharmacopeia guidance and bioanalytical method validation from ICH. Specifically, selectivity was evaluated by endogenous interference peaks at the retention time of VAN, MEM, LZN, and IS of blank samples from six individual whole blood samples. Linearity was validated by calibration curves. Peak area ratios to IS were taken as the ordinate, and the nominated concentrations of the three antibiotics were taken as the abscissa and fitted to a 1/x2 weighted linear regression. For the intra- and inter-batch precision and accuracy, six replicates of QC and LLOQ samples were determined at three analytical batches. Accuracy was accepted within the range of 85%–115% while the precision coefficient of variance (CV) was below 15%. The matrix effects were carried out at low, medium, and high concentrations. Blank plasma and DBS samples from six different individuals were prepared, and a working solution was then added to the extractant in order to evaluate the matrix effects. The stability of plasma and DBS samples in different storage conditions was evaluated. Samples were stored at room temperature, 2–8°C, 35°C, and −80°C, and the post-treated stability of the autosampler was evaluated by examining two levels of QC samples (low and high). Samples were considered stable if concentrations were within the range of 85%–115%. The dilution integrity of the plasma samples was processed. Extra-high QC plasma samples at a nominal concentration of 125 µg/ml for VAN, 50 µg/ml for MEM, and 25 µg/ml for LZN (1.25 times the ULOQ concentration) were diluted 10:1 with blank plasma. Six replicates of the diluted samples were processed and analyzed.For the CMS accuracy and adsorption test, six replicates of QC samples were determined. The impact of the hematocrit (Hct) on antibiotic DBS measurements was also tested. Blood samples with Hct levels of 15, 20, 25, 30, 35, and 40% were obtained from whole blood, and plasma was then added or removed. QCL and QCH DBS samples were prepared from the blood and analyzed in triplicate at a fixed Hct level of 30%. The influence of the Hct percentage on VAN, MEM, and LZN determinations was expressed as the percentages of nominal concentrations measured in DBS samples. Acceptance criteria were values between 85% and 115%.From the drug concentration in DBS samples and the Hct, an estimated plasma concentration (EPC) was calculated using the formula:EPC was evaluated using both the individual and average Hct of the group of patients. Agreement between methods was evaluated using Deming regression and the Bland–Altman difference plot. Statistical analyses were performed with R4.04 software.A large whole blood sample set was drawn, for routine TDM purposes, from pediatric patients receiving VAN, MEM, and LZN for the treatment of various infections at the ICU of the Children's Hospital of Fudan University, Shanghai, China. The patients were not selected in advance, and samples were included as they were received in our laboratory for routine TDM. DBS and CMS samples were produced from these whole blood samples, which were maintained at –−80°C until analysis. This study was in compliance with the Declaration of Helsinki and was approved by the local ethics committee at the hospital.Typical chromatograms obtained from plasma and DBS samples of VAN, MEM, and LZN in double blank, blank with internal standards, and LLOQ are illustrated in There was no endogenous substance interference between analytes and the internal standard. Linearity was acceptable using a 1/x2 weighting over a range of 1–100, 0.4–40, and 0.2–20 µg/ml for VAN, MEM, and LZN, respectively. The results of the two matrixes (plasma and DBS) for the intra- and inter-assay precision and accuracy tests were all within the acceptable ranges . The matThe results of the stability testing of VAN, MEM, and LZN in plasma are shown in The capillary tube absorption test and the Hct effect on DBS samples are shown in r > 0.98, p < 0.01). However, CMS to DBS drug concentration ratios were highly variable. VAN ranged from 1.03 to 1.95 with an average of 1.39, MEM from 1.04 to 1.93 with an average of 1.34, and LZN from 0.77 to 1.08 with an average of 0.94, indicating that LZN could be accurately determined in DBS cards (Deming regression: y = 0.99 x −0.32)  . Howeverr > 0.98, p < 0.01). The slope value from the Deming regression was 1.04 vs. 1.06 (p < 0.01) for VAN and 1.04 vs. 1.04 (p < 0.01) for MEM. However, the EPC estimated using individual Hct performs better at fitting than the EPC estimated using average Hct by the Bland-Altman difference plots. As shown in the diagram, 83.8% (52/62) of the VAN EPC estimated using individual Hct did not differ by more than 20% from the CMS samples when compared to 77.4% (48/62) using average Hct. In addition, the MEM EPC showed the same trend (92.5% (49/53) vs. 84.9% (45/53).Critically ill children commonly experience anemia and hemodilution due to medical interventions. The average Hct in these children (17%–39%) was markedly lower than that of normal children (35%–45%). Deming regression and Bland-Altman difference in concentrations of VAN and MEM obtained in CMS samples and EPCs were compared in Here, we developed a microsampling TDM platform for the simultaneous quantification of empirical antibiotics commonly prescribed in critically ill pediatric patients. In comparison with outpatients, critically ill children in an ICU could have a venous catheter, which is suitable for venous microsampling. A total of 72.9% (43/59) of pediatric patients in our study received MEM treatment in combination with VAN or LZN. Microsampling and combined detection appear to reduce the blood volumes drawn from patients and provide plasma concentrations of multiple drugs.n = 9), where the LZN was homogeneously distributed in the blood. Alternatively, the CMS samples were not affected by Hct and were considered the closest microsamples to the “gold standard” of plasma sampling  , 23, it Here, we developed and validated a microsampling TDM platform for the accurate quantification of VAN, MEM, and LZN. CMS and DBS sampling can be used for TDM and the personalization of antibiotic therapy in critically ill children or even in neonates.


Paeonia suffruticosa) is a traditional Chinese flower that is not resistant to high temperatures, and the frequent sunburn during summer limits its normal growth. The lack of understanding of the molecular mechanisms in tree peony has greatly restricted the improvement of novel heat-tolerant varieties. Therefore, we treated tree peony cultivar “Yuhong” (P. suffruticosa “Yuhong”) at normal (25°C) and high temperatures (40°C) and sequenced the transcriptomes, to investigate the molecular responsive mechanisms to heat stress. By comparing the transcriptomes, a total of 7,673 differentially expressed genes (DEGs) were detected comprising 4,220 upregulated and 3,453 downregulated genes. Functional annotation showed that the DEGs were mainly related to the metabolic process, cells and binding, carbon metabolism, and endoplasmic reticulum protein processing. qRT-PCR revealed that three sHSP genes  were upregulated in the response of tree peony to heat stress. Tissue quantification of the transgenic lines  showed that all three genes were most highly expressed in the leaves. The survival rates of transgenic lines  restored to normal growth after high-temperature treatment were 43, 36, and 31%, respectively. In addition, the activity of superoxide dismutase, accumulation of free proline, and chlorophyll level was higher than those of the wild-type lines, while the malondialdehyde content and conductivity were lower, and the membrane lipid peroxidation reaction of the wild-type plant was more intense. Our research found several processes and pathways related to heat resistance in tree peony including metabolic process, single-organism process, phenylpropane biosynthesis pathway, and endoplasmic reticulum protein synthesis pathway. PsHSP17.8, PsHSP21, and PsHSP27.4 improved heat tolerance by increasing SOD activity and proline content. These findings can provide genetic resources for understanding the heat-resistance response of tree peony and benefit future germplasm innovation.Tree peony ( Paeonia suffruticosa Andrews, Paeoniaceae) is a type of perennial deciduous subshrub originating in China as one of the 10 traditional Chinese flowers , kinases, transcription factors (TFs), and hormones to regulate changes in the transcriptome, proteome, and metabolome (2+ accumulation and flow from the extracellular matrix into the cell (2−), and hydroxyl (−OH), which results in peroxidative damage to lipids, nucleic acids, proteins, and cell membranes  to protect cells and transduce signals . HSPs caP. suffruticosa “Yuhong” seedlings treated at normal and high temperatures were sequenced to scan differential HSP genes. The functions of the three PsHSP genes were analyzed using transgenic Arabidopsis thaliana. Our results will provide genetic resources for germplasm innovation of tree peony and the future breeding of high heat-resistant cultivars.Since few studies have focused on the response of tree peony to heat stress, the mechanisms underlying the response to heat stress remain largely unknown. To explore the molecular mechanism of heat resistance in tree peony, the transcriptomes of Paeonia suffruticosa “Yuhong” is one of the high heat-resistant cultivars in the Jiangnan group grown in the intelligent experimental shed of Zhejiang A&F University . Six annual seedlings with the same growth were individually transplanted into single flowerpots with normal water and fertilizer management and then placed into the growth cabinet RXZ-100A  at 25°C, with 12 h/12 h light/dark, 4,000 lx light intensity, and 80% humidity for 2 weeks for pretreatment. Subsequently, according to previous research , and the quality was checked by an ultraviolet spectrophotometer and 1.5% agarose gel electrophoresis. The pair-end complementary DNA (cDNA) library was constructed and then sequenced using Illumina HiSeq2500 , with an insert size of 250 bp based on sequencing by synthesis (SBS) technology to generate the raw data. Trimmomatic v0.39  and Perl2|fold change (FC)| ≥ 1 and false discovery rate (FDR) < 0.01 and then enriched in GO function and the KEGG pathway. DNAMAN v7PsHSP genes, and BioXM v2.7.1 , the reaction mix contained 3.2 