Boswellia (Burseraceae), are known for their anti-inflammatory and potential antitumor efficacy. Although boswellic acids generally occur as α-isomers (oleanane type) and β-isomers (ursane type), 11-keto-boswellic acid (KBA) was found only as the β-isomer, β-KBA. Here, the existence and natural occurrence of the respective α-isomer, 11-keto-α-boswellic acid (α-KBA), is demonstrated for the first time. Initially, α-KBA was synthesized and characterized by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy, and a highly selective, sensitive, and accurate high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed by Design of Experiments (DoE) using a pentafluorophenyl stationary phase. This method allowed the selective quantification of individual 11-keto-boswellic acids and provided evidence for α-KBA in Boswellia spp. oleogum resins. The contents of α-KBA as well as further boswellic acids and the composition of essential oils were used to chemotaxonomically classify 41 Boswellia oleogum resins from 9 different species. Moreover, α-KBA exhibited cytotoxicity against three treatment-resistant triple-negative breast cancer (TNBC) cell lines in vitro and also induced apoptosis in MDA-MB-231 xenografts in vivo. The respective β-isomer and the acetylated form demonstrate higher cytotoxic efficacies against TNBC cells. This provides further insights into the structure-activity relationship of boswellic acids and could support future developments of potential anti-inflammatory and antitumor drugs.Boswellic acids, and particularly 11-keto-boswellic acids, triterpenoids derived from the genus  Boswellia Roxb. ex Colebr. (Burseraceae) secrete oleogum resins (frankincense), which have been known for centuries for their anti-inflammatory, antibiotic, analgesic, and antitumor activities  cell lines in vitro and in vivo. With 30%, breast cancers represented the most common cancer type of all new female cancer cases in 2019, causing 15% of all female cancer deaths . TNBC shBoswellia oleogum resins, initially, 11-keto-α-boswellic acid -3-hydroxy-11-oxo-olean-12-en-24-oic acid) was synthesized and characterized. Starting from acetyl-α-boswellic acid (α-ABA), firstly, acetyl-11-keto-α-boswellic acid (α-AKBA) was synthesized by radical-type reaction with bromine and characterized as previously described [To develop an analytical method for selective quantification of individual keto-boswellic acids in escribed . Afterwa30H46O4 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) with an exact mass at m/z 469.3323 for [M-H]− . Investigation of the compound´s isotopic mass pattern corresponded well to the calculated pattern of the formula (see m/z 469.3 ([M-H]−) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) exhibited characteristic fragments at m/z 353.3, 376.3, 391.4, 407.4, and 451.4 . Its molecular formula was determined as Cmula see . Collisind 451.4 .1H NMR spectrum of α-KBA showed seven singlet signals  accounting for methyl groups attached to quaternary carbons (H 0.88 (CH3-29) and δH 0.89 (CH3-30). The presence of a keto function at C-11 (δC 198.9) was confirmed by long-range heteronuclear correlations with two methine signals, namely the adjacent angular H-9 (δH 2.35) and the olefinic H-12 (δH 5.47), which represents the most downfield signal in the 1H NMR spectrum. The remaining signals were assigned by homo- and hetero-nuclear correlated NMR spectroscopy, 1H, 1H COSY (correlated spectroscopy), 1H, 13C HSQC (heteronuclear single quantum coherence spectroscopy), and 1H, 13C HMBC (heteronuclear multiple bond correlation). The stereochemistry was elucidated based on correlations in the 1H, 1H ROESY (rotating frame Overhauser enhancement spectroscopy) spectrum , which served as projections for two-dimensional (2D) spectra.Using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, the structure of α-KBA was elucidated . The 1H  carbons . Charactspectrum b. SpectrFor selective and simultaneous quantification of the constitutional isomers of 11-keto-boswellic acids (KBAs), i.e., 11-keto-α-boswellic acid (α-KBA) and 11-keto-β-boswellic acid (β-KBA), as well as acetyl-11-keto-boswellic acids (AKBAs), i.e., acetyl-11-keto-α-boswellic acid (α-AKBA) and acetyl-11-keto-β-boswellic acid (β-AKBA), a novel chromatographic separation method was developed.Since the chromatographic separation of the constitutional isomers of KBAs and AKBAs with the help of C18 phases was insufficient ,25,26,27R, between the individual constitutional isomers served as dependent response variables. R was calculated with the following formula:R is the respective retention time and w0,5 is the respective peak width at half-height. Whereby, two peaks with a resolution R ≥ 1.5 were considered as sufficiently separated for individual quantification hexane (hashishene). Hashishene is formed by photo-oxidation of myrcene and was given its common name from hashish, resins of s sativa . As in hBoswellia species overlap and taxonomic specifications are rarely clearly defined, the botanic assignment of individual specimen has always been hampered [Boswellia species flourish [Boswellia species.As the distribution areas of the more than 25 known hampered . This isflourish . FurtherB. sacra is mostly distributed at the Arabian Peninsula (Oman and Yemen), but occasionally, there have also been specimens described in Somalia  and coincidently, a low proportion of α-KBA (1.1–6.6%) , but relatively high total contents of AKBAs with 21.7–41.8 µg/mg  a. Furthe.8 µg/mg b. This cα-AKBA 0.–1.8%, bum resins .B. serrata (syn. B. thurifera Colebr. and B. glabra Roxb.) grow in the central and northern parts of India and in Pakistan [B. serrata from other Boswellia species [B. serrata represents probably the best investigated Boswellia oleogum resin and it has been used in Ayurvedic medicine for centuries [Trees of Pakistan . The geo species . Frankinenturies ,7,15,43.B. serrata differed from those of B. sacra with higher values of 3.0–22.9 µg/mg. The proportion of α-KBA was low. In contrast to B. sacra, B. serrata contained lower total AKBA contents (8.0–18.7 µg/mg). Thus, this confirms previous results showing that the composition of oleogum resins from B. serrata differ from that of B. sacra due to a high percentage of deacetylated boswellic acids [B. serrata oleogum resins.The total KBA contents of ic acids ,16. ThisB. papyrifera is Ethiopia, Eritrea, and Sudan [B. papyrifera is mostly used as incense in churches and it is potentially endangered by over-harvesting [The growth region of nd Sudan . Frankinrvesting .B. papyrifera exhibits a similar boswellic acid composition compared to B. sacra [B. papyrifera contained higher total KBAs contents (3.1–4.8 µg/mg) and a higher proportion of α-AKBA (3.4–6.4%) than B. sacra.Although B. sacra , the KBAB. papyrifera clearly distinguishable from other Boswellia species [Furthermore, GC analysis of the essential oil revealed exceptionally high amounts of octanol (7.0%), octanol acetate (77.7%), and the potential psychoactive ,45 diter species ,46.Boswellia species, B. dalzielli  is distributed in West and Central Africa, especially in Nigeria, Senegal, and Burkina Faso [B. dalzielli trees that is used [Unlike other ina Faso ,19. In t is used .B. papyrifera resemble those of B. sacra with regard to the contents of acetylated boswellic acids [B. dalzielli is unique  post hoc Fisher´s least significant difference (LSD), whereby the proportion of α-AKBA is higher too compared to B. sacra. Furthermore, the total KBAs content (9.8–13.8 µg/mg) is higher than in most other Boswellia species.At first glance, oleogum resins of ic acids  and of αs unique c. The toB. carterii (syn. B. carteri or B. bhaw-dajiana Birdw.) thrives in North Somalia, especially in the regions of Bari, Puntland, and Somaliland [B. carterii is a prime example for the difficulty of taxonomic and botanic classification of Boswellia species. After the discovery of B. sacra in 1867 by Friedrich August Flückiger, George Birdwood redefined the species as B. carterii in 1870 [B. sacra Flueck. and African B. carterii Birdw. [B. sacra and B. carterii are the same or two different species [maliland ,19. Its  in 1870 . In 1969i Birdw. . Until n species ,33,42,50B. carterii samples contained a very low total KBA content (0.08–0.87 µg/mg) with a relatively high proportion of α-KBA (20.3–25.5%)  a. Likewi3–25.5%) b. Howeve samples c. MoreovB. sacra . A studyα-pinene . FurtherB. frereana is located in Northern Somalia and in contrast to all other Boswellia species, its trees prefer rocky terrain  and AKBAs (0.03–0.14 µg/mg) and high proportions of the respective α-isomers, α-KBA (18.5–32.9%) and α-AKBA (15.9–18.4%). Considering the similar contents of further boswellic and lupeolic acids, the differentiation between B. neglecta and B. carterii species is hampered. Only the high contents of acetyl-lupeolic acid, acetyl-α-boswellic acid, and acetyl-β-boswellic acid in some B. carterii gum resins might serve as a reference point for differentiation [α-KBA 18.–32.9% anB. neglecta from Kenya as well as B. rivae are characterized by very low total contents of KBAs (<LOQ 0.06 µg/mg) and AKBAs (0.002–0.055 µg/mg). Remarkably, B. rivae is the only sample that exhibited a higher proportion of the α-isomer α-KBA with 57.7%, which makes it unique. Further investigations of B. neglecta (from Kenya) and B. rivae revealed very low contents of all boswellic and lupeolic acids [Commiphora, such as the rather unknown species Commiphora confusa [Commiphora specific compounds, e.g., E- and Z-guggulsterone, could provide further information on that.Differently, oleogum resins of 0.002–0.05 µg/mg.  confusa ,54. Anal50) of 42.0 ± 4.6 µM  more sensitive to α-KBA compared to human PBMC, the non-cancerogenic control group (IC50 = 75.2 ± 9.0 µM). However, comparing the cytotoxicity of KBA α- and β-isomers, β-KBA is more effective against MDA-MB-231 cells  cell line MDA-MB-231 in vitro with a half maximal inhibitory concentration (IC± 4.6 µM a. We hav31 cells . Compari31 cells b. In fac50 = 34.6 ± 1.9 µM) and CAL-148 cells (IC50 = 36.1 ± 1.9 µM), indicating cytotoxic efficacy against different treatment-resistant TNBC cells.Further, the cytotoxic efficacy of α-KBA was investigated on two additional TNBC cell lines, CAL-51 and CAL-148. Likewise, α-KBA exhibited considerable cytotoxicity against CAL-51 cells  of fertilized chick eggs. Moreover, the influence of acetylation on cytotoxicity in vivo was analyzed. After treatment for 3 consecutive days with the individual compounds at different doses, the tumor volumes were measured and specimens were analyzed immunohistochemically regarding proliferation and apoptosis by Ki-67 antigen staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, respectively