μl ddHt method  using thPsHSP17.8, PsHSP21, and PsHSP27.4, which were amplified by PCR with designed primers. To further investigate the role of these three sHSPs in tree peony resistance to heat stress, double digestion (Xho I and Cla I) was used to construct the expression vectors (AY562547.1) of the three sHSPs (Agrobacterium-mediated transformation (GV3101) was used for transgenic A. thaliana. With reference to −1 kanamycin +50 mg·mL−1 timentin +50 mg·mL−1 ampicillin in 1/2 MS solid medium  sequences of ee sHSPs . Agrobacd medium , S3. The3 transgenic lines were stressed at 40°C for 12 h, and then, leaves from the same position were immediately collected to measure relevant physiological indices. Chlorophyll content was extracted by macerating in 80% acetone and determined with a spectrophotometer , and the conductivity of the leaves was measured by a conductivity bridge  in accordance with The MT and TObvious phenotypic changes in the leaves were observed with an increasing time under heat stress at 40°C . At 0 h,1, CK2, and CK3) and the treatment group . The number of clean bases in each sample was 6.86–9.08 Gb, and the count of clean reads ranged from 22,967,108 to 30,430,627. The GC content was stable at 43.68–44.96%, and the proportion of Q30 bases in each sample was ≥96%. All unigenes were blasted against the eight databases to obtain annotations. A total of 24,309 unigenes were annotated, of which 9,731 (40.03%) unigenes were 300–1,000 bp, and 14,578 (59.97%) were over 1,000 bp. Among the annotated unigenes, the most annotations were obtained in NR , Eggnog , and Swiss-Prot , while the least were from COG . There were 15,108 (62.15%), 14,568 (59.93%), 13,669 (56.23%), and 8,416 (34.62%) unigenes annotated in Pfam, GO, KOG, and KEGG, respectively  were 459, 714, and 693 bp, respectively  phosphorylation site and four casein kinase 2 (CK2) phosphorylation sites. An HSP20 feature alpha crystal domain (ACD) was found at 18–135 aa. PsHSP20 contained one PK phosphorylation site and three CK2 phosphorylation sites, and the ACD was 129–237 aa. PsHSP21 was predicted to contain three N-linked glycosylation sites, one cyclin-dependent kinase (CDK) phosphorylation site, two CK2 phosphorylation sites, and six PKC phosphorylation sites, and an sHSP domain was detected at 116–230 aa. All three PsHSP genes contained the feature domain ACD (HSP20) of the sHSP family, which was the same as the predicted results in NCBI (PsHSP17.8 was a cytosolic (CI) sHSP homologous to sHSP-Class I in other species, while PsHSP21 and PsHSP27.4 were homologous to chloroplast (CP) sHSPs, and both were organelle sHSP genes. The prediction of protein structures showed that PsHSP17.8, PsHSP21, and PsHSP27.4 contained 3, 4, and 4 α-helix structures, 10, 14, and 14 β-sheet structures, 28, 21, and 37 turn structures, and 24, 18, and 25 curvilinear loop structures, respectively (sHSPs contained HSP20 conserved domains with nine conserved β-sheets (β2–β10) at the variable N-terminal and the conserved C-terminal, which were consistent with the α crystallin conserved domains (PsHSP17.8 close to QlHSP17.8 and QlHSP18.1 (Quercus fabri) within the CI clade. Within the CP clade, PsHSP21 was close to CasHSP , while PsHSP27.4 was closest to DzsHSP (Durio zibethinus) in another clade that diverged from PsHSP27.4  was detected using qRT-PCR, which elucidated that the three sHSPs were expressed more in leaves and less in flowers and siliques. In addition, the expression levels of PsHSP21 and PsHSP27.4 in the stem were also higher than those of PsHSP17.8 , PsHSP21 (0.78 ± 0.08 cm), and PsHSP27.4 (1.32 ± 0.09 cm) were 2.1, 1.31, and 2.2 times higher than that of the WT (0.59 ± 0.10 cm), respectively, and the leaf widths of PsHSP17.8 (0.61 ± 0.15 cm) and PsHSP27.4 (0.60 ± 0.08 cm) were also higher than those of PsHSP21 (0.36 ± 0.03 cm) and the WT (0.34 ± 0.04 cm). There was no significant phenotypic difference between the leaves of the overexpressed PsHSP17.8 and PsHSP27.4 lines, but they were both longer than those of the PsHSP21 line , conductivity (18.18 ± 1.00–19.78 ± 1.21), SOD activity (198.18 ± 4.70–209.77 ± 9.73 U·g−1FW), proline content (2.13 ± 0.04–2.57 ± 0.10 μmol·g−1FW), and chlorophyll content (4.95 ± 0.19–5.52 ± 0.02 mg·g−1FM) remained relatively stable in both the WT and the three transgenic lines  and conductivity  of the PsHSP17.8, PsHSP21, and PsHSP27.4 transgenic lines were significantly lower than those of the WT (0.69 ± 0.02 μmol·g−1FW and 86.23 ± 3.10), indicating that the WT experienced more intense membrane lipid peroxidation (−1FW) in the transgenic lines was significantly enhanced compared to MT  compared to the WT  and passion fruit  showed that PeCPK10 could act as an effective heat-tolerant gene to reduce damage and protect organic tissues during heat stress  signaling pathway at high temperatures. Downstream TFs, such as ZATs, WRKYs, and others, can stimulate the expression of antioxidant enzymes to reduce ROS signaling, and multiprotein bridging factor 1 (MBF1) acts as a transcriptional co-activator  in unstressed cells and plays an important role in targeting the chloroplast .The data presented in the study are deposited in the GenBank repository .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Staphylococcus aureus isolates for antibacterial, antibiofilm, and antihemagglutination activities is the aim of this study. Therefore, the potential clinical resistance of the strains was evaluated by their ability to form biofilms, antibiotic resistance, and agglutinate erythrocytes macroscopically and microscopically; besides, the bacterial biofilm was screened for any association with the patient's serum immunoglobulin levels and complements. Despite the effective concentration for C5 and C6 compounds, which is ≤ 31.25 μg/ml, the reduction rate is not concentration-dependent; it depends on the molecular docking of the hydantoin compounds. Hence, the effect of the minimal inhibitory concentrations (MICs) is variable. In this study, the results for the compounds (with the concentration of 31.25–62.5 μg/mL for C5 and 62.5–125 μg/mL for C6) significantly manifest the antibacteria, antibiofilm, and antihemagglutination effects against the virulent strains of S. aureus due to the high percentage of biofilm inhibition that was caused by the new hydantoin compounds. Besides, time-kill kinetics studies showed that these compounds pose bactericidal action. Overall, this study revealed that the new hydantoin derivatives have an interesting potential as new antibacterial drugs through the inhibition of bacterial adhesion. The infections of these isolates activate the complement system through the lectin pathway. Nevertheless, these compounds can be improved in order to be used at even lower concentrations.Although the structure-activity relationship indicates that the 4-thioxoimidazolidin ring is essential for antibacterial activities and pharmaceutical applications, there were no enough studies on the derivatives of this compound. Evaluating the new hydantoin compounds C5 (3-((2-bromobenzylidene) amino)-2- thioxoimidazolidin-4-one) and C6 (3-((4- methoxybenzylidene) amino)-2-thioxoimidazolidin-4-one) that were prepared against clinical  Staphylococcus aureus is one of the crucial pathogenic bacteria that needs to be studied in healthcare or in the community as it is a major cause of both nosocomial and community-acquired infections since it overcomes the immunity and invades deeper tissues; consequently, the infections no longer respond to antibiotics treatment  that were prepared in the BHI broth . S. aureS. aureus cells at a density 1.5 × 108 CFU/ml and new hydantoin compounds C5 and C6 at concentrations of 1×, 4×, 8, and 16× the minimum inhibitory concentration (MIC) was incubated at 37°C. Control was only the broth inoculated with the same cell number of bacteria. Each treatment as well as the control (100 μl) was inoculated onto nutrient agar plates, and measuring colony counts were at 0, 6, 12, and 24 h. The kill measurement and half-maximal inhibitory concentration (IC50) were calculated by plotting the viable colony counts as log10 (CFU/ml) versus time [The nutrient broth medium with sus time .S. aureus biofilm formation, the M.T.P assay was carried out accordingly by [µL BHI broth containing 1% (w/v) glucose followed by inoculation with 20 µL of overnight bacterial culture. To this, 100 µL hydantoin compounds were added in concentrations of 31.25, 62.5, 125, 250, and 500 µg/mL and covered by parafilm along with control (without hydantoin compounds) and incubated at 37°C for 24 h.Then, the wells' content was removed, washed twice with sterile water, and left to dry at room temperature for 15 min. To evaluate the adherence of sessile bacteria, they were stained with crystal violet  and left for 20 min. The excessive stain was removed by deionized water and kept for drying. Furthermore, dried plates were washed with 95% ethanol and then optical density was determined using a microtiter plate reader (BIORAD) at 595 nm. The biofilm inhibition rate (Percentage Inhibition %) was calculated using the formula:To evaluate the efficacy of the new hydantoin derivative compounds in interrupting ingly by , 33 usin% Biofilm Inhibition = 1– ×100 .The activity of the two new hydantoin derivatives on hemagglutination was assessed correspondingly as previously described above . The newStatistical Analysis System - SAS (2012) program was used to analyse the difference between the study factors .Those compounds that were synthesized are described in The formation of the new compounds was confirmed by FTIR.−1, 3244 cm−1, 3402 cm−1, and 3292 cm−1, respectively, denoting υas and υs (NH2) vibrations [υ (C=S) seems to peak at 1093 cm−1 and 1082 cm−1, respectively [In Figures brations , 32, 36;ectively , 38.υ (NH) vibrations appear like weak bands at 3068 cm−1 and 3117 cm −1, respectively [The FTIR peak characters of C5 and C6 compounds appear in Figures ectively .