a.Boswellia extracts and their cytotoxicity against MDA-MB-231 cells [Treatment of breast cancer xenografts with 50 µM α-KBA significantly reduced the tumor volume and cancer cell proliferation, and induced apoptosis b–d. Howe31 cells . Moreove31 cells . In addi31 cells ,11,12. N50 = 0.71 ± 0.05 µM) [Boswellia extracts and boswellic acids, no adverse effects were observed in mouse studies [As a positive control for in vitro and in vivo experiments, the non-halogenated anthracycline doxorubicin was analyzed. Doxorubicin is used as a chemotherapeutic agent for the treatment of breast cancer patients. Doxorubicin exhibited higher toxicity against MDA-MB-231 cells in vitro (IC0.05 µM)  and anti0.05 µM) ,57, wher studies ,12,58 anB. serrata by their β-KBA and β-AKBA contents [Boswellia oleogum preparations.The European pharmacopoeia (Ph. Eur.) recommends the characterization of extracts from contents . Howevercontents . Considecontents ,24,26,27All solvents and chemicals were of analytical reagent grade. The solvents used for the extraction, sample preparation, and HPLC-MS/MS analysis were MeOH, acetic acid , and ultrapure water ) coupled to a Milli-Q station . The compounds 11-keto-β-boswellic acid (β-KBA) and acetyl-11-keto-β-boswellic acid (β-AKBA) were purchased from Extrasynthese . Acetyl-11-keto-α-boswellic acid (α-AKBA) was synthesized and characterized as previously described .Boswellia oleogum resins were purchased from commercial sources or were obtained from cooperation partners. All samples were thoroughly examined and numbered as previously detailed [Boswellia samples are deposited at the Herbarium of the Botanical Garden of Ulm University, Institute of Systemic Botany and Ecology, Germany (voucher: ULM-24224). For more precise sample information, please see the previous article and corresponding detailed . Samplesm/v). For base hydrolysis, the solution was heated to 75 °C for 25 min with continuous stirring. The reaction was stopped by adding 420 µL concentrated acetic acid yielding a pH value of 4.5. The reaction product was precipitated by adding 1000 µL warm water and subsequently cooling down for 30 min at 4 °C. Afterwards, the product was washed three times with 1000 µL cold water and dried by lyophilization yielding 5.73 mg raw product.α-KBA was synthesized by saponification of α-AKBA. For this purpose, 6.99 mg (13.6 µmol) of α-AKBA was dissolved in 420 µL of 10% KOH in MeOH (v/v), and eluent B, MeOH, both acidified with 0.2% acetic acid. Initial conditions were 30% eluent A and 70% eluent B followed by a linear gradient to 90% eluent B over 15 min, then 90% eluent B until 25 min. Thereafter, followed a linear gradient to initial conditions until 25.5 min and re-equilibration continued until 30 min. In order to stabilize the chromatographic system, the column was kept at a temperature of 28 °C.The raw product was purified by semi-preparative HPLC using a LC-9A HPLC pump , an IWN CH100 column oven , an UVD 340 U photodiode array detector , and a fraction collector . Instruments were controlled and data processed by Chromeleon software version 6.6 . For purification, a semi-preparative column  with a precolumn  was used. The raw product was dissolved in 1000 µL DMSO and filtered through a 0.45 µm regenerated cellulose filter. The flow rate was 5000 µL/min and the injection volume was 250 µL. The mobile phase consisted of eluent A, MeOH/water . Purity control by high-performance liquid chromatography with photodiode array detection (HPLC-DAD) at 210, 254, and 280 nm exhibited a purity > 99%.The collected fractions were concentrated with a nitrogen stream first, and then dried by lyophilization, yielding 3.94 mg (8.37 µmol) α-KBA. This corresponds to a total yield of 61.8% ) was infused into the MS with a constant flow rate of 120 µL/h. Spectra were acquired with a time-domain of 4 megawords and 30 scans were accumulated within an m/z range from 147 to 750. Calibration of the MS and ion source settings were the same as recently described [m/z 469.332267 and superb mass accuracy in the ppb range.A high-resolution mass spectrum of α-KBA was recorded with a 12 Tesla solariX FT-ICR-MS  equipped with an APOLLO II electrospray source  and operated in the negative ionization mode. A diluted sample  spectra of α-KBA were recorded on a 500 MHz Bruker Avance III HD spectrometer equipped with a CryoPlatform and a 5 mm TCI CryoProbe . Spectrometer control and data processing were accomplished with Bruker TopSpin Ver. 3.6.1. The sample was measured in DMSO-All HPLC-MS/MS experiments were carried out on an Agilent 1260 Infinity HPLC system  coupled with an AB API 2000 triple quadrupole mass spectrometer  using an electrospray ionization ion source (ESI). Devices were controlled and data were processed by means of Analyst 1.6.1 software .N is the total number of experiments and CN is the number of experiments of the cube, i.e., the full two-level factorial design [The quantification of total contents of KBAs and AKBAs was executed by a HPLC-MS/MS method developed, validated, and implemented as previously published ,27. To ql design . The expv/v), and eluent B, MeOH, both acidified with 0.2% acetic acid. The injection volume was 20 µL and the column was kept at a stable temperature of 28 °C. On the basis of the results obtained by the DoE, the method herein was developed: Initial conditions were 92% eluent A and 8% eluent B, followed by a linear gradient to 62% eluent B over 44.5 min, and subsequent linear gradient to 95% eluent B until 45.0 min. Thereafter, 95% eluent B until 54.5 min, followed by linear gradient to initial condition until 55.0 min, and re-equilibration until 60.0 min. Here, the flow rate of the mobile phase was 690 µL/min.The mobile phase consisted of eluent A, MeOH/water  detection. The atmospheric pressure electrospray ion source was heated to 400 °C and the ion spray voltage was set to −4250 V. Collision energy was optimized to −40 V and the dwell time to 250 ms. The precursor ion at β = 1 mg/mL or β = 10 mg/mL). To determine the individual contents, the total content was divided with regard to the ratio of the areas under curves (AUC) of the individual compound’s peak within the MRM chromatograms. To ensure a proportional ratio, the linearity was investigated for each individual compound at 10 concentration levels. Thus, this procedure ensured a linear proportion (p < 0.001) between concentration and AUC in a range of 1–1000 ng/mL for α-KBA, β-KBA, α-AKBA, and β-AKBA. All samples were diluted according to this linear range and analyzed in duplicates to ensure an accurate quantification.For sample preparation, the samples were freshly dissolved in MeOH , B. serrata (sample # 18), B. carterii (sample # 25), B. frereana (sample # 38), B. dalzielli (sample # 12), B. papyrifera (sample # 15), B. neglecta (sample # 35), or B. rivae (sample # 37) were added to 250 mL water and mixed for several minutes until a thick homogenous dispersion was formed. For hydrodistillation, the mixtures were subsequently heated for 6–7 h at 120–125 °C with continuous stirring. The obtained essential oils were carefully separated from the aqueous phase with the help of a separating funnel, yielding 1.2–9.7% essential oils (w/w), respectively.Essential oils were obtained by hydrodistillation of GC-FID analysis of essential oils was performed on an Agilent 7890A system equipped with a DB-5 capillary column (10 m × 0.10 mm × 0.10 µm) and a DB-Wax capillary column (10 m × 0.10 mm × 0.10 µm). Hydrogen was used as carrier gas . Oven temperature started at 35 °C for 1 min, followed by linear gradient to 260 °C by 9 °C/min, and then held for 10 min. Essential oil samples were diluted at roughly 20 mg/mL in GC-grade pentane and were injected with a volume of 1 µL and a split of 50:1 with an inlet temperature of 250 °C.n-alkanes and mass spectra comparisons with NIST14 libraries, in-house libraries, and specialist literature [GC-MS analysis of essential oils was performed on an Agilent 7890B/5977B system equipped with a HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Helium was used as a carrier gas . Oven temperature started at 40 °C for 2 min, followed by linear gradient to 270 °C by 4 °C/min, and then held for 5 min. Essential oil samples were injected neat with a volume of 0.5 µL and a split of 50:1, with an inlet temperature of 250 °C. Compounds were identified using their retention indexes as calculated from a homologous series of terature ,62.2 (MDA-MB-251 und CAL-51) or 10% CO2 (CAL-148) atmosphere. Cells were sub-cultured according to the supplier’s recommendations after reaching 80% confluence. For the experiments, 5000 cells/well of the respective cancer cell lines or 300,000 PBMC/well were seeded into 96-well plates in medium supplemented with 10% FCS overnight and treated 24 h after seeding. Respective concentrations of the samples were applied by means of a Tecan D300e Digital Dispenser . After a 72 h incubation period, cell viability was analyzed by addition of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt . Absorbance of the formed orange formazan dye was measured using an Infinite M1000 PRO Tecan plate reader (Tecan) at λ = 450 nm with a λ = 630 nm reference filter. For quantification of cell viability, the blank values containing the respective compounds in the according concentration were subtracted and the percentage of viable cells was calculated by normalization to the vehicle control.Triple-negative human breast cancer cells MDA-MB-231 , CAL-51 , and CAL-148 (DSMZ), as well as peripheral blood mononuclear cells (PBMC) were analyzed. PBMC were isolated from whole venous blood from healthy male donors via density gradient centrifugation using Biocoll . Informed consent of the volunteers and approval of the institutional Ethics Committee (#177/18) were obtained. MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% FCS , 0.1 mM MEM  non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. CAL-51 cells were cultured in Dulbecco´s Modified Eagle Medium  supplemented with 10% FCS, 100 U/mL penicillin, and 100 mg/mL streptomycin. CAL-148 cells were cultured in Dulbecco´s Modified Eagle Medium  supplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10 ng/mL epidermal growth factor (EGF). All cancer cell lines were cultured in a humidified incubator at 37 °C and 5% CO6 MDA-MB-231 cells in medium/matrigel  were xenografted onto the chick chorioallantoic membrane (CAM) of fertilized chick eggs 7 days after fertilization. For the next three consecutive days, cells were treated topically with 10 or 50 µM of α-KBA and α-AKBA respectively, or 10 µM doxorubicin dissolved in 0.9% NaCl (vehicle control: 0.5% DMSO). Tumors were collected, imaged, fixed, and embedded in paraffin