δ (ppm), singlet at 4.06 due to CH2 of imidazolidine, signal at 7.38–7.97 (multiplate for aromatic ring), singlet at 8.59 due to CH=N group, and at 12.07 (singlet for NH) (see δ (ppm), singlet at 2.34 due to the methyl group, singlet at 4.08 due to CH2 of imidazolidine, signal at 6.92–7.91 (multiplate for aromatic ring), singlet at group, and at 11.25 (singlet for NH) (see The 1H-NMR spectrum of the C5 compound shows  NH) see . The 1H- NH) see .S.aureus isolates from nosocomial-infected patients was 20%; all of these isolates were observed to form biofilms by the M.T.P technique. Strong biofilms were formed by 6 and moderate biofilms by 3 (both strong and moderate accounted for 75%). Weak biofilms were seen in 3 (25%) isolates [S. aureus is one of the major virulence factors that contributes to the environmental success of this pathogen in hospitals [S. aureus isolates [The percentage of isolates   from this study were multidrug-resistant with different antibiotic-resistant patterns. Multiple antibiotic resistance patterns were observed for Ceftazidime (CAZ30 µg), Piperacillin (PRL30 µg), Azithromycin, (AZM30 µg), and Amoxicillin/Clavulanic acid (AMC 20/10 µg), while 75% were sensitive to Imipenem (IPM 30 µg) (see Tables µg), Levofloxacin (Lev5 µg), Amikacin (AK 30 µg), Topromycin (TOB5 µg), and Vancomycin (VA 30 µg). The results were determined by describing the resistance of the bacteria (R) and the sensitivity of the bacteria (S). The inhibition zone diameter was measured and compared to that stated in CLSI.All isolated S. aureus vancomycin resistance was 58.33%, and variable levels of vancomycin resistance were indicated. The variability among countries is attributed to the nature of the healthcare system, the method of use, and the number of years used [S. aureus was mainly observed in biofilm producer strains [. aureus clinical isolates can easily acquire the antibiotic resistance gene from the environment [Saphylococcus aureus (VISA) (VRSA), which can cause chronic infections; in addition to that, S. aureus can spread easily in the environment. Therefore, these bacteria bring attention to find new antibacterial therapies [ars used . Antibio strains , 46. In  strains . Moreoveironment , 49. Thuherapies , 51. Accherapies , 2% of pherapies .P > 0.001), while the IgG level was significantly elevated in patients infected with strains (produced and non-produced biofilms) (see P > 0.001), while the levels of complement components (C4) were elevated for both groups (formed and non-formed biofilms)  that binds and stabilizes C3 convertase [S. aureus biofilms escape the immune detection by evading Toll-like receptors (TLRs); TLR4 acts as a ligand for bacterial lipopolysaccharide, and TLR9 for oligonucleotides of cytosine and guanine with phosphodiester backbone (CpG); oligonucleotide and bacterial DNA sugar backbone cause complement activation [β and TLR families is critical to initiate an immune response; IL-1β play a role in T-cell-dependent antibody production and increases the bacterial biofilm formation [S.aureus blocks IgA and inhibits all three pathways of complement activation by producing a superantigen like-protein (SSL7), which binds with the C5 protein complement in the serum [S. aureus biofilm formations, which can be assumed is due to the immune response against S. aureus biofilm infection triggering lectin pathway-dependent C4 turnover, which is initiated by lipoteichoic acid complexes with serum L-ficolin [S. aureus has become extremely problematic in the treatment of infections caused by the strains as they can evade the immune system.The level of IgM and IgA appeared normal in the sera of patients infected with strains (produced and non-produced biofilms) (lms) see . Also, tlms) see . Generalnvertase , 58. Besnvertase –61. S. ativation . The TLRormation –65. In gormation . The immormation . S.aureuhe serum , 69, The-ficolin , 70, and-ficolin , 72. Sub-ficolin . The IgG-ficolin . Althougponents C were ele-ficolin . This waHemagglutinin plays a chief part in the devotion of this organism adhesion to a polymer comprising biomaterials [aterials .  Table aterials . The mecaterials . Hemagglaterials , 44 or materials , the extaterials , 78. Thuaterials , and conS. aureus clinical isolates have been evaluated. The compounds' effective dose that reduced the bacterial growth was from 31.25 μg/ml to 500 3 μg/mL.To assess the clinical potential of the compounds, their effects and antibacterial activity against virulent in vitro against the 12 virulent S. aureus isolates are reported in Tables S. aureus at a concentration of 125 μg/mL; C5 inhibited 7 isolates and C6 inhibited 8 isolates.C5 and C6 compounds showed excellent activity against virulent μg/mL in 2 isolates, and the MIC was 62.5 μg/mL in 3 isolates.The MIC value for compound C5 was 31.25 μg/mL in one isolate, and 62.5 μg/mL in 2 isolates. The MIC was 250 μg/mL in 2 isolatesThe MIC value for C6 compound was 31.25 μg/mL and the MIC 250 μg/mL for C5 was not assessed.For both the compounds, the MICs were 500 Finally, the new hydantoin compounds were not concentration dependent.The MIC value of the new hydantoin derivatives (C5 and C6) Generally, the compounds showed potentiating properties at concentrations from 31 to 125 µg/mL. Thus, these compounds have more potential than previously reported anti-S.aureus activity MIC values –82; in aµg/ml (1X) to 500 µg/ml (16X)) on the growth of bacteria. All treated cultures were reduced in the CFU count of bacteria in comparison with the initial inoculum. The time-kill kinetics of the new hydantoins against the test organisms (S. aureus at test concentrations) showed a reduction in the number of viable cells over the 6, 12, and 24 hours compared to the control. Furthermore, the resultant effect of the compounds from incubating the bacteria at 1, 2X MICs was a rapid reduction in the average of the 7log10 reduction (99.99% inhibition). The reduction in the cell counts between 12 and 24 h of incubation period showed that the compounds were highly bactericidal. Regarding the C5 compound, the bacterial colonies were totally wiped out after incubating for 6 h at concentration of 8, 16X MICs and for 12h at 4X MICs, and the IC50 of the compound was 46.7892 ± 2.51e + 04 (5.365e + 04%). With the C6 compound, bacterial colonies were totally wiped out after incubation for 6, 12, and 24 h at the concentration of 16X, 8X, and4 MICs, respectively, and the IC50 of the compound was 55.0085 ± 4.758e + 04 (8.649e + 04%). The results of the time-kill profiles for the tested bacteria and antibacterial assays determined that time-kill depends on the compounds' molecular docking.The data demonstrated in Figures 9The antibiofilm activity of the compounds, C5 and C6, were determined toward six strains, selected in the present study due to their strong biofilm-forming ability.μg/mL in one isolate, followed by 91.63% and 90.74% at 31.25 μg/mL, while two isolates had an inhibition close to 89% at 62.5 μg/ml  between the inhibition rate of biofilms and untreated biofilms. The biofilm formation decreased significantly as compared with untreated biofilms of isolates 9 and 8 at concentration 62.5 μg/mL, and isolate 11 at 31.25 μg/mL. There were no significant differences in the biofilm inhibition of C5 concentration of isolates 2, 6 at 62.5 μg/mL, and isolate 3 at 31.25 μg/mL.The highest level of inhibition was 99.19% at 62.5 g/ml see . The lowμg/mL and 62.5 μg/mL  at 125 g/mL see ; howeverα error of 0.05. Also, the results showed that the effect value of subMICs was different in compound C6 against S. aureus isolates, which ranged from 62.5 µg/mL to 125 µg/mL. Hence, the new hydantoin compound was not concentration dependent. Moreover, the results illustrated a significant difference (P < 0.05s) between the inhibition rate of treated biofilms and untreated biofilms. Consequently, the potential of exhibiting antibiofilm activity against biofilms of S. aureus is more by these compounds compared to the previous reports (it inhibited 40% S. epidermidis ATCC 12228 biofilm) [This was validated by ANOVA with a significance level of 95% and mean comparison with an biofilm) . So far,µg/mL concentrations, while C6 showed effects in the range of 62.5–125 µg/mL concentrations. However, the compounds' concentration of the hemagglutination reduction was not uniform, which was determined using six biofilm strong formation strains that were selected in the present study based on their subMIC biofilm inhibition ability. S. aureus strain number 6 has no antihemagglutination effect at a concentration 125 µg/mL in the presence of compound C6, which may be due to the inhibition effect that depends on appropriate compound structure with bacterial strain type. Erythrocytes' hemagglutination is an essential character of S. aureus pathogenesis, which is associated with biofilm creation adherence and it is critical for human bacteremia [S. aureus express binding proteins, fibronectin and fibrinogen, and the clumping factor responsible for bacterial attachment, blood clumping, intravascular hemolysis, and damaged tissue [In this study, the C5 and C6 hydantoin compounds evidenced hemagglutination inhibition against five of the six virulent bacteria , quinolones, verapamil, and omeprazole, in addition to biocides, dyes, quaternary ammonium compounds, and antiseptics [S. aureus MICs, and were referred to interreact with the resistance mechanisms, which are mediated mostly by efflux pumps [Moreover, MRSA uses 30 efflux pumps that encode chromosomally and on plasmids to resist antibiotics. Particularly, the NorA efflux pump in iseptics –92. Theriseptics –98. The iseptics –98 showeiseptics . Other siseptics , 100. Thux pumps . Additioux pumps  the studStaphylococcus aureus biofilm-producing strains produce extracellular polysaccharides that play an important role in the recognition by immunoglobulins to activate complement factors and elicit strong hemagglutination that consequently becomes more difficult to be treated with antibiotics.The results demonstrate that all the tested compounds (C5 and C6) prevented biofilm formation, but their effectiveness varied. Overall, all the compounds were able to strongly prevent hemagglutination; besides, the effectiveness of the compounds has not depended on concentration.  noted th