for analysis by immunohistochemistry on the fourth day after treatment initiation. Tumor volumes (mm3) were calculated with the formula length (mm) × width2 (mm2) × π/6 [For investigation of antitumor activity against TNBC xenografts in vivo, 0.7 × 102) × π/6 . For immt-test. Results with 0.001 ≤ p < 0.05 were considered as statistically significant and p < 0.001 as highly significant. Principal component analysis (PCA) was derived from a correlation matrix of the raw data yieldingExperiments for HPLC-MS/MS quantifications were carried out in duplicates and all in vitro and in vivo experiments in at least three independent experiments. All in vitro experiments were additionally performed in triplicates. The data are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated. Statistical analysis was performed with Minitab 18 software , SigmaPlot 14.0 software , and Valoo 2.10 software . All data were tested for normal distribution by the Anderson-Darling test and equality of variances by Levene´s test. Sample groups were compared by one-way analysis of variance (ANOVA) and post hoc by Dunett´s test or by Fisher’s least significant difference (LSD) test. Comparison of two sample groups was executed by Student´s Boswellia spp., a novel triterpenoid, 11-keto-α-boswellic acid (α-KBA), was identified. α-KBA was synthesized and characterized by 1D and 2D NMR and HR-MS and a highly sensitive and accurate HPLC-MS/MS method for selective evidence and quantification of individual keto-boswellic acids was developed. By means of this method, 41 Boswellia oleogum resins were investigated and it could be demonstrated that in addition to the well-known 11-keto-β-boswellic acid (β-KBA), its α-constitutional isomer, α-KBA, exists in Boswellia spp. On the basis of the individual keto-boswellic acid contents and with inclusion of the chemical composition of essential oils and the composition of the oleogum resins’ boswellic acids, a chemotaxonomic characterization of different Boswellia species could be achieved. Moreover, α-KBA exhibited cytotoxicity against human treatment-resistant, triple-negative breast cancer (TNBC) cell lines in vitro and in vivo. However, the β-isomer, β-KBA, and its acetylation enhanced the lipophilicity as well as the cytotoxic efficacy. These results provide a deeper insight into the structure–activity relationship of boswellic acids and point to the necessity for further studies aiming at the development of new anticancer drugs.In oleogum resins of


The resulting miktoarm star polymers were investigated by 1H NMR spectroscopy and gel permeation chromatography. The critical micellar concentration value of the micelles increases with an increase in PNAM block length. As revealed by transmission electron microscopy and dynamic light scattering, the amphiphilic miktoarm star block copolymers can self-assemble to form spherical micellar aggregates in water. The anticancer drug doxorubicin (DOX) was encapsulated by polymeric micelles; the drug-loading efficiency and drug-loading content of the DOX-loaded micelles were 81.7% and 9.1%, respectively. Acidic environments triggered the dissociation of the polymeric micelles, which led to the more release of DOX in pH 6.4 than pH 7.4. The amphiphilic PLGA-b-PNAM2 miktoarm star block copolymers may have broad application as nanocarriers for controlled drug delivery.Owing to their unique topology and physical properties, micelles based on miktoarm amphiphilic star block copolymers play an important role in the biomedical field for drug delivery. Herein, we developed a series of AB To date, few reports have been published for the preparation of AB2-type miktoarm-block copolymers and their drug-delivery application  undec-7-ene (DBU) as a catalyst at room temperature. The 1H NMR spectra of the PLGA macro initiator is presented in 1H NMR investigation confirms that the PLGA macroinitiator was successfully prepared. The integrated peak areas “b” of the PDLLA backbone chain and ‘‘a’’ of the PGA backbone chain were compared by the integrated peak area of ‘‘k’’ of the chain-end xanthate moiety of the miktoarm initiator, to estimate the Mn (NMR) of the PLGA copolymer, which was calculated to be 4800 g mol−1. The gel permeation chromatography (GPC) study of the copolymer specified that the molecular weight (Mn) and dispersity (Đ) were 5300 g mol−1 and 1.20 , 3.0–4.0 (r) and 1.70–1.90 (p) ppm, due to the presence of PNAM backbone chains. The GPC chromatogram shows that the elution times of the three miktoarm-block copolymers are shorter than that of the PLGA macroinitiator as can be seen in The PLGA-b-PNAMIt is also noticeable that the GPC curves of M3 display shoulders to higher molecular weights while the shapes of M1 and M2 are unimodal. The bimodal-like shape of M3 may be attributed to the star–star coupling of the propagating species of miktoarm copolymers at the high ratio of NAM with respect to the macroinitiator . In othe1HNMR spectrum of M2 in D2O clearly shows the appearance of PNAM and the peaks attributed to PLGA are suppressed in comparison with the 1H NMR spectrum obtained in chloroform-d which dissolves both polymers  at various concentration  was purified by distillation under reduced pressure. Doxorubicin hydrochloride (DOX.HCl) was kindly supplied by Boryung Pharmaceutical Co., Ltd. . AIBN , dichloromethane , tetrahydrofuran , benzoic acid , dimethylformamide  and 1,8-diazabicyclo [5.4.0] undec-7-ene  were purchased from Sigma-Aldrich (Korea).Glycolide and D,L-lactide  were recrystallized in ethyl acetate. A detailed methodology for the synthesis of the miktoarm initiator is described in the Supporting Information.−2 mol) of D, L-lactide and 4.0 mL of dry DCM was placed in a 100-mL dry RB flask under nitrogen atmosphere in a glove box. Then, 20 mg (1.20 × 10−4 mol) of the miktoarm initiator and 10 μL  of DBU were added to the above RB flask. The reaction mixture was stirred at room temperature. Then, the solution of 0.3 g of glycolide in 2.0 mL of THF was dropwise added to the reaction mixture and the reaction continued for 5 min. Then, the polymerization was stopped by adding 39 mg (3.9 × 10−5 mol) of benzoic acid. The obtained copolymer was purified by precipitating in cold methanol (−10 °C). The copolymer was precipitated in methanol twice and finally dried under vacuum at room temperature for 24 h.The PLGA copolymer was prepared by ROP of D, L-lactide and glycolide using the miktoarm initiator and DBU catalyst. In brief, 1.0 g : δ (ppm) = 5.1–5.25 (1Hb), 4.88–478 (2Ha), 4.65–4.58 (4Hk), 4.4–4.3 (2Hd), 4.30–4.26 (2Hh), 4.1–4.0 (2He), 1.62–1.20 (3Hc + 6Hi + 6Hl), 0.87–0.85 (3Hf).Mn(NMR) = 4800 g mol−1, Mn(GPC) = 5300 g mol−1 and Đ = 1.20.2 miktoarm star block copolymers were prepared by the RAFT method. Typically, into a dried reaction flask, 0.1 g (0.020 mmol) of the PLGA macro initiator, 0.15 g (1.04 mmol) of NAM and 0.7 mg (0.010 mmol) of AIBN in 4 mL of dry DMF were added. After purging with N2 gas for 30 min, the reaction started at 80 °C with magnetically stirring for 8 h. The polymerization was quenched by exposing it to air in an ice bath. The product was purified by precipitation from cold diethyl ether (−10 °C) and dried in a vacuum oven at 40 °C for 24 h, yielding a white solid .The PLGA-b-PNAM1H NMR :δ (ppm) = 5.25–5.12 (1Hb), 4.88–478 (2Ha) 4.65 (4Hk), 3.9–3.1 (8Hr), 2.78–2.4 (2Hq), 1.92–1.65(1Hp), 1.58–1.20 (3Hc + 6Hi + 6Hl), 0.85–0.83 (3Hf). n(NMR) = 10,670 g mol−1, Mn(GPC) = 11,600 g mol−1 and Đ = 1.38.M2 was dissolved in 2.0 mL of DMSO and the mixture was stirred at room temperature for 6 h. Further, 3.0 mg of DOX.HCl was added dropwise, followed by the addition of 3.5 μL of triethylamine. The reaction mixture was left to stir at room temperature for 24 h. Then, the resultant solution was dialyzed (MW cut off = 3500 Da) against of distilled water, which was renewed periodically for every 2 h during the course of initial 15 h, then every 4 h to remove the unloaded drug for 24 h. After the dialysis, dialyzed drug-loaded polymer solution was filtered and lyophilized. In order to determine the DLC and DLE, lyophilized drug-loaded polymer was dissolved in PBS at pH 7.4 and analyzed by UV absorbance at 485 nm, using a standard calibration curve which was experimentally obtained with different concentrations of DOX/PBS (pH = 7.4) solutions. DLC and DLE were calculated according to the following formula:Drug loading and drug release of M2 micelles were carried out by following the reported methods ,43. In bFor the drug-release study, 5.0 mg of the DOX-loaded polymer was dissolved in 1.0 mL of PBS (pH = 7.4) and transferred into a dialysis tube (MW cut off = 3500 Da). Then the tube was placed into 10 mL of PBS. The system was kept under stirring at 37 °C. At a definite interval, 3.0 mL of the PBS solution was taken out, and the solution was replenished with 3 mL of a fresh PBS solution after each sampling. The amount of drug released was estimated by UV spectroscopy at 485 nm.Mn and Đ of the prepared miktoarm star block copolymers were measured by 1H NMR  and GPC . The micellar solution of the miktoarm-block copolymer was prepared by dialysis technique. The cmc was determined using a dynamic laser light scattering . A series of solutions ranging from 1.0 to 1 × 10−6 mg/mL was prepared from an aqueous/PBS stock solution of miktopolymer at a concentration of 1% (w/v). For transmission electron microscopy , samples were prepared by dropping micellar solutions on carbon coated copper grids. No staining was applied to the sample.The chemical structure,


Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed  Induction therapy is administered peri-transplant to minimize the risk of acute rejection and, ideally, to decrease the need for maintenance immunosuppression (mIS) long-term. Commonly, antibodies are used as part of induction therapy to induce target cell depletion and/or inhibit immune cell activation. Autoimmune conditions are characterized by unwanted immune responses against autologous tissue. Similar to transplantation, monoclonal antibody therapy for treatment of autoimmune conditions aims at depletion and/or inhibition of disease-mediating cells.per se; however, memory T cells (Tmem) are more resistant to depletion than naïve T cells (Tn) leading to a relative enrichment of Tmem post-depletion (Depletion of antibody-bound cells can be mediated through complement-dependent cytolysis (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and apoptosis induction. While depletory antibodies have more side effects than non-depletory induction antibodies, they have been shown to be superior in preventing acute rejection . Depletiepletion . Furtherepletion . Tmem arepletion . Moreoveepletion . Theoretin vitro (+ cells with previous in vitro evidence suggesting ADCC as one mechanism that is elicited by siplizumab as MLR suppression by siplizumab was contingent on the presence of NK cells (in vivo. CD2 expression is increased on Tmem relative to naïve T cells (Tn), and on activated T cells relative to resting T cells (One form of activation inhibition is costimulatory blockade (CoB). CoB agents have been shown to reduce the risk of acute rejection while having less side-effects compared to commonly used depletory antibodies . Siplizuin vitro , 7, in cin vitro , and phain vitro . In addi T cells . Thus, a T cells  showed t T cells . This in T cells , 13.Alemtuzumab is a humanized monoclonal anti-CD52 IgG1 antibody  approved for treatment of multiple sclerosis . It is fThe goal of this study was to further explore the mechanistic profile of siplizumab and to contrast its effects in allogeneic mixed lymphocyte reaction with Alemtuzumab and rATG.Rabbit anti-thymocyte globulin  and Alemtuzumab  were purchased from Apoteket Hjärtat AB . Siplizumab  is an investigational drug and was provided by the manufacturer.6 effector cells per ml once all reagents had been added. Serial dilutions of siplizumab or Fc-silent siplizumab (negative control) in assay buffer or pure assay buffer (untreated controls) were added at the indicated concentrations. The reaction mixtures  were incubated at 37°C 5% CO2 for 23 h. After equilibration to room temperature , 70 µl of freshly thawed Bio-Glo Luciferase assay substrate  was added. After 15–20 min of incubation at RT and in the dark, luminescence was measured using a Synergy HTX Multi-Mode plate reader . Jurkat reporter cells express no or very low levels of luciferase in the absence of target-bound antibodies. If reporter cells bind a target-bound IgG antibody with their respective FcγR, downstream NFAT signaling induces luciferase expression. Bio-Glo Luciferase assay substrate solution induces cell lysis and contains luciferase substrate. Therefore, increased luminescence indicates agonistic FcγR signaling.Jurkat effector cells expressing Fcγ receptor I (FcγRI), FcγRIIA, or FcγRIIIA  were resuspended in assay buffer 3% ultra-low IgG FBS  in RPMI-1640 ATCC Modification  and added to white cell culture plates at a final concentration of 1.1 × 10via Ficoll® Paque Plus  density gradient centrifugation from buffy coats. Buffy coats were obtained from anonymous healthy donors via Uppsala University Hospital blood bank  or Karolinska University Hospital blood bank  and PBMC isolation was carried out within 24 h of blood collection.Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation and incubated with siplizumab or positive control rat anti-CD2 IgG2b mAb  in 10% ultra-low IgG FBS in PBS at the indicated concentrations for 30 min at 1 × 107 cells per ml at RT with shaking. Subsequently, an equal volume of rabbit complement  was added followed by 60 min of incubation at RT with shaking. Cells were subsequently washed twice in saline solution, blocked with Fc-receptor binding inhibitor  and then stained with 7-Aminoactinomycin D , anti-CD3 APC (Clone HIT3a), anti-CD56 BV421 (Clone NCAM16.2), and anti-CD19 APC-H7 (Clone SJ25-C1) for 30 min on ice and shielded from light. All antibodies were purchased from BD Biosciences . Subsequently, samples were washed twice in saline solution and acquired using a BD Celesta flow cytometer . Positive staining of lymphocytes for 7-AAD was used as a read-out for complement-dependent cytolysis .PBMC were isolated 7 cells per ml and stained with violet proliferation dye 450  according to the manufacturer’s instructions. This study employed a two-way allogeneic MLR setup where PBMC from neither donor were inactivated via irradiation or chemical treatment. This means that PBMC from both donors functioned as responders to foreign HLA on PBMC from the other donor and stimulators providing foreign HLA against which PBMC from the other donor can respond. As this study investigated the effect of antibody agents on T and NK cell responses in general and not responsiveness of a specific donor, this setup was deemed satisfactory.Equal amounts of PBMC from each donor were mixed in PBS at a concentration of 1.5–2.0 × 106 cells per ml . For Treg phenotyping cells were kept in culture at 37°C, 5% CO2 for 7, 10, and 14 days with 100 µl fresh culture medium  being added after 7 days of culture.For Treg analysis, stained PBMC were washed and resuspended in 10% heat-inactivated fetal bovine serum  in RPMI-1640 ATCC Mod. supplemented with 50 U/ml Streptomycin and Penicillin . Resuspended PBMC were dispensed into round-bottom 96-well cell culture plates (for flow cytometry) and pure medium (no antibody controls) or antibody solution  in culture medium was added to a final concentration of 2 × 106 cells per ml . MLRs were incubated at 37°C, 5% CO2 for 1, 2, 4, and 7 days, respectively. On day 6, 100 µl fresh culture medium  was added to each well.For all other MLRs, stained PBMC were washed and resuspended in 10% heat-inactivated FBS  in AIM V medium . Resuspended PBMC were dispensed into round-bottom 96-well cell culture plates (for flow cytometry) and pure medium (no antibody controls) or antibody solution  was added to a final concentration of 2 × 106 cells/ml without addition of antibody (untreated controls) or addition of 10 µg/ml of the respective antibody agent . Cells were analyzed via flow cytometry prior to culture and on days 1, 3, 5, and 7.T cells were isolated from PBMC using the Pan T cell Isolation Kit  according to the manufacturer’s instructions. T cells were kept in autologous culture at a concentration of 1 × 10via CD28/CD3 beads  for 48 h  and target antigen expression was investigated.Resting PBMC were stained and analyzed for target antigen expression (See below). Subsequently, PBMC in autologous culture were activated Before staining, cells were washed twice in saline solution. All antibodies listed below were purchased from BD Biosciences  if not specified otherwise.+ CD56− and CD3− CD56+, respectively. CD69+ and HLA-DR+ T cells were identified using fluorescence minus one controls , days 1, 2, 4, and 7.Samples for analysis of T cell activation, T cell proliferation, T cell subpopulations and NK cell activation were blocked with Fc-receptor binding inhibitor  and then stained with anti-CD3 VioGreen , anti-CD45RA BV650 (Clone HI100), anti-CD69 BV786 (Clone FN50), anti-CD8 BB550 (Clone RPA-T8), anti-CD56 PE , anti-HLA-DR PerCP-Cy5.5 (Clone G46-6), anti-CCR7 APC (Clone G43H7), and anti-CD4 APC-Vio770 . T and NK cells were defined as CD3+ CD127− CD25+ FoxP3+ , anti-CD25 PE (Clone M-A251), anti-CD45RA APC-H7 (Clone HI100), anti-CD127 PerCP-Cy5.5 (Clone HIL-7R-M21), and anti-FoxP3 Alexa647 (Clone 259D/C7). They were permeabilized using BD Human FoxP3 Buffer Set  according to the vendor’s protocol. Tregs were identified as CD4Samples from Pure T cell cultures were analyzed with anti-CD69 APC (Clone FN50) and anti-CD3 PerCP-Cy5.5 (Clone SP34-2).Samples investigating antigen expression were incubated with Fc-receptor binding inhibitor  and then washed twice in FBS staining buffer  followed by incubation with 10 µg/ml siplizumab, Alemtuzumab or rATG for 20 min. Unbound antibody was removed before staining with secondary anti-human IgG Fc BV421 . Unbound antibody was removed before surface staining for T cell subpopulations, regulatory T cells or B and NK cells. T cell subpopulation and Treg panels were the same as described above. NK/B cell panel consisted of anti-CD16 FITC (Clone 3G8), anti-CD56 PE , anti-CD3 APC (Clone SK7) anti-CD14 APC-H7 (Clone M5E2), and anti-CD20 PerCP (Clone SJ25C1). MFI values for T and NK cell populations were adjusted for background signal using an isotype control. More specifically, the MFI value observed for each T/NK cell population in PBMC stained with Rituximab and secondary antibody were subtracted from values of samples incubated with siplizumab, Alemtuzumab or rATG.via flow cytometry on days 7, 10, and 14 for Treg analysis. Cell proliferation was assessed using VPD450 .Treg analysis samples, pure T cell culture samples and antigen expression samples were stained in the dark at 4°C and were acquired using a FACSVerse flow cytometer . MLR samples were phenotyped ® 10.5.3 software .Post-acquisition editing and data analysis of all flow cytometry data was conducted using FlowJo+ CD25+ FoxP3+ cells were sorted via fluorescent-activated cell sorting (FACS) using a FACSAria III flow cytometer . Samples were frozen in liquid nitrogen as minimal fluid pellets and stored at −80°C until analysis by EpigenDx  using direct bisulfite modification with pyrosequencing .After 7 and 14 days of MLR as described above—supplemented with no antibody (Untreated control) or 10 µg/ml of the respective antibody agent—CD4Visualization of results and statistical analysis of underlying data were carried out using GraphPad Prism 8 software . Data displayed in graphs and flow cytometry gating strategies can be found in supplementary information. Data were analyzed using two-way ANOVA followed by Dunnett’s multiple comparison test with no antibody controls serving as the comparison data set. Significant differences are indicated as * (p < 0.05) in figures.via surface plasmon resonance (data not shown). While siplizumab did not induce CDC, the positive control anti-CD2 mAb (BTI-322) induced a dose-dependent increase in the percentage of 7-AAD+ lymphocytes (As displayed in via violet proliferation dye 450 (VPD450) tracking as measured by flow cytometry after 1, 2, 4, and 7 days of allogeneic MLR . Siplizumab significantly reduced T cell count after 7 days of allogeneic MLR at 0.1 µg/ml (p = 0.0014), 1 µg/ml (p = 0.0035), and 10 µg/ml (p = 0.0082). Alemtuzumab induced significant T cell depletion on all time points at 0.1, 1, and 10 µg/ml (p < 0.0001). At 0.01 µg/ml Alemtuzumab induced mild but significant T cell depletion after 1 (p = 0.0157) and 2 (0.0036) days of allogeneic MLR. rATG induced significant T cell depletion after one and 2 days of allogeneic MLR at 0.01 µg/ml , 0.1 µg/ml , 1 µg/ml , and 10 µg/ml (p < 0.0001). Only 10 µg/ml rATG led to sustained depletion through days 4 (p = 0.0001) and 7 (p = 0.0030).To complement proliferation data, T cell count was analyzed . Tregs were identified as CD4+ CD127− CD25+ FoxP3+ and were analyzed after 7, 10, and 14 days of allogeneic MLR.Expansion of alloreactive Tmem can facilitate allograft rejection, especially since they have a lower activation threshold and decreased sensitivity to immunosuppressive agents. Further, Tregs are commonly seen as promoting peripheral tolerance. To investigate whether mAb-induced changes in T cell proliferation and count influenced T cell phenotype, T cell subpopulations were analyzed with regards to naïve, memory and regulatory phenotypes using flow cytometry. Naïve T cells (Tn) were defined as CCR7+ Tn at all tested concentrations after 1 day of allogeneic MLR (p ≤ 0.0342) as well as at 0.001–10 µg/ml after 2 (p ≤ 0.0077) and 4 (p ≤ 0.0187) days of allogeneic MLR. After 7 days, statistically significant enrichment of CD4+ Tn was observed in samples treated with 0.01 µg/ml (p = 0.0423) and especially at 0.1–10 µg/ml (p < 0.0001) siplizumab. Similarly, siplizumab enriched CD8+ Tn at all tested concentrations after 1 day (p ≤ 0.0398), at or above 0.001 µg/ml after 2 days (p ≤ 0.0039), at or above 0.01 µg/ml after 4 days (p ≤ 0.019) and at 0.1 µg/ml or higher after 7 days (p ≤ 0.0044) of allogeneic MLR. Notably, enrichment of Tn by siplizumab at concentrations of 0.1 µg/ml or higher tended to be more pronounced than at 0.01 µg/ml. In comparison, Alemtuzumab increased CD4+ Tn at 0.0001–1 µg/ml on day 1 (p ≤ 0.0362), 0.01–10 µg/ml on day 2 (p ≤ 0.0255), and 0.01–0.1 µg/ml on day 4 (p ≤ 0.0005) of allogeneic MLR. No notable enrichment of Tn among CD8 T cells was detected in Alemtuzumab-treated samples. Further, rATG enriched CD4+ Tn at 0.001 µg/ml (p = 0.0411) and 0.1–10 µg/ml (p ≤ 0.0285) on day 1, at 0.01–10 µg/ml (p = 0.0411) on day 2 (p ≤ 0.0338), and at 0.01 µg/ml on day 4 (p = 0.0470) of allogeneic MLR. Moreover, 10 µg/ml rATG induced a significant depletion of CD4+ Tn on day 7 (p = 0.0273). No clear dose-dependent enrichment or depletion of CD8+ Tn occurred in rATG-treated samples.Untreated controls (no antibody) tended to display a continuous decline in the frequency of Tn among CD4 and CD8 T cells  and 14 (p < 0.0001) days as well as among proliferated CD4+ T cells after 7 (p = 0.0004), 10 (p < 0.0001) and 14 (p < 0.0001) days of allogeneic MLR. Treg phenotyping could not be performed in Alemtuzumab-treated samples due to an insufficient number of Treg events upon flow cytometric analysis (severe T cell depletion). Similar to siplizumab, rATG significantly enriched Tregs among total CD4+ T cells and proliferated CD4+ T cells after 7 , 10 (p < 0.0001 for both), and 14 (p < 0.0001 for both) days of allogeneiec MLR.Untreated controls (no antibody) displayed approximately 7.5% Tregs among total CD4bona fide Tregs vs. activation-induced FoxP3 expression), FoxP3 promoter methylation in FACS-sorted Tregs was measured at baseline (day 0) as well as after 7 and 14 days of allogeneic MLR  Tregs. FoxP3 promoter methylation in Tregs isolated from siplizumab-treated samples after 7 days of allogeneic MLR was significantly lower than in Tregs isolated from untreated controls (p = 0.0190). Further, the decrease in FoxP3 promoter methylation measured in Tregs isolated from siplizumab-treated samples after 14 days of MLR trended toward significance (p = 0.0523). In contrast, rATG did not induce a significant decrease in FoxP3 promoter methylation after 7 and 14 days of MLR.To further investigate the identity of Tregs (Given that T cell proliferation and differentiation tended to occur during later time points (after day 4), activation marker expression (CD69 and HLA-DR) on T cells in allogeneic MLRs was analyzed at baseline and after 1, 2, 4, and 7 days of alogeneic MLR to detect potential early effects of mAb treatment on T cell activation , 1 µg/ml (p = 0.0279), and 10 µg/ml (p = 0.0216). Significant inhibition of T cell CD69 expression was maintained on day 4 in samples treated with 10 µg/ml siplizumab (p = 0.0351). Additionally, HLA-DR expression on T cells was significantly inhibited after 2, 4, and 7 days of allogeneic MLR in samples treated with 0.1 µg/ml (p ≤ 0.0373), 1 µg/ml (p ≤ 0.0189), and 10 µg/ml (p ≤ 0.0190) siplizumab. In contrast, Alemtuzumab significantly increased the percentage CD69+ T cells and HLA-DR+ T cells after 1 day of allogeneic MLR at 0.01 µg/ml , 0.1 µg/ml , 1 µg/ml , and 10 µg/ml . Alemtuzumab induced a significant increase in HLA-DR expression after 2 days of allogeneic MLR at 0.1 µg/ml (p = 0.0167) and 1 µg/ml (p = 0.0157). Increased HLA-DR expression at 10 µg/ml on day 2 trended toward significance (p = 0.0731). After 4 days HLA-DR expression on T cells remained significantly elevated in allogeneic MLRs treated wth 1 µg/ml (p = 0.0261) and 10 µg/ml (p = 0.0010) Alemtuzumab. A significant increase in CD69+ T cells was detected after 1, 2, and 4 days of allogeneic MLR in samples treated with 0.001 µg/ml (p ≤ 0.0467), 0.01 µg/ml (p ≤ 0.0024), 0.1 µg/ml (p ≤ 0.0017), 1 µg/ml (p ≤ 0.0007), and 10 µg/ml rATG (p < 0.0001). After 7 days, rATG induced a significant increase in CD69 expression on T cells at 0.01 µg/ml, 0.1 µg/ml, 1 µg/ml, and 10 µg/ml . A decrease in HLA-DR expression in rATG-treated samples was noted at 0.01–10 µg/ml; however only, 0.01 µg/ml (0.0170) and 10 µg/ml (p = 0.0048) reached significance.The percentage of CD69To investigate the effects of target antigen-binding in isolation, untouched T cells were isolated using negative magnetic bead-selection and kept in autologous culture with or without the addition of antibody drugs. Expression of CD69 on T cells was measured at baseline (day 0) and after 1, 3, 5, and 7 days of autologous culture  and after 1, 2, 4, and 7 days of allogeneic MLR (CD69 is an early activation marker on T and NK cells. As depletory mAbs can induce ADCC + NK cells at 0.001 µg/ml (p = 0.0284), 0.01 µg/ml (p = 0.0001), and 0.1–10 µg/ml (p < 0.0001). On days 2, 4, and 7 of allogeneic MLR, siplizumab significantly increased CD69+ NK cells at 0.01 µg/ml (p ≤ 0.0377), 0.1 µg/ml (p ≤ 0.0100), 1 µg/ml (p ≤ 0.0248), and 10 µg/ml (p ≤ 0.0115). Similarly, Alemtuzumab significantly increased CD69 expression on NK cells on day 1 at 0.001 µg/ml (p = 0.0248), 0.01 µg/ml (p = 0.0001), and 0.1–10 µg/ml (p < 0.0001), on day 2 at 0.01 µg/ml (p = 0.0024) and 0.1–10 µg/ml (p < 0.0001), on day 4 at 0.1 µg/ml (p = 0.0005), 1 µg/ml (p = 0.0003) and 10 µg/ml (p = 0.0007), as well as on day 7 at 0.1 µg/ml (p = 0.0003) and 1 µg/ml (p = 0.0076). In comparison, rATG significantly increased CD69+ NK cells at 0.01–10 µg/ml (p ≤ 0.0102) on day 1, 0.1–10 µg/ml (p ≤ 0.0022) on day 2, 1–10 µg/ml (p ≤ 0.0266) on day 4 as well as 1–10 µg/ml (p ≤ 0.0038) on day 7 of allogeneic MLR. Interestingly, rATG inhibited CD69 expression on NK cells on day 4 at 0.001 µg/ml (0.0034) and 0.01 µg/ml (p = 0.0001).The percentage of CD69+ NK cells in allogeneic MLR increased from on average 1.95% at baseline to 15.29% on day 7. On day 1, siplizumab induced a significant increase in CD69via flow cytometry. Moreover, target antigen expression was investigated on both resting and activated PBMC to detect any potential up- or downregulation associated with cell activation.Heterogenous target antigen expression on T and NK cell subpopulations may lead to certain subpopulations being more or less affected by mAb treatment. Therefore, target antigen expression on T and NK cell subpopulations was measured via an isotype control (Rituximab). Tn express CD2 at lower levels than Tmem, with the highest expression seen on Tem. CD56Bright NK cells had a higher expression of CD2 compared to CD56Dim and CD56NegCD16+ NK cells. CD52 was highly expressed on cell types investigated although somewhat lower on NK cells and CD8+ Temra compared to other T cell populations and B cells. CD25 is relatively highly expressed on CD4+ T cells and Tregs in particular. rATG is a polyclonal antibody mixture with some of the most common clones binding CD2, CD5, CD8, CD11a, and CD18  may explain the rapid clearance of target cells from peripheral blood  Tregs. Interestingly, anti-CD2 antibodies are among the most abundant clones in rATG . Therefore, Tregs may be more resistant to siplizumab-mediated depletion than other T cell subpopulations and/or less reliant on CD2/LFA3 costimulation for activation and differentiation. The fact that siplizumab enriches aTregs despite their high CD2 expression further suggests that depletion of CD2+ cells may not be the sole mechanism of action of siplizumab. The exact mechanisms by which siplizumab enriches Tregs remain incompletely characterized and should be investigated more deeply in future research. Notably, it should be investigated whether Fc-receptor-mediated crosslinking of siplizumab-bound CD2 has effects on T cell activation, proliferation, differentiation and interaction with APCs which are independent of Fc receptor-mediated depletion and CD2/LFA3 CoB.Previous evidence has shown that Tregs enriched by siplizumab are CD45RAWhen using depletory antibodies, the phenotypes of both depleted and non-depleted cells are important to consider. Reconstitution from lymphopenic states is achieved by both thymic and bone marrow output of naïve lymphocytes as well as homeostatic proliferation of non-depleted lymphocytes . DepletiWhile non-depletory antibody agents would avoid the potential problem of homeostatic proliferation of Tmem and depletion of pathogen-reactive T cells, they do not eliminate alloreactive cells. Thus, while these agents may contribute to preventing rejection, they are unlikely to make significant contributions to immunological engraftment as they merely prevent activation of alloreactive T cells instead of actively modelling the T cell repertoire toward selective tolerance. Given that CD2 is upregulated on Tmem and upon T cell activation, siplizumab may be an optimal depletory agent to use for skewing the post-depletion T cell repertoire toward Tn and Treg phenotypes.T cell malfunction characterizes many autoimmune and chronic inflammatory pathologies, for example multiple sclerosis (MS), rheumatoid arthritis (RA), and type-1 diabetes. In these cases, the use of a T cell depletory agent could be of great interest. Investigators using the anti-CD2 biologic Alefacept for treatment of type-1 diabetes have reported promising results . AlemtuzIn conclusion, each of the tested antibody agents has displayed a unique functional profile. When considering desired characteristics for an antibody agent used during organ transplant induction therapy or treatment of autoimmune diseases , siplizumab has a particularly attractive mechanistic profile. None of the tested agents is likely to create an optimal immunological environment in patients by itself. However, the informed use of these agents as part of more elaborate treatment protocols can contribute to minimizing the risk of rejection and need for immunosuppression long-term as well as maximizing the chance of patients developing durable immune tolerance.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CB and FS carried out experiments and wrote the first manuscript. All authors contributed to the article and approved the submitted version.All authors are employees of ITB MED AB. EB and DB own shares in ITB MED AB.