Results shown herein are focused on this RD. Two patients had DLTs . Grade ≥ 3 hematological toxicity  was manageable and did not result in treatment discontinuation. Transient and manageable grade 3 ALT increase (26%) was the most common biochemical abnormality. At the RD cohort, overall response rate was 22.2% , including one stringent complete response, one very good partial response, and two partial responses in r/r patients to BTZ and/or lenalidomide. The clinical benefit rate was 77.8% . No major pharmacokinetic drug–drug interaction was found. In conclusion, the triple combination of plitidepsin, BTZ, and DXM showed an acceptable safety profile and had moderate activity in adult patients with r/r MM.Previous studies showed antitumor activity for plitidepsin plus dexamethasone (DXM) in relapsed/refractory multiple myeloma (r/r MM), and in vitro synergism with bortezomib (BTZ) or DXM against MM cells. This phase I trial evaluated plitidepsin (3‐h intravenous infusion Day 1 and 15), BTZ , and DXM , every 4 weeks in 36 r/r MM patients. Twenty‐two patients were treated using a standard dose escalation design (10 at the recommended dose [RD] cohort), and 14 additional patients were treated to expand the RD cohort. No dose‐limiting toxicities (DLTs) occurred during dose escalation. The highest dose level evaluated  and dexamethasone (DXM) in relapsed/refractory multiple mieloma patients (r/r MM). This triple combination of plitidepsin, BTZ, and DXM showed an acceptable safety profile and had moderate activity in adult patients with r/r MM that have failed to standard common therapeutic options, including BTZ and lenalidomide. The study protocol was approved by the Independent Local Ethics Committee of each center. Signed written informed consent was obtained for each patient prior to any procedure.2.19/L; hemoglobin ≥8.0 g/dL; absolute neutrophil count ≥1.0 × 109/L ; and adequate hepatic and renal function. Patients pretreated with BTZ or another proteasome inhibitor could be included if they had achieved a minor response (MR) >2 months at least once .Inclusion criteria were age ≥ 18 years; r/r MM diagnosed according to Durie‐Salmon criteria; ≥1 previous treatment lines; recovery from previous toxicities to grade ≤ 1 ; Eastern Cooperative Oncology Group performance status score ≤ 2; normal left ventricular ejection fraction (LVEF); platelet count ≥50 × 102.2Treatment consisted of escalating doses of plitidepsin 3‐h intravenous (i.v.) infusion Day 1 and 15, and BTZ subcutaneous bolus injection Day 1, 4, 8, and 11, with fixed DXM dose 40.0 mg orally Day 1, 8, 15, and 22, all every 4 weeks (q4wk), for a maximum of 8 cycles. If response or disease stabilization was observed, treatment after Cycle 8 could continue with plitidepsin and DXM at the same doses. Antiemetic prophylaxis was given to all patients, consisting of ondansetron 8 mg i.v. or equivalent, diphenhydramine hydrochloride 25 mg i.v. or equivalent, and ranitidine 50 mg i.v. or equivalent administered 30 min before each plitidepsin infusion. If necessary, 10 mg of metoclopramide every 8 h could be administered after the end of plitidepsin infusion or the duration of treatment with serotonin antagonists could be extended.2.32) was 80% of the RD for plitidepsin alone as a 3‐h i.v. infusion Day 1 and 15 q4wk.2) was about 80% of the approved dose (1.3 mg/m2) in relapsed MM as a twice‐weekly schedule, alone or in combination with DXM.Dose escalation followed a standard 3 + 3 design. The starting plitidepsin dose  and laboratory abnormalities were graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI‐CTCAE) v.4. Antitumor activity was evaluated according to the International Myeloma Working Group (IMWG) criteria.2.5Sixteen samples were collected from each patient to quantify plitidepsin whole blood and BTZ plasma concentrations at baseline and at different times 1 week after the first infusion. In patients treated at the RD, 15 additional samples were taken 1 week after the second plitidepsin infusion.2.6Continuous variables were presented with summary statistics and categorical variables in frequency tables. Time‐to‐event variables were calculated using Kaplan–Meier approach. Binomial exact distribution was used to calculate 95% confidence intervals (95%CIs) for categorical variables. Individual PK parameters were tabulated. Statistical analyses were performed using SAS v.9.2, (SAS Institute Inc), and PK non‐compartmental analysis (NCA) was performed using Phoenix WinNonlin v.6.3.33.12, BTZ 1.0 mg/m2 and DXM 40.0 mg), four at DL2 , and 10 at DL3 (recommended dose [RD] for this combination)  plus 14 additional treated patients to expand the RD cohort. An additional three patients at the RD cohort were included but never treated because they unmet the required criteria before starting treatment on Cycle 1 Day 1. Twelve of the 22 treated patients at the dose escalation stage were evaluable for the determination of the RD .A total of 39 patients were enrolled between June 2014 and August 2018. Of these, 36 patients were treated at three dose levels: eight at DL1 , 19 (49%) had previously received hematopoietic stem cell transplantation (HSCT)  ; 111 of these cycles were given at the RD cohort, with a median of 2 cycles per patient . Median relative dose intensity at the RD was 73% for both plitidepsin and BTZ, and 68% for DXM.3.2Twelve of the 22 patients treated at the escalation stage were evaluable for dose‐limiting toxicities (DLTs).The other10 patients were non‐evaluable for RD. Six of these patients did not receive a complete Cycle 1 . Of the other four patients, two received incorrect BTZ or DXM doses at Cycle 1, one received treatment on Days 4 and 11 despite not fulfilling treatment criteria, and one had no laboratory tests done in Cycle 1.2, and DXM 40.0 mg) was defined as the RD for this combination, and then 14 additional patients were treated to expand the RD cohort.No DLTs occurred during dose escalation. Dose escalation beyond DL3 was not allowed by the study protocol; hence, DL3  had DLTs during Cycle 1; these DLTs consisted of grade 3 diarrhea, and a combination of grade 3 nausea and grade 3 vomiting refractory to antiemetic therapy, in one patient each.3.3n = 3 patients; 8%), diarrhea and vomiting , and constipation, nausea, plitidepsin extravasation, hypersensitivity, and respiratory tract infection .The most common treatment‐related AEs found among the 36 patients treated at all dose levels were nausea (36% of patients), fatigue (28%), diarrhea (25%), and peripheral neuropathy and vomiting (19% each). Most of these AEs were grade 1/2. Grade ≥ 3 treatment‐related AEs at all dose levels comprised fatigue , diarrhea (25%), fatigue (21%), and vomiting (17%). Most of these AEs were grade 1/2  at all dose levels was anemia . Most cases were grade 1/2. Grade ≥ 3 anemia was found in 11 of 36 treated patients (31%). Thrombocytopenia was observed in 86% of patients reaching grade ≥ 3 in 17 patients. Neutropenia was found in 47% of patients with grade ≥ 3 in five patients. No episodes of febrile neutropenia were reported.At the RD cohort, grade ≥ 3 anemia was present in eight of 24 treated patients . Thrombocytopenia was found in 83% of patients with grade ≥ 3 in 11 patients . Neutropenia was found in 54% of patients, with grade 3 in four patients  at baseline, including six of 11 with grade 3/4 thrombocytopenia, three of four with grade 3 neutropenia, and seven of eight with grade 3/4 anemia. No treatment discontinuations, cycle delays, or dose reductions occurred at the RD due to hematological toxicity, which was managed with dose omissions.The most frequent biochemical abnormality regardless of relationship was alanine aminotransferase (ALT) increase (94%) at all dose levels, followed by aspartate aminotransferase (AST) increase (80%), creatine increase (71%), and creatine phosphokinase (CPK) increase (35%). Most transaminase increases at all dose levels were grade 1/2, and none reached grade 4. Grade 1/2 ALT increases and AST increases were reported in 26 patients each (46% of patients had grade 1 ALT increase and 57% had grade 1 AST increase). Grade 3 ALT increases were reported in seven patients (20%) and grade 3 AST increase occurred in two patients (6%).Among the 24 patients treated at the RD cohort, the most frequent biochemical abnormality regardless of relationship was ALT increase (91%), followed by AST increase (83%), creatine increase (68%), and CPK increase (36%). Most of these abnormalities at the RD cohort were grade 1/2 and none reached grade 4; grade 3 were ALT increase in six patients, and grade 3 AST increase, creatinine increase and CPK increase in one patient each . Six patients had dose reductions in 7 cycles. Of these, five dose reductions occurred due to toxicity, with four of them involving plitidepsin  and only one involving BTZ (grade 1 paresthesia). Two dose reductions were due to reasons unrelated to treatment. Ten patients required packed red blood cell transfusions and six were given platelet transfusions.3.4Twenty‐eight patients treated at all dose levels (18 at the RD cohort) were evaluable for efficacy as per IMWG criteria. Eight patients were not evaluable because they did not receive at least one complete cycle. Overall, 10 patients showed PR or better, including two sCRs, one CR, three VGPRs, and four PRs . The clinical benefit rate was 82.1%  . Both PRs were observed in refractory patients . The clinical benefit rate was 77.8%   and the median PFS was 4.1 months . At the RD cohort, the median DoR was 12.3 months  and the median PFS was 2.8 months .3.5Vss) for plitidepsin at DL1, DL2, and DL3 were 5.67, 5.59, and 9.41 L/h and 169, 192, and 341 L, respectively (Table max) were 11.0, 16.9, and 17.0 μg/L, respectively; for total clearance after extravascular administration (CL/F) they were 23.4, 17.9, and 24.9 L/h; and for volume of distribution at terminal phase (Vz/F) they were 1308, 1208, and 1658 L. The reference for BTZ PK results was a phase I study in patients with MM.All patients were sampled for PK analysis, but only 17 and 18 of them were suitable for NCA of plitidepsin and BTZ, respectively. Mean total body clearance (CL) and volume of distribution Vss for pl42 on Day 1 and 15, BTZ 1.3 mg/m2 on Day 1, 4, 8, and 11, and DXM 40.0 mg on Day 1, 8, 15, and 22 q4wk. This triple combination was generally well tolerated, with no DLTs observed during dose escalation. At the expanded cohort, two patients had DLTs: one with grade 3 diarrhea, and one with grade 3 nausea/vomiting refractory to antiemetic therapy.The RD for phase II studies for this triple combination was plitidepsin 5.0 mg/m2 on Day 1 and 15 q4wk plus DXM in phase II and III trials.The most common hematological abnormality  was anemia, which