N-Dealkylation, N-deacylation, hydroxylation, and N-oxidation were the most abundant metabolic reactions and the corresponding metabolites are expected to be promising analytical targets for toxicological analysis. Isozyme mapping revealed the main involvement of CYP3A4 in the phase I metabolism of 4F-Cy-BAP and in terms of Fu-BAP additionally CYP2D6. Therefore, drug-drug interactions by CYP3A4 inhibition may cause elevated drug levels and unwanted adverse effects. MTC experiments revealed malformations and changes in the behavior of larvae after exposure to 100 µM Fu-BAP. Both substances were only able to produce a weak activation of MOR and although toxic effects based on MOR activation seem unlikely, activity at other receptors cannot be excluded.The two fentanyl homologs cyclopropanoyl-1-benzyl-4´-fluoro-4-anilinopiperidine (4F-Cy-BAP) and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have recently been seized as new psychoactive substances (NPS) on the drugs of abuse market. As their toxicokinetic and toxicodynamic characteristics are completely unknown, this study focused on elucidating their in vitro metabolic stability in pooled human liver S9 fraction (pHLS9), their qualitative in vitro (pHLS9), and in vivo (zebrafish larvae) metabolism, and their in vitro isozyme mapping using recombinant expressed isoenzymes. Their maximum-tolerated concentration (MTC) in zebrafish larvae was studied from 0.01 to 100 µM. Their µ-opioid receptor (MOR) activity was analyzed in engineered human embryonic kidney (HEK) 293 T cells. In total, seven phase I and one phase II metabolites of 4F-Cy-BAP and 15 phase I and four phase II metabolites of Fu-BAP were tentatively identified by means of liquid chromatography high-resolution tandem mass spectrometry, with the majority detected in zebrafish larvae. The online version of this article (10.1007/s00204-020-02726-1) contains supplementary material, which is available to authorized users. More and more compounds intended to be consumed as substitutes and/or alternatives to classic opioids such as heroin are brought into the drugs of abuse market (Beardsley and Zhang N-(1-benzylpiperidin-4-yl)-arylacetamides, structurally related compounds, were described to be potent agonists at the sigma receptor -arylacetamide at the sigma1 and sigma2 receptor of 3.9 and 240 nM, respectively. Fu-BAP and related compounds were also identified as antagonists at the acetylcholine M2 and M3 receptor with Ki values of 794 and 100 nM for Fu-BAP, respectively  and furanoyl-1-benzyl-4-anilinopiperidine (Fu-BAP) have been seized in Europe and were intended to be brought onto the market as NSO EMCDDA . Their cTo close the knowledge gap concerning their toxicokinetics and toxicodynamics, the present study aimed to elucidate the toxicokinetics of these compounds, including in vitro metabolism in pooled human liver S9 fraction (pHLS9) incubations in comparison to in vivo metabolites identified using the zebrafish larvae model, isozyme mapping, and the determination of plasma protein binding (PPB). Toxicodynamic properties should include characterizing the MOR activity in engineered human embryonic kidney (HEK) 293 T cells as well as maximum-tolerated concentration (MTC) studies in zebrafish larvae.3, isocitrate, isocitrate dehydrogenase, superoxide dismutase, 3′-phosphoadenosine-5′phosphosulfate (PAPS), S-(5′-adenosyl)-l-methionine (SAM), dithiothreitol (DTT), reduced glutathione (GSH), magnesium chloride (MgCl2), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), tris hydrochloride, fetal bovine serum (FBS), and poly-d-lysin were from Sigma Aldrich  and NADP + from Biomol . Centrifree devices were obtained from Merck . Dulbecco’s Modified Eagle’s Medium , Opti-MEM® I Reduced Serum Medium, penicillin–streptomycin (5.000 U/mL) and amphotericin B (250 µg/mL) were purchased from Thermo Fisher Scientific . The Nano-Glo® Live Cell reagent, which was used for the readout of the MOR bioassay, was procured from Promega . Acetonitrile (LC–MS grade), methanol (LC–MS grade), ammonium formate , formic acid (LC–MS grade), and all other reagents and chemicals  were from VWR . Zebrafish embryos were obtained from in-house bred adult zebrafish of the AB wild-type line. The baculovirus-infected insect cell microsomes (Supersomes) containing human cDNA-expressed flavin-containing monooxygenase 3 (FMO3) (5 mg protein/mL), CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP3A4 (1 nmol/mL), CYP2C9, CYP2E1, or CYP3A5 (2 nmol/mL), as well as pooled human liver microsomes , pooled human liver S9 fraction , UGT reaction mixture solution A (25 mM UDP-glucuronic acid), and UGT reaction mixture solution B  were supplied by Corning . After delivery, the enzymes were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at − 80 °C until use.4F-Cy-BAP and Fu-BAP were provided as citrate salts for research purposes from the EU-project ADEBAR/State Bureau of Criminal Investigation Schleswig–Holstein . Chemical purity and identity of the compounds were verified by mass spectrometry (MS) and nuclear magnetic resonance analysis. Stock solutions in methanol (1 mg/mL) or DMSO were freshly prepared before each experiment. Hydromorphone was purchased as hydromorphone HCl from Fagron . Fentanyl was obtained as a free base from LGC Chemicals . Trimipramin-d2+, 2.5 mM isocitrate, 0.6 mM NADP + , 0.8 U/mL isocitrate dehydrogenase, 100 U/mL superoxide dismutase. Thereafter, 2.5 mM UDP-glucuronic acid (UGT reaction mixture solution A), 40 μM PAPS, 1.2 mM SAM, 1 mM DTT, 10 mM GSH was added. To ensure a linear metabolism during incubation, the compound concentrations were set at 2.5 µM  . In order to identify metabolites formed by NADP+ independent enzymes, incubations without NADP+ were also performed. Blank incubations without substrate and control incubations without enzyme (pHLS9) were prepared to examine whether interfering or non-metabolically formed compounds were present.Reactions were initiated after addition of 4F-Cy-BAP or Fu-BAP and continued for 360 min. Meanwhile, 30 µL samples were taken after 1, 15, 30, 45, 60, 75, 90, 180, and 360 min, respectively. Reactions were stopped by adding 10 µL ice-cold acetonitrile. Afterwards, the samples were cooled for 30 min at − 20 °C, centrifuged at 18,407×t-test was done to confirm that there was no significant difference between the compound concentration at 360 min in control incubations and the initial concentrations in the pHLS9 incubations at 1 min. GraphPad Prism 5.00  was used for statistical calculations with the following defined settings: unpaired; two-tailed; significance level, 0.05; confidence intervals, 99%.Metabolic stability was determined by declining substrate concentration  and intrinsic clearance  was predicted using parallel tube model with (Eq. fu) and well-stirred model with  was transferred to a new reaction tube. All samples were precipitated by adding a volume of 50 µL of ice-cold acetonitrile containing trimipramine-d3 (2.5 µM) as internal standard (IS). This was done as there was no deuterated 4F-Cy-BAP or Fu-BAP available and trimipramine-d3 was shown in be suitable as IS. Afterwards, they were cooled for 30 min at − 20 °C, centrifuged for 2 min at 18,407×g, and measured by LC-HRMS/MS. Ultrafiltration was done in triplicate.PPB studies were done as described earlier . Reactions were stopped after 1, 5, 10, 15, 20, 25, and 30 min. Blank incubations without substrate and negative control incubations without enzymes were conducted to examine whether interfering or non-metabolically formed compounds were present. All incubations were done in duplicate.As described elsewhere 2, 1.5 mM HEPES, and 1.2 µM methylene blue. MTC studies were performed by placing the collected embryos in 6-well plates with 10 embryos per well in 2 mL Danieau’s medium. Zebrafish larvae at 4 days post-fertilization (dpf) were exposed to 4F-Cy-BAP and Fu-BAP dissolved in Danieau’s medium containing 1% (v/v) DMSO (waterborne exposition). Final compound concentrations were 0.01, 0.1, 1, 10, 50, and 100 µM. A negative control without drug was prepared, to exclude morphological malfunctions caused by DMSO  formic acid, shaking for 2 min, and cooling for 30 min at − 20 °C. Before analysis, the samples were centrifuged at 18,407×g for 2 min and the supernatant was transferred to an autosampler vial. Twenty larvae (one tube) were extracted with 50 µL methanol and shaken for 2 min. After centrifugation at 18,407×g for 2 min, the supernatant was transferred to an autosampler vial. All above described experiments were prepared and analyzed in triplicate. Blank zebrafish larvae (n = 2) were incubated in the medium without drugs and analyzed along with their blank medium to identify interfering compounds. Furthermore, a control medium sample containing only the drug in Danieau´s medium was prepared, respectively, for the detection of compound degradations during incubation.Extraction of the medium was conducted by precipitation of 50 µL medium with 50 µL acetonitrile containing 0.1%  following agonist-induced recruitment of a β-arrestin 2 (βarr2) protein  to MOR (fused to a large part of NanoLuc). Details regarding the development of the stable cell line used here have been reported elsewhere . Luminescence was monitored during the equilibration period until the signal stabilized (15 min). We added 20 µL per well of test compounds, present as 6.75 × stocks  in Opti-MEM® I. The luminescence was continuously measured for 120 min. Solvent controls were run in all experiments. Curve fitting and statistical analyses were performed using GraphPad Prism. The concentration–response curves were generated from experiments performed in triplicate, the data points representing the mean area under the curve (AUC) ± standard error of mean (SEM). All results were normalized to the maximal activity (Emax) of hydromorphone (= 100%), used as the reference compound. Curve fitting of