occurred in all patients (grade ≥ 3 in 33% at the RD). Most cases were grade 1/2, and at the RD the majority of patients with grade ≥ 3 anemia while on treatment already had grade 2 anemia at baseline. The most common severe hematological toxicity at the RD was thrombocytopenia, which was grade ≥ 3 in half of patients with most of them (6 of 11) already having grade ≥ 2 thrombocytopenia at baseline. Lower rates of severe thrombocytopenia (21%–22%) have been previously observed in patients with r/r MM treated with the double combination of plitidepsin 5.0 mg/mThe most common biochemical abnormality was transaminase increases. Grade 3 ALT increase was most frequent at the RD (26% of patients), but it was transient and manageable with dose delays, omissions, or reductions.Treatment‐related AEs at the RD for this triple combination were mostly mild/moderate; grade 3 diarrhea, nausea, vomiting, or fatigue were observed in less than 10% of patients. Severe muscular toxicity was not observed with the triple combination in this study. In contrast, incidences of 5.4% and 3.6% were reported for severe myalgia and muscular weakness, respectively, in MM patients treated with plitidepsin plus DXM.At the RD cohort, only three patients discontinued treatment due to treatment‐related events, thereby suggesting an acceptable safety profile for plitidepsin combined with BTZ and DXM in this heavily pretreated r/r MM population .This triple combination showed modest antitumor activity  in this population of heavily pretreated r/r MM patients, most of them having received BTZ and with disease refractory to the last prior therapy. Comparing the efficacy of the 18 evaluable patients during the escalation stage with an ORR of 55.6% ,The antitumor activity found in patients treated with the combination of plitidepsin, BTZ, and DXM  was higher than that observed in the ADMYRE trial evaluating plitidepsin plus DXM (ORR: 13.8%),Results of the VISTA trial were further supported by those of phase II trials: RETRIEVE (ORR: 40%) and SUMMIT , and of the phase III APEX trial (crossover from DXM to BTZ), in which the overall and complete response rates with BTZ were 43% and 9%, respectively.Of note, the median DoR  was longer than that observed with most other BTZ and DXM‐containing combinations, including panobinostat (6.0 months),2 administered subcutaneously and with the same sampling schedule duration of 72 hours.The similarity of PK parameters found in the present study compared to reference values, and the lack of overlapping metabolic routes of plitidepsin and BTZ, suggest that no major PK interaction between these two drugs is expected. Compared to the values reported herein, patients with MM treated with plitidepsin in other trials had a similar median CL (4.4 L/h) and a somewhat larger median peripheral volume 513 L) (unpublished data). The plitidepsin concentrations found in this study fitted within the 90% prediction interval simulated for patients with MM, although most were above the 50th percentile for the typical values expected during the first 48 hours after dosing  in patients with r/r MM that have failed to standard common therapeutic options, including BTZ and lenalidomide.In summary, the triple combination of plitidepsin 5.0 mg/mMaría Victoria Mateos: Conceptualization, Investigation, Resources, Writing—original draft, Writing—review & editing. Felipe Prosper: Investigation, Resources, Writing—review & editing. Jesús Martin Sánchez: Investigation, Resources, Writing—review & editing. Enrique M. Ocio: Methodology, Investigation, Resources, Writing—review & editing. Albert Oriol: Investigation, Resources, Writing—review & editing. Cristina Motlló: Investigation, Resources, Writing—review & editing. Jean‐Marie Michot: Investigation, Resources, Writing—review & editing. Isidro Jarque: Investigation, Resources, Writing—review & editing. Rebeca Iglesias: Investigation, Resources, Writing—review & editing. María Solé: Investigation, Resources, Writing—review & editing. Sara Martínez: Conceptualization, Methodology, Writing—original draft, Writing—review & editing, Supervision. Carmen Kahatt: Conceptualization, Methodology, Writing—review & editing, Supervision. Salvador Fudio: Conceptualization, Methodology, Formal analysis, Writing—original draft, Writing—review & editing, Supervision. Gema Corral: Methodology, Formal analysis, Writing—review & editing. Ali Zeaiter: Methodology, Writing—review & editing, Supervision. Lola Montilla: Methodology, Writing—original draft, Writing—review & editing. Vincent Ribrag: Investigation, Resources, Writing—review & editing.The study was funded by Pharma Mar, S.A.María Victoria Mateos has received honoraria for lectures and participation in advisory boards from Janssen, Celgene, Takeda, Amgen, GSK, AbbVie, Pharma Mar, Pfizer, Regeneron, Roche, Sanofi, Oncopeptides, and Seagen. Enrique M. Ocio has received honoraria or consultation fees from Janssen, BMS/Celgene, Sanofi, GSK, Oncopeptides, Takeda, Amgen, Pfizer, Karyopharm, and Pharma Mar. Jean‐Marie Michot has declared Principal/sub‐Investigator role of clinical trials from AbbVie, Agios, Amgen, Argen‐x, Astex, AstraZeneca, Beigene, Blueprint, BMS, Boeringer Ingelheim, Celgene, Chugai, Clovis, Daiichi Sankyo, Debiopharm, Eisai, Eos, Exelixis, Forma, Gamamabs, Genentech, Gortec, GSK, H3 biomedecine, Incyte, Innate Pharma, Janssen, Kura Oncology, Kyowa, Lilly, Loxo, Lysarc, Lytix Biopharma, Medimmune, Menarini, Merus, MSD, Nanobiotix, Nektar Therapeutics, Novartis, Octimet, Oncoethix, Oncopeptides AB, Orion, Pfizer, Pharmamar, Pierre Fabre, Roche, Sanofi, Seattle Genetics, Servier, Sierra Oncology, Taiho, Takeda, Tesaro, and Xencor. He has also received personal fees such as honoraria, royalties or fees for consulting, lectures, speakers bureau, expert testimony, employment, advisory boards, among others from Roche, AstraZeneca, and Amgen. In addition, he has also received non‐financial support such as, equipment supplied by the entity, travel paid by the entity, writing support, administrative support, among other from Celgene, Bristol‐Myers Squibb, and GSK. Isidro Jarque has declared speakers bureau and consultancy role by AbbVie, Amgen, AstraZeneca, Bristol‐Myers Squibb, Gilead, Janssen, Novartis, Pfizer, Roche and Takeda. Carmen Kahatt, Salvador Fudio, Gema Corral and Ali Zeaiter report personal fees for salary as full time employee and stock ownership from Pharma Mar, outside the submitted work. Sara Martínez and Lola Montilla report personal fees for salary as full time employee from Pharma Mar, outside the submitted work. No disclosures were reported by the other authors.Appendix S1Click here for additional data file.


Sanghuangprous vaninii is a wood-inhabiting fungus, and its mycelium and fruiting body show excellent medicinal values. Mulberry is one of the major hosts of S. vaninii, however, the mechanism of mulberry affecting the growth of S. vaninii has not been reported. In the present study, a mulberry-inhabiting strain of S. vaninii was selected to explore the effects of mulberry branch extracts (MBE) on the growth of the strain. Results showed that MBE could significantly promote the growth of S. vaninii mycelium at the concentration of 0.2 g/l. After 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium was higher by three times compared with that in the control. The non-targeted metabonomic analysis of the culture medium at different culture times and concentrations was conducted to find the key components in MBE that promoted the growth of S. vaninii mycelium. Under the different concentrations of MBE culture for 10 and 16 days, 22 shared differential metabolites were identified. Next, in accordance with the peak value trend of these metabolites, HPLC–MS and liquid culture validation, four components derived from MBE  could significantly increase the growth rate of mycelium at the concentration of 2 mg/l. Transcriptomic and qRT-PCR analyzes showed that MBE could upregulate hydrolase-related genes, such as serine–glycine–asparaginate–histidine (SGNH) hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase, glycosyl hydrolase family 61, cerato-platanin protein and Fet3, which might enhance the nutrient absorption ability of S. vaninii. Importantly, MBE could significantly increase the content of harmine, androstenedione and vesamicol, which have been reported to possess various medicinal effects. Results suggested that MBE could be an excellent additive for liquid culture of S. vaninii mycelium, and these hydrolase-related genes also provided candidate genes for improving the nutrient absorption capacity of S. vaninii. Sanghuangprous vaninii (Ljub.) L.W. Zhou and Y.C. Dai., formerly named Phellinus gilvus (Schwein.) Pat, is an important wood-inhabiting fungus that has been widely utilised in traditional medicine in China and adjacent countries  for 2 h. The filtered aqueous extracts were added with absolute ethanol at a ratio of 1:3. The supernatant after centrifugation was concentrated to 1/5 volume by rotary evaporator  and lyophilised by vacuum freeze drier  to obtain MBE powders. The dried powders were stored at −20°C before use.The non-targeted metabonomic analysis was conducted by Guangzhou Genedenovo Biotechnology Co., Ltd. (China). The mycelia (M) and culture media (CM) at different sampling times were collected. Mycelia (100 mg) were washed thrice by PBS buffer and ground with liquid nitrogen, and the homogenate was resuspended with prechilled 80% (v/v) methanol and 0.1% (v/v) formic acid by well vortex. CM (1 ml) were freeze-dried and resuspended with prechilled 80% (v/v) methanol and 0.1% (v/v) formic acid by well vortex. Samples were incubated on ice for 5 min and centrifuged at 15000 g and 4°C for 15 min. A certain amount of the supernatant was diluted to final concentration containing 53% (v/v) methanol by LC–MS-grade water. Samples were centrifuged at 15000 g and 4°C for 15 min. Finally, the supernatant was injected into the HPLC-MS/MS system .HPLC-MS/MS analyzes were performed using the Vanquish UHPLC system  coupled with the Orbitrap Q ExactiveTMHF-X mass spectrometer  in Guangzhou Gene Denovo Co., Ltd. (China). Samples were injected onto the Hypesil Gold column  by using a 17 min linear gradient at a flow rate of 0.2 ml/min. The eluents for the positive polarity mode were eluents A  