concentration − effect curves via nonlinear regression was employed to determine the potency (EC50) and the efficacy (Emax).Engineered HEK 293 T cells were routinely maintained at 37 °C, 5% COv/v, pH 3, eluent A) and 2 mM ammonium formate solution with acetonitrile:methanol , water , and formic acid . At first, the flow rate was set to 500 μL/min for a period of 10 min followed by 800 µL/min for 10–13.5 min. The gradient was stepped from 0 to 1 min hold 99% A, 1–10 min to 1% A, 10–11.5 min hold 1% A, and 11.5–13.5 min hold 99% A. HESI-II source settings were: heater temperature, 320 °C; ion transfer capillary temperature, 320 °C; spray voltage, 4.0 kV; ionization mode, positive; sheath gas, 60 arbitrary units (AU); auxiliary gas, 10 AU; sweep gas, 0 AU; and S-lens RF level, 50.0. External mass calibrations were done in advance before analysis as recommended by the manufacturer. Identification and quantification of parent compounds and metabolites were performed using full scan data and a subsequent data‐dependent MS2 (dd-MS2) mode with an inclusion list containing the exact masses of the respective parent compound and its presumed metabolites. Expected phase I metabolites such as hydroxy, dihydroxy or N-dealkyl metabolites and phase II e.g. sulfates, glucuronides were the inclusion lists. Full scan data acquisition was conducted as follows: resolution, 35,000; microscans, 1; automatic gain control (AGC) target, 1e6; maximum injection time (IT), 120 ms; and scan range, m/z 50–750. The following settings for the dd-MS2 mode were defined: option “pick others”, enabled; dynamic exclusion, disabled; resolution, 17,500; microscans, 1; isolation window, 1.0 mass-to-charge ratio (m/z); loop count, 5; AGC target, 2e5; maximum IT, 250 ms; high collision dissociation cell with stepped normalized collision energy, 17.5, 35.0, 52.5; exclude isotopes, on; spectrum data type, profile; and underfill ratio, 1%. Chemical structure drawings of presumed metabolites and exact mass calculations were prepared by ChemSketch 2010 12.01 . Data handling was performed by TF Xcalibur Qual Browser software version 2.2. Automated peak integration settings were as follows: mass tolerance, 5 ppm; peak detection algorithm, INCOS; baseline window, 40; area noise factor, 5; and peak noise factor, 10.The used Thermo Fisher Scientific  Dionex UltiMate 3000 RS pump was composed of a degasser, a quaternary pump, and an UltiMate autosampler and connected to a TF Q-Exactive Plus system equipped with a heated electrospray ionization source (HESI)-II. A volume of 1 µL was injected for all samples. Gradient elution was performed as described earlier  and 95% (Fu-BAP). CLh predictions of Fu-BAP in consideration of fu resulted in 0.7 mL/min/kg in both models. In disregard of fu, CLh values were 10.6 mL/min/kg, calculated with the parallel tube and 8.6 mL/min/kg with the well-stirred model, which gave ERh values of 0.5 and 0.4, respectively.Metabolic stability data are summarized in Table S1 in the Electronic Supplementary Material (ESM). Non-metabolic compound degradation during the pHLS9 incubations could be excluded by control incubations as the 2 mode were compared to that of the respective parent compound. All metabolites are listed in Table S2 in the ESM along with their metabolite ID, PI recorded in MS1, characteristic fragment ions (FIs) in MS2, relative intensities, calculated exact masses, elemental composition, mass deviation errors of the most abundant FIs, and retention time (RT). The in vitro and in vivo metabolic pathways of 4F-Cy-BAP and Fu-BAP are depicted in Fig. 2 spectra of 4F-Cy-BAP or Fu-BAP and their three most abundant in vitro and in vivo metabolites are given in Figs. 2 spectra of the lower abundant metabolites are represented in Fig. S1 (4F-Cy-BAP) and Fig. S2 (Fu-BAP) in the ESM.To avoid redundancies and to ease readability, results of in vitro and in vivo metabolite identification will be combined in the following section. Metabolites were identified by mining the data recorded in full-scan mode for their on beforehand calculated exact precursor ions (PIs). Subsequently, the spectra of the tentative metabolites obtained from the dd-MSm/z 353.2023) as well as Fu-BAP (PI at m/z 361.1910) were FIs at m/z 174.1277 and at m/z 91.0542. The former fragment originated from the benzyl piperidine part of the compounds and the latter of the phenyl coupled to the methyl spacer after piperidine cleavage. A distinctive fragment of 4F-Cy-BAP was the FI at m/z 246.1288, which was generated after the separation of the piperidine nitrogen plus benzyl part. Another prominent FI at m/z 69.0334 contained the cyclopropyl and carbonyl moiety formed after amide cleavage. Equally, distinguishing FIs of Fu-BAP were the less abundant FI at m/z 254.1175 and the FI at m/z 95.0127, which differed from the MS2 fragments of 4F-Cy-BAP through substitution of the cyclopropyl with the furanyl group.In the following section, only exact masses will be used for the characterization of parent compounds and their respective metabolites. High abundant but less characteristic FIs of 4F-Cy-BAP (PI at m/z 263.1554), which originated from N-dealkylation at the piperidine nitrogen. A characteristic FI was FI at m/z 180.0819, which consisted of the fluorophenyl linked to the cyclopropyl moiety. M2 (PI at m/z 285.1761), showed a similar fragmentation pattern as the parent compound, except for the missing FI at m/z 69.0334, which represented the cyclopropyl and carbonyl moiety. N-oxidation of the piperidine nitrogen led to the formation of M7 (PI at m/z 369.1972). The characteristic FI at m/z 98.0600 correlated with FI at m/z 84.0807 varying in one oxygen and two missing hydrogen atoms.One of the most abundant metabolites of 4F-Cy-BAP was M1 (PI at m/z 267.1855) emerged from N-deacylation at the amide. Its MS2 spectrum was similar to that of the parent compound, except for the FI at m/z 95.0127, which originated from the furanyl part. M10 (PI at m/z 271.1441) was formed by N-dealkylation at the piperidine nitrogen and specified by FI at m/z 188.0706, which was generated after separation of the piperidine. M15 (PI at m/z 377.1859) was one of two hydroxy isomers, with the hydroxy group located at the phenyl part, which was part of the benzyl moiety. The FI at m/z 107.0491 corresponded to the FI at m/z 91.0548, which was altered by one oxygen atom. Both hydroxy isomers  were distinguishable from each other by different RT and intensities.The Fu-BAP metabolite M9  was formed by N-deacylation at the amide and followed by hydroxylation at the phenyl, which was represented by the prominent FI at m/z 107.0491. N-Deacylation followed by N-oxidation of the linker nitrogen between piperidine and fluorophenyl led to the formation of M4 (PI at m/z 301.1710). A characteristic FI of M4 was FI at m/z 193.1135, which was matched with the fluorophenyl part linked to the piperidine ring with one double bond indicating loss of water. The two hydroxy isomers M5 and M6 (PI at m/z 369.1972) were formed by hydroxylation at the phenyl part. As already described for the Fu-BAP metabolite M15, M5 and M6 were characterized by the same FI at m/z 107.0491. A differentiation between both isomers was possible by different RT. The phase II metabolite M8 (PI at m/z 545.2293) was formed by glucuronidation of M5 or M6, which was also identified by the FI at m/z 107.0491.The m/z 283.1804) were two Fu-BAP metabolites formed by N-deacylation plus hydroxylation. M12 was the equivalent N-deacyl metabolite of M15 or M16, which was characterized by FI at m/z 107.0491. The hydroxylation of M11 (PI at m/z 283.1804) occurred at the phenyl, which was designated by FI at m/z 192.1258 originating from elimination of the benzyl part. M13 (PI at m/z 305.1495) was formed by N-dealkylation and dihydrodiol formation by epoxidation of one double bond at the furanyl, followed by a non-enzymatic hydrolysis. Identification of M13 followed FI at m/z 166.0862, which contained the phenyl linked to a remaining part of the furanyl. M14 (PI at m/z 363.1373) stemmed from M11 through sulfation of the hydroxy group and their MS2 spectra were in accordance to each other. The second hydroxy isomer M16 (PI at m/z 377.1859) showed a similar fragmentation pattern as M15. M17 (PI at m/z 377.1859) originated from N-oxidation of the piperidine nitrogen, which was also specified with FI at m/z 98.0600, as already described for M7 of 4F-Cy-BAP. The 2,5-dihydroxypent-2-enal metabolite M18 (PI at m/z 381.2172) occurred through oxidative opening of the furan ring, which was identified by FI at m/z 363.2067 and FI at m/z 174.1277. The former fragment indicated a loss of water and due to the presence of the latter, which was unchanged compared to parent compound, the water loss was located at the opened furan ring. Aromatic dihydroxylation led to the formation of M19 (PI at m/z 393.1808), with the hydroxy groups located at the phenyl being part of the benzyl. The specific FI at m/z 123.0440 represented the dihydroxylated benzyl moiety, which differed in two oxygen atoms to the FI at m/z 91.0548. The dihydrodiol metabolite M20 (PI at m/z 395.1965) was characterized due to the absence of FI at m/z 95.0127, which was assigned to the furanyl part. M21 (PI at m/z 407.1965) occurred through methylation of one hydroxy group at the catechol structure of M19. The pronounced FI at m/z 137.0597 corresponded to FI at m/z 107.0491, which was altered by one additional methoxy group. The dihydroxy-5-oxopent-3-enoic acid metabolite M22 (PI at m/z 411.1914) originated from oxidative furan ring opening, oxidation of the terminal hydroxy group to carboxylic acid, and an additional hydroxylation at the opened side chain as described for the saturated furan ring of a fentanyl analogue  were three dihydrodiol-hydroxy isomers. In case of M23 and M25 the hydroxy group was determined at the phenyl, which belonged to the benzyl moiety, by means