and B (methanol). The eluents for the negative polarity mode were eluents C  and D (methanol). The solvent gradient was set as follows: 2% B, 1.5 min; 2–100% B, 12.0 min; 100% B, 14.0 min; 100–2% B, 14.1 min and 2% B, 17 min. The Q ExactiveTM HF-X mass spectrometer was operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320°C, sheath gas flow rate of 40 arb and aux gas flow rate of 10 arb.t-test were used to screen significantly different metabolites between different comparison groups, and the threshold of significant difference was as follows: VIP ≥ 1 and t-test p < 0.05  to perform peak alignment, peak picking and quantitation for each metabolite. The main parameters were set as follows: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal/noise ratio, 3 and minimum intensity, 100,000. After that, peak intensities were normalised to the total spectral intensity. Normalised data were used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. Peaks were matched with the mzCloud,p < 0.05 .Q-value ≤20) bases. The HISTAT2.2.4 was used to map clean reads to the genome with default parameters  following the manufacturer’s instructions. RNA integrity, purity and concentration were assessed using the 2,100 Bioanalyzer , NanoPhotometer spectrophotometer  and Qubit 2.0 fluorometer , respectively. The construction of libraries and the RNA-Seq on the Illumina sequencing platform were performed by Guangzhou Genedenovo Biotechnology Co., Ltd. Raw reads were trimmed to remove adaptors and enhance quality by fastp  and SYBR® Fast qPCR Mix  were used for the CFX96 real-time PCR system . All operations were performed in accordance with the manufacturer’s instructions. The thermocycling conditions consisted of initial denaturation at 95°C for 30 s followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. β-Actin was used as internal reference gene, and gene expression was quantified using the comparative 2q method . PCR pripost-hoc test. p < 0.05 indicated a significant difference.Data were expressed as mean ± SD. Statistical analysis was performed using the SPSS 16.0 software . One-way ANOVA was used to analyze statistical differences between groups under different conditions followed by Tukey’s S. vaninii mycelium were observed. As shown in S. vaninii mycelium, and the mycelium became dense. At the concentration of 0.2 g/l, MBE did not inhibit the expansion of mycelium and could significantly increase the fresh weight of mycelium on PDA plate . As shown in S. vaninii mycelium.The effects of different concentrations of MBE on the growth of DA plate . Next, wS. vaninii mycelium. A total of 2,229 differential metabolites, including 1,509 in positive ion mode and 720 in negative ion mode, were identified. Under the different concentrations of MBE culture for 10 days  vs. control group culture for 10 days (CM10d_0), respectively, and CM16d_1, CM16d_2 and CM16d_5 vs. control group CM160, respectively. A total of 22 shared differential metabolites in the six groups above, including 17 in positive ion mode and 5 in negative ion mode  and concentrations  was conducted to find the key components in MBE that promoted the growth of ion mode . Next, aion mode , the peascreened . To confS. vaninii mycelium were verified. Three concentrations  were set to check the effects on growth rate and fresh weight of S. vaninii mycelium on PDA. At concentrations of 2 mg/l, scopoletin, kynurenic acid, 3,5-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and salicylic acid showed better growth potential than other concentration gradients. However, the growth rate of mycelium showed no difference from that of PDA, but the fresh weight was significantly higher than PDA except salicylic acid  was conducted. According to the growth trend of mycelium, the parameters of screening differential expression genes (DEGs) were as follows: all upregulated DEGs in M10d_1 vs. M10d_0, M10d_2 vs. M10d_0, M16d_1 vs. M16d_0, M16 d_2 vs. M16 d_0 and M16 d_0 vs. M10 d_0  hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase and glycosyl hydrolase family 61 genes showed high expression levels at M16d_0 than M10d_0, whereas Fet3 and Cytochrome oxidase complex assembly protein 1 (COA1) were downregulated at M16d_0 compared with M10d_0  was carried out. KEGG analysis showed that DEGs were enriched in the 2-oxocarboxylic acid metabolism pathway and biosynthesis of amino acids pathway , and difS. vaninii has the characteristics of slow growth and weak competitiveness, and the improvement of growth rate of mycelium can largely overcome these shortcomings. In the present study, the extracts from host of S. vaninii can significantly promote the growth of S. vaninii mycelium in liquid culture. S. vaninii mycelium at the concentration of 0.06 g/l. Findings imply that MBE may have similar effects. In the present study, after 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium reaches 1.82 g per 300 ml, which is thrice that of the control. Metabonomic analysis is conducted to identify key active compounds in MBE. Components derived from PDA are excluded by screening the differential shared metabolites of CM10d_0 vs. CM10d_1, CM10d_0 vs. CM10d_2, CM10d_0 vs. CM10d_5, CM16d_0 vs. CM16d_1, CM16d_0 vs. CM16d_2 and CM16d_0 vs. CM16d_5 . PI is an important secondary messenger that can affect diverse cellular processes, including protein transport, cell polarity, cytoskeletal organisation, ion-channel function and gene expression  liquid culture may possess improved effects. Androstenedione, a steroidal hormone, is thought to be an enhancer for athletic performance, build body muscles, reduce fats, increase energy, maintain healthy red blood cells and increase sexual performance (S. vaninii mycelium has been reported. In the MBE (0.2 g/l) group, vesamicol, a selective vesicular acetylcholine transporter inhibitor, increases, whereas acetylcholine decreases, implying that the mycelium in MBE (0.2 g/l) group can also treat cholinergic dysfunction-associated disorders. Pearson correlation analysis showed that harmine has strong positive correlation (>0.99) with 58 genes. Except several hydrolase family genes, majority of genes are cytochrome p450s and genes of unknown function  can remarkably increase the contents of harmine, androstenedione and vesamicol by more than 10-fold . Harmineformance . No relefunction , which mS. vaninii mycelium at the concentration of 0.2 g/l and identified four key active compounds in MBE, which were primarily responsible for the growth-promoting effects. In addition, MBE promoted the mycelial growth by upregulating hydrolase-related genes. Finally, MBE could significantly increase several bioactive ingredients in mycelium. Results suggested that MBE is an excellent additive for the liquid culture of S. vaninii mycelium.In the present study, MBE could promote the growth of https://doi.org/10.6084/m9.figshare.20524158.v1.The raw RNA-seq data for this study can be found in the NCBI database – BioProject ID: PRJNA871986. The non-targeted metabonomic datasets can be found in Figshare – YL and SZ conceived and designed the study. JH performed the experiments with the help of YS, MP, HM, TL, and ZL. YL, SZ, and JH analyzed the data and prepared the manuscript. All authors contributed to the article and approved the submitted version.This work was supported financially by Science and Technology Department of Zhejiang Province (LQ21C150002 and 2018C02003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1024987/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.Click here for additional data file.Click here for additional data file.


HAECs were evaluated by measuring their entrapment efficiency (EE%), particle size (PS), and polydispersity index (PDI). Optimal hyaluronic acid enriched cerosomes (OHAECs) were subjected to further in vitro and ex-vivo and in-vivo studies. The in silico study concluded better interactions between SP and phosphatidyl choline in presence of hyaluronic acid (HA) and high stability of their binding in water. The prepared HAECs had acceptable EE%, PS, and PDI values. The statistical optimization process suggested OHAEC containing 10.5 mg ceramide III and 15 mg HA, utilizing Kolliphor® RH40. OHAEC had EE% and PS of 89.3 ± 0.3% and 261.8 ± 7.0 nm, respectively. OHAEC was stable for up to 3 months. It also showed a mixed tubular and vesicular appearance under transmission electron microscope. The ex vivo and in vivo studies concluded better skin deposition and accumulation of SP from OHAEC. The histopathological study demonstrated the safety of OHAEC for topical application. Therefore, OHAEC could be considered as effective system for topical application of SP to manage hirsutism, with prolonged action, coupled with minimized side effects.Spironolactone (SP) is a potassium sparing diuretic with antiandrogenic properties. This study aimed at formulating SP into hyaluronic acid enriched cerosomes (HAECs) for topical management of hirsutism. HAECs were prepared by ethanol injection method, according to D-optimal design, after a proper  The aim of this study was to formulate and evaluate hyaluronic acid enriched cerosomes (HAECs) that are nanostructures composed of ceramides, phospholipid, hyaluronic acid and edge activator (Kolliphor RH40 or Kolliphor EL). Each of these components had one or more function. In more details, ceramides may provide protective activity and skin hydration. Addition of edge activators in the formulations enhances the drug deposition and aids in the production of highly stable unaggregated vesicles of the double lipidic, phosphatidylcholine–ceramide mixture. Both components are expected to augment the topical effect of SP. Hyaluronic acid was used to enhance the nanovesicles bioadhesion. In silico studies are computer aided studies used to predict the interaction between molecules and their behavior in different media, respectively. So, this study was targeted to formulate SP-HAECs, that might have acceptable dermal deposition profile for topical management of hirsutism.Hirsutism is a common disorder in females, usually linked to polycystic ovarian syndrome and other causes. It is characterized by the abnormal presence of hair in unwanted areas . Hyaluronic acid (HA), kolliphors RH 40 and EL were purchased from Acros Organics, Belgium. L-α phosphatidyl choline (PC) was purchased from Sigma Aldrich, USA. Ceramide III was provided by Evonik Co. (Germany). Chloroform, ethanol, and methanol were purchased from El-Nasr Pharmaceutical Company, Cairo, Egypt.2.2.2.2.1.in silico study used to predict the binding of SP and PC in presence of HA using a molecular docking study. Additionally, the thermodynamic stability of SP-PC-HA (SPH) system was investigated throughout an all-atom MDS to choose suitable solvent(s) for preparation of HAECs and predict their stability in water. The molecular docking workflow involved 3 D construction of SP, PC, and HA using the builder module within the Molecular Operating Environment (MOE) software . Partial charges were assigned, and minimization was proceeded using the Amber10: EHT forcefield to an energy gradient of 0.1 RMS Kcal mol−1 A−2. SP and HA were docked on the PC. The docked ligand conformations were developed through bond rotation, lodged within in the active site guided by triangular-matching approach, then conformations were ranked via the London_dG scores. The top ten docked poses (n = 10) were retained for refinement then an energy minimization stage, within the target pocket, before they were rescored by Generalized-Born solvation-VI/Weighted-Surface Area_dG (GBVI/WSA) force field scorings. The MOE incorporated scoring system relies on Coulomb’s electrostatics using protein-ligand van der Waals score, current-loaded charges, exposure-weighted surface area, and solvation electrostatics  as well as favored polar and hydrophobic binding interaction. The cutoff values for hydrogen bond  distance and angle were assigned at 3.0 Å and 20°, respectively  containing water, chloroform, or both ethanol and chloroform using the explicit Amber10:EHT forcefield. The periodic boundary conditions were applied in all dimensions maintaining a 10 Å non-bounded cutoff distances. The composition of each established system is presented within An 2.2.2.® software    to studylis, MN) .2.2.3.SP-HAECs were prepared using ethanol injection method . The EE % was calculated by the direct method , followed by lysis using methanol. The UV absorbance was measured using spectrophotometer , at the predetermined 2.2.5.Zetasizer  was used to measure the PS and PDI. The formulae were diluted before measurement  V.17 at p < 0.05 before and after storage.OHAEC formula was stored for 3 months in refrigerator. The formula was observed for changes in visual appearance. EE%, PS, ZP, and PDI were compared using Student 2.2.7.2.Transmission electron microscope (TEM) was utilized for the examination of the morphology of optimal hyaluronic acid enriched cerosomes (OHAECs). The formula was diluted then deposited in a carbon-coated copper grid and stained by 2% w/v phosphotungestic acid  at 37 ± 0.5 °C stirred at 100 rpm. Samples of 1 mL were withdrawn at 1, 2, 4, 6, 8 and 24 h and then analyzed using HPLC. The cumulative amounts permeated per unit area were calculated  were obtained  , with an average age of 7 weeks were used. The study protocol was approved by Research Ethics Committee (REC) for experimental and clinical studies at Faculty of Pharmacy, Cairo University. For the administration of SP suspension and OHAECs through 2.2.9.2.The rats were divided into three groups with two rats each. Group I acted as control. Rats in group II were subjected to SP suspension, while rats in group III had OHAECs applied to their skin, three times daily for 1 week. The rats were then sacrificed, and the skin was excised. Skin samples were fixed in 10% formaline, embedded in paraffin. Sections from the paraffin blocks were cut using a microtome . The obtained sections were stained, then examined by light microscope . AUC0–10 for both treatments was compared using student t-test by SPSS® software 22.0.Animals were divided into 2 groups of 18 animals each. One group received SP suspension and the other obtained the OHAECs topically. Half mL of each test formula  was applied to rat skin. After treatment, three animals from each group were sacrificed at 1, 2, 4, 6, 8, and 10 h. The excised skin was cut into pieces and sonicated in 5 mL methanol for 30 min. The extract was then filtered, and the concentration of SP was determined by HPLC. AUC3.3.1.n = 5), only single polar interaction (hydrogen bonding) was depicted toward the PC since the latter only furnish a single hydrogen bond donor at its phosphate functionality (–OPO(O)OH). The latter predicted docking pose came in good agreement with reported literatures showing preferential binding of several drugs: including metformin and rosuvastatin, at the phosphate head of the PC molecule  and the carbonyl group of the hydrophobic acyl tail (2.1 Å/135.8°). An additional polar interaction was furnished between the HA C6-OH at the N-acetyl-d-glucosamine scaffold and oxygen atom of the phosphate group (2.3 Å/152.0°). The d-glucopyranosyl moiety of the HA further mediated strong polar contacts with the acetylthio group and C3 carbonyl functionality of the SP skeleton . On the other hand, proximity hydrophobic contacts were illustrated by SP spiro rings and acetylthio substitution at the PC non-polar tails (<5 Å). Based on the extended polar network and strong hydrophobic interactions among the formulation main three components, higher docking score was obtained with a combined value of −6.566 kcal/mol suggesting higher drug binding stability on the PC. The above obtained docking findings could explain the importance of the introduction of HA. In addition, HA would serve as a carrier mediating the loading of SP on the PC molecule for optimized SP EE%. The dispersion behavior and thermodynamic stability of the SPH tertiary complex within water, chloroform, ethanol, and 1:1 stoichiometric combination of chloroform: ethanol was investigated through an explicit MDS. Moreover, MDS within 100% chloroform showed significant instability of the SPH complex (1–OH and C2–OH of the acid sugar in one hand and the carbonyl group of the drug acetylthio substitution at the other hand (average 1.9 ± 0.31 Å/139.5 ± 0.22° and 1.6 ± 0.12 Å/155.1 ± 0.65°). Moreover, strong hydrogen bond was illustrated between the carboxylic group of HA nucleus and the 3-oxo group of SP (average 1.5 ± 0.08 Å/153.9 ± 0.73°). These polar contacts maintained tight closeness between the formulation sugar additive and SP nucleus. On the other hand, the hydroxyl groups at C3 of the glucuronic acid and C1 of the N-acetyl d-glucosamine portion of the HA-mediated alternating polar contacts with the phosphate group  as well as the acetyl moiety of one of the hydrophobic tails (average 2.6 ± 0.07 Å/153.3 ± 0.27°) across the MDS. Such polar contacts allowed pulling both PC hydrophobic acyl tails away from each other furnishing an opened compass conformation which would have increased the volume of the hydrophobic chain. On the contrarily, small surface area was maintained along the simulation where the HA-SP complex furnished several strong compact hydrogen bonding with the PC polar head. Such type of packing permitted the SPH complex to acquire an inverted cone structure with maintained micellar configuration. Finally, it was concluded that the composition of solvents could modify the packing parameters , HA amount (X2) and EA type (X3) significantly affected the EE% of the prepared formulae (p = 0.0002 for ceramide amount and p < 0.0001 for HA amount and type of EA). The direct relation between ceramide amount and EE% could be attributed to the increased viscosity of formulation accompanying the increase in the amount of ceramide included and the consequent hinderance of diffusion of SP, ensuring that SP stay entrapped within the vesicles  and EA type (X3) significantly affected the PS (p < 0.0001 for both factors). On the other hand, HA amount (X2) had no significant effect (p = 0.3541). The direct relation between ceramide amount and PS can be explained in the light of two reasons. The first of which is that ceramides tend to aggregate in formulation. The second one is the ceramide induced changes in the vesicles’ membranes. Ceramides have limited ability to cross between the vesicle membrane leaflets, leading to its accumulation within them. The overall outcome is an increase in PS . The observed EE% and PS results were 89.3 ± 0.3% and 261.8 ± 7.0 nm, respectively. The predicted values were 88.9% and 266.7 nm, respectively. The bias percent was 0.45 and 1.87%, for EE % and PS, respectively. The high similarity between the observed and predicted responses of OHAEC could conclude the validity of the design to predict the responses.SP-HAECs were to be optimized according to the constraints listed in 3.7.3.7.1.Figures S3 and S4 demonstrate the PS and ZP of OHAEC, respectively after storage. No statistical difference was found in EE%, PS, ZP, and PDI . Moreover, there was no change in the physical appearance of OHAEC.The values of EE%, PS, ZP, and PDI for fresh and stored OHAEC are listed in 3.7.2.It is obvious in 3.8.Q24 (cumulative amount permeated per unit area after 24 h) and Jmax values and significantly higher Dep24 value leading to LAEI value 18.5 times higher than suspension (p < 0.05)  . Formula < 0.05) . On the  < 0.05) .3.9.3.9.1.3.9.2.0–10, obtained from the deposition profiles was 402.9 µg h/cm2 for OHAEC, which was significantly higher than that of suspension (248.3 µg h/cm2), meaning a 1.62 folds increase in SP-deposition. These findings agree with findings of the ex vivo study and signify the role of formulation components in localizing SP within the skin.The deposition profile of SP from OHAEC, compared to SP suspension is illustrated in 4.in silico study that exhibited the importance of hyaluronic acid in increasing the extent of binding between SP and phospholipid. Optimal HAECs (OHAEC) contained 10.5 mg ceramide III, 15 mg HA and Kolliphor RH40 as an edge activator. OHAEC was stable for up to 3 months. Transmission electron microscopy showed a mixed tubular and vesicular appearance of OHAEC. Both ex vivo and in vivo dermatokinetic studies concluded better deposition of SP from OHAEC, compared to its suspension. Histopathology demonstrated the safety of OHAEC. Hence, it could be concluded that OHAEC could be effective formula that could treat hirsutism.SP-loaded HAECs were prepared by ethanol injection, according to D-optimal design, following an Click here for additional data file.


To compare inpatient treated patients with idiopathic (ISSNHL) and non-idiopathic sudden sensorineural hearing loss (NISSNHL) regarding frequency, hearing loss, treatment and outcome.All 574 inpatient patients  with ISSNHL and NISSNHL, who were treated in federal state Thuringia in 2011 and 2012, were included retrospectively. Univariate and multivariate statistical analyses were performed.p < 0.001). The mean interval between onset of hearing loss to inpatient treatment was shorter in ISSNHL (7.7 days) than in NISSNHL . The initial hearing loss of the three most affected frequencies in pure-tone average (3PTAmax) scaled 72.9 dBHL ± 31.3 dBHL in ISSNHL and 67.4 dBHL ± 30.5 dBHL in NISSNHL. In the case of acute otitis media, 3PTAmax (59.7 dBHL ± 24.6 dBHL) was lower than in the case of varicella-zoster infection or Lyme disease . Mean absolute hearing gain (Δ3PTAmaxabs) was 8.1 dB ± 18.8 dB in patients with ISSNHL, and not different in NISSNHL patients with 10.2 dB ± 17.6 dB. A Δ3PTAmaxabs ≥ 10 dB was reached in 34.3% of the patients with ISSNHL and to a significantly higher rate of 48.8% in NISSNHL patients (p = 0.011).ISSNHL was diagnosed in 490 patients (85%), NISSNHL in 84 patients (15%). 49% of these cases had hearing loss due to acute otitis media, 37% through varicella-zoster infection or Lyme disease, 10% through Menière disease and 7% due to other reasons. Patients with ISSNHL and NISSNHL showed no difference between age, gender, side of hearing loss, presence of tinnitus or vertigo and their comorbidities. 45% of patients with ISSNHL and 62% with NISSNHL had an outpatient treatment prior to inpatient treatment (ISSNHL and NISSNHL show no relevant baseline differences. ISSNHL tends to have a higher initial hearing loss. NISSHNL shows a better outcome than ISSNHL.The online version contains supplementary material available at 10.1007/s00405-021-06691-y. So far, many studies have analyzed epidemiological data for idiopathic sudden sensorineural hearing loss (ISSNHL) , 2. In aIn contrast, not much is known on the outcome of non-idiopathic sudden sensorineural hearing loss (NISSNHL), because in all studies analyzing hearing loss and recovery these patients are excluded , 10. TheIn the federal state of Thuringia, there are eight hospitals with departments for ears-nose-throat (ENT) medicine. These have formed a network for the scientific evaluation of ENT diseases , 12. RecWe performed a retrospective analysis in all eight ENT departments of the federal state Thuringia. All patients who were hospitalized in 2011 and 2012 due to acute hearing loss with the ICD codes  H91.0, H91.1, H91.2, H91.3, H91.8 and H91.9 were included in the study. A positive ethical vote for the evaluation of the underlying data was obtained . A total of 723 patients with the above-mentioned diagnoses were treated as inpatient patients and were included in the primary dataset. It made no difference whether the patients had comorbidities or had already received prior outpatient treatment. 58 patients were initially excluded from the evaluation due to missing data sets  and 91 patients were excluded due to a lack of initial hearing loss, inpatient treatment due to middle ear surgery or cochlear implantation or lack of follow-up hearing examinations. Of the remaining 574 patients, 490 had an idiopathic sudden sensorineural hearing loss (ISSNHL) while the remaining 84 patients had a disease underlying the hearing loss  and were classified as NISSNHL. All 574 patients, ISSNHL as well as NISSNHL, were examined in the present study .The follow-up was recorded and evaluated until August 2013. Patient data such as age and gender, clinical and functional examination, medical and surgical treatment were recorded and the treatment of hearing loss in the case of ISSNHL compared to NISSNHL was evaluated.abs = PTApre minus PTApost in dB) [rel = 100*(PTApre minus PTApost)/PTApre and relative hearing improvement compared to the contralateral ear  ΔPTArelcontral = 100* (PTApre minus PTApost)/. ΔPTAabs ≥ 10 dB, ΔPTAabs ≥ 15 dB, ΔPTArel ≥ 50% and ΔPTArelcontral ≥ 50% in a dichotomous distribution (yes / no) were considered as criterions for a successful improvement [The extent of the initial hearing loss was described using the pure-tone average (PTA) in decibels hearing level (dB HL). The average hearing loss of the three most affected frequencies 3PTAmax), 10 frequencies , 9 frequencies , 4frequencies , low- , middle- , and high frequency  hearing loss were calculated PTAmax, 1, 14. Heat in dB) . Furtherrovement . Kanazakrovement .www.tls.thueringen.de).The epidemiological statistics were calculated on the basis of the annual average population of Thuringia from 2011 and 2012, which are published in the online database of the statistical office of the federal state of Thuringia (The patients affected by ISSNHL were treated according to the German guidelines for the treatment of sudden hearing loss: All patients received intravenous prednisolone therapy. Prednisolone was administered in a dose of 250 mg/d (range of 100–500 mg/d) , 8. The p < 0.05.Unless otherwise noted, data were presented with mean values ± standard deviation (SD). All statistical analyses were performed using IBM SPSS, version 24.0.0.0. The non-parametric Mann–Whitney-U-test for independent metric data was applied to compare different subgroups of patients. The Chi-square test was applied for independent nominal data. The non-parametric Wilcoxon test for dependent metric data was applied to analyze differences between initial hearing loss and final hearing loss on the affected ear at the end of the follow-up. A multivariate binary logistic regression was performed including the significant associations. Nominal p-values of two-tailed tests are reported. The significance level was set at p = 0.218). There was no side predominance of acute hearing loss neither in ISSNHL nor in NISSNHL (p = 0.197). The accompanying symptoms like tinnitus  or vertigo  occurred equally frequently in both groups (Table p = 0.117), coronary heart disease (p = 0.601), diabetes mellitus type II (p = 0.414), hypercholesterolemia (p = 0.755) and arterial hypertension (p = 0.754) occurred equally frequently in ISSNHL and NISSNHL (Table p < 0.001). The time from the onset of hearing loss to hospital admission was less for NISSNHL (7.7 days ± 12.2 days) than for ISSNHL . The majority of the patients (98.1%) received prednisolone therapy during the inpatient stay . Patients with NISSNHL had to undergo surgery during the hospital stay more often (52%) than patients with ISSNHL  . 12% of the patients had an acute deafness and 2% had a combined vestibulocochlear lesion . Considering the 10PTA, 9PTA, 4PTA, LF-3PTA and MF-3PTA ISSNHL had more severe hearing loss than NISSNHL (p < 0.05)  . In LF-3PTA 27.4% of NISSNHL and 42% of ISSNHL reached ΔPTAabs ≥ 10 dB (p = 0.011) and 17.9% of NISSNHL and 32.2% of ISSNHL reached ΔPTAabs ≥ 15 dB. Other parameters showed no difference in NISSNHL and ISSNHL  at NISSNHL was 67.4 dBHL ± 30.5dBHL and showed no statistical difference to hearing loss at ISSNHL with a 3PTAmax of 72.9 dBHL ± 31.3 dB (p = 0.033)   and that more patients with prior outpatient treatment showed a hearing impairment of Δ3PTAmaxabs ≥ 10 dB (p = 0.032). There was no difference for the tinnitus (p = 0.325), as well as for the comorbidities of coronary heart disease (p = 0.531), hypercholesterolemia (p = 0.439), diabetes mellitus type II (p = 0.653) and arterial hypertension (p = 0.850)  inhabitants in 2011 and 2012. In total, an average of 16.61 inpatients per 100,000 habitants was treated in Thuringia per year. Of these, 11.25 inpatients per 100,000 habitants had an ISSNHL and 1.93 per 100,000 inhabitant people per year had an NISSNHL . There was no difference in gender distribution. The incidence of the ISSNHL was 21.6/100,000 in women and 23.4/100,000 in men. The incidence of NISSNHL was 3.5/100,000 in women and 4.2/100,000 in men.abs [In the current study, causes for sensorineural hearing losses were found in 14.6%, which corresponds to the numbers reported so far . There wabs . An explabs , 18, whiabs .Studies that directly compare ISSNHL and NISSNHL are not known. Therefore, the retrospective study presented here with a total of 490 patients with ISSNHL and 84 patients with NISSNHL is the largest study of this type published to date. One disadvantage of the current study is that only patients with acute hearing loss were included here. Patients treated in hospital with the ICD codes H65.0, H65.1, H65.2, H65.3 H65.4 H65.9, H66.0, H66.1, H66.2, H66.3, H66.4, H66.9, H67.0* H67.1*, H67.8*, H83.0, H73.0, J11.8,H70.0, H70.2, H70 0.8, H70.9, B02.8 and A69.2 for any underlying disease were not included. Therefore, there is an unreported number of patients with other diseases combined with sensorineural hearing loss. Likewise, the true incidence of diseases is underestimated here because only patients who have been hospitalized are included in this evaluation. This is associated with a selection bias in favor of the more severe cases. The evaluation of hearing loss and hearing gain is handled inconsistently in most studies –21. So fThe data on the occurrence, extent and recovery of ISSNHL have already been discussed in detail elsewhere . TherefoIn addition to potentially life-threatening complications, acute otitis media can lead to a permanent impairment of the patient due to hearing loss . A zosteIn the present study, it was found that acute otitis media and Menière disease showed significantly less absolute hearing loss compared to the other subgroups. Many evaluations consider hearing loss, but often no distinction is made between the appearance of conductive hearing loss and sensorineural hearing loss. Occasionally the absolute extent of hearing loss (in dBHL) is not described. For acute otitis media hearing loss is reported between 25 and 40 dBHL , 35–37. So far, there is no guideline for the treatment of acute otitis media with sensorineural hearing loss in Germany. The German Society for General Medicine and Family Medicine published a S2k-guideline "Earache": In the guideline, initially symptomatic treatment and, in the event of a lack of improvement or indications of a complicated course, antibiotics are used . In acutThe current retrospective study examines inpatients with ISSNHL and NISSNHL in Thuringia in 2011 and 2012. It can be seen that ISSNHL tends to have a higher initial hearing loss than patients with NISSNHL. ISSNHL and NISSNHL show no difference in the degree of absolute or relative hearing improvement. However, patients with NISSNHL are more likely to show successful hearing improvement considering the 3PTAmax. The data are not sufficient to show prognostic differences of subgroups at NISSNHL. However, we were able to show that patients with acute otitis media and Menière disease show an initially lower hearing loss compared to patients with varicella zoster or Lyme disease or other underlying diseases.Supplementary file 1 (TIF 12771 KB) Supplemental Digital Content 1. Inclusion and exclusion of patients´ datasets. 574 patients were analyzed. 490 patients had an ISSNHL , 84 patients had a NISSNHL .Below is the link to the electronic supplementary material.