of FI at m/z 107.0491. Although the hydroxy group of M24 was located at the furanyl part, due to the presence of FI at m/z 267.1855, which resulted from the cleavage of the furanyl moiety, the precise structure of the furanyl residue was not determinable. Therefore, the most likely ring-closed structure is given for M24. The phase II metabolite M26 (PI at m/z 473.1376) was formed by sulfation of one hydroxy group of M21. It was specified by the FI at m/z 203.0008, which correlated to the FI at m/z 123.0440, differing in one sulfate group. M27 (PI at m/z 553.2180) was the corresponding phase II metabolite of M15 or M16, which was formed by glucuronidation of the hydroxy group. The prominent FI at m/z 107.0491 was used for identification. In the negative control incubations of all in vitro models and in the zebrafish larvae control media, the N-deacyl-metabolites M2 and M9 were also present with similar peak intensities as in the corresponding incubations with enzymes. However, in incubations without NADP+ solely the peak intensity of M9 increased. Blank incubations confirmed the absence of interfering compounds.M11 and M12  was present in incubations of CYP1A2, CYP2C19, CYP3A4, and CYP3A5. Furthermore, CYP2C19 catalyzed the formation of the two hydroxy isomers . The former (M5) was also formed by CYP2D6. CYP3A4 and CYP3A5 contributed to the emergence of the N-oxide (M7). Equally, several isozymes catalyzed the formation of the N-dealkyl Fu-BAP (M10), namely CYP1A2, CYP2C8, CYP2C19, CYP2D6, and CYP3A4. Apart from that, CYP2C8 was only involved in the formation of the N-oxide (M17). CYP2C19 contributed to numerous different steps, amongst them the formation of the two hydroxy isomers , the N-oxide (M17), and the furan ring opened 2,5-dihydroxypent-2-enal metabolite (M18). CYP2D6 was involved in the formation of the dihydrodiol metabolite (M20), the furan ring opened dihydroxy-5-oxopent-3-enoic acid metabolite (M22), as well as the dihydrodiol-hydroxy metabolite (M24). CYP3A4 was another considerable isozyme, catalyzing, besides M10 the formation of the hydroxy isomer (M15), the N-oxide (M17), and the dihydrodiol-hydroxy metabolite (M24).v/v) DMSO. Visible morphological changes manifested in a spinal curvature (1), abnormal pericardial edema (2), and a dark brown colored yolk sac (3), which has already been described e.g. for environmental toxins  and fentanyl (Emax of 180%), as presented in Fig. 50 values.Analysis of the in vitro MOR activation potential of 4F-Cy-BAP and Fu-BAP showed an t1/2, CLint, CLh, and ERh. CLint differs from CLh in the independence of physiological factors, such as hepatic blood flow and drug binding  had its peak already within the first minute of incubation and declined afterwards most likely due to further biotransformation to M3. However, its formation was also observed in stock solutions after long-term storage and negative control incubations and thus, it may be also of artificial nature. Its subsequent metabolites  were also marked to be possible artifacts e.g. in Table +, M9 and all metabolites derived thereof were presented as metabolites. Some metabolites  were solely detected in single isozyme incubations and therefore considered as minor metabolites. Moreover, it can be assumed that the origin of the Fu-BAP metabolite M19 was artificial because the retention time correlated with that of M26. Nevertheless, M19 was obliged to be a precursor of the phase II metabolites M21 and M26, but most probably with another RT. Suitable analytical targets for toxicological urine screenings should be the N-dealkyl metabolites  and the N-deacyl metabolites  of both compounds, and additionally 4F-Cy-BAP N-oxide (M7), as well as hydroxy Fu-BAP (M15).As shown in Fig. Isozyme mapping is essential for the prediction of possible interactions, e.g. drug-drug interactions, or interindividual variations by different expressions of isozymes. Figure 50 values of both compounds were strongly reduced compared to hydromorphone and fentanyl.The receptor activation was evaluated via the interaction between βarr2, a cytosolic protein, and the G-protein coupled MOR. Both βarr2 and MOR are fused to an inactive part of nanoluciferase. When MOR is activated by a ligand, βarr2 is recruited to the receptor, allowing interaction of the complementary nanoluciferase subunits, yielding a functional enzyme that generates a bioluminescent signal in the presence of the substrate furimazine  was originally listed in the US as a Scheduled I controlled substance, but was removed from the list as the Drug Enforcement Administration (DEA) indicated that this compound was inactive at MOR  could be observed in vitro but strong agonism or antagonism at other receptors such as the sigma1, sigma2 or acetylcholine M2 and M3 receptors cannot be excluded and should be investigated.The current study focused on the toxicokinetic and toxicodynamic properties of the fentanyl homologs 4F-Cy-BAP and Fu-BAP. As 4F-Cy-BAP was metabolically much more stable with an in vitro tSupplementary file1 (DOCX 1680 kb)Below is the link to the electronic supplementary material.


The crystal structure of the title heterocyclic amine was determined in the presence of water. The compound co-crystallizes with three water molecules in the asymmetric unit, which leads to the formation of hydrogen bonding in the crystal. 10H13N·3H2O, a heterocyclic amine, was determined in the presence of water. The compound co-crystallizes with three water molecules in the asymmetric unit, which leads to the formation of hydrogen bonding in the crystal.The crystal structure of the title compound, C The structure is comparable with those of other tetrahydroisochinoline derivatives such as 2-(2-chloroacetyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline  Å. The isoquinolines on the other side of the infinite water channel are invertedly aligned along the c axis. This also results in the formation of alternating hydrophilic and hydrophobic phases of the hydrogen-bonded water framework and organic phases of the heterocyclic amines along the a axis.The crystal water forms a matrix in the c axis shows that the heterocyclic amines are alternately connected to the hydrogen-bonding system along the axis, which leads in the formation of syndiotactic polymer chains in this dimension  and the graph-set motifs of the hydrogen-bonding network. The infinite hydrogen-bonded network is formed along the c axis by chains of connected five-membered . This can be highlighted by an Hirshfeld surface analysis, shown in Fig. 5A⋯O3 of 168.8 (2)° , the ideal tetrahedral environment  and the pure amine could be obtained as a colourless oil (94% yield).1,2,3,4-Tetrahydroisoquinoline  was dissolved in 30 mL of formic acid (99%). After adding formaldehyde  the solution was stirred under reflux for 6 h and stirred at room temperature for an additional 12 h. Subsequently KOH was added to adjust to pH 13. In the next step, the two-phase system was extracted with diethyl ether (3 x 50 mL). The combined organic phases were dried with MgSO2O, mixing the two phases and then separating again to obtain a moist organic phase. Storage of the organic phase at 243 K results in crystallization of the title compound in colourless needles, which are stable up to 273 K before they start melting. The crystals were therefore selected for measurement with help of a X-Temp 2 low-temperature stage : δ (ppm) 2.48 , 2.72 , 2.95 , 3.61 , 7.11–7.15 .13C NMR : δ (ppm) 29.1 , 46.0 , 52.8 (NCH2CH2), 57.9 (CarCH2N), 125.6 , 126.4 , 128.6 , 133.7 , 134.5 .m/z (intensity %): 146 (100) [M–H]+ 131 (9) [M–CH3–H]+, 104 (51).GC/MS (EI) 10H13Ni: C 81.6, H 8.9, N 9.5; found: C 81.2, H 9.0, N 9.6.Elemental analysis calculated (%) for C1H NMR spectrum, which overlaps with other signals in d-acetonitrile.Because of the low stability of the crystals of the trihydrate, no further analysis of the trihydrate was carried out, except for NMR spectroscopy of the crystals, which reveals a broadened water signal in the A and H6B, were included in calculated positions with C—H = 0.95 Å, Uiso(H) = 1.2Ueq(C) for aromatic hydrogen atoms, C—H = 0.99 Å, Uiso(H) = 1.2Ueq(C) for CH2 hydrogen atoms and with C—H = 0.98 Å, Uiso(H) = 1.5 Ueq(C) for methyl hydrogen atoms. All other protons were located in the difference-Fourier maps and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020000730/zl2766sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020000730/zl2766Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020000730/zl2766Isup3.cmlSupporting information file. DOI: 1979129CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Group A was treated with levo-thyroxine (LT4) at the dose of 1 µg/kg/day for six months, whereas patients of the group B did not receive any treatment. Thyroid stimulating hormone (TSH), free-thyroxine (FT4), peak systolic velocity (PSV), International Index of Erectile Function 5-item version (IIEF-5) score, mean platelet volume (MPV), and total cholesterol were evaluated at enrollment (T0) and after six months (T1). Patients without hypertension, diabetes mellitus, dyslipidemia, not on drugs, and with normal total testosterone (TT) values were included in this study. Results: Group A and B did not differ for age (61.2 ± 4.8 vs. 60.3 ± 5.6 years), body-mass index (28.7 ± 2.5 vs. 28.3 ± 2.6 Kg/m2), and serum TT levels (481.2 ± 54.0 vs. 492.1 ± 59.7 ng/dL). At T0, serum TSH levels (6.5 ± 1.2 vs. 6.0 ± 1.0 µIU/mL), FT4 (8.8 ± 0.6 vs. 8.8 ± 0.6 pmol/L), PSV (26.5 ± 1.4 vs. 25.8 ± 2.1 cm/s), IIEF-5 score (8.2 ± 1.7 vs. 9.0 ± 1.7), and total cholesterol (167.8 ± 21.7 vs. 171.6 ± 21.3 mg/dL) did not significantly differ in patients of Group A vs. those of Group B. MPV was significantly higher in Group A than in Group B (12.3 ± 0.3 vs. 11.8 ± 0.7 fL). At T1, Group A showed significantly lower TSH (2.26 ± 0.5 µIU/mL), MPV (9.5 ± 0.3 fL), and total cholesterol (137.8 ± 29.2 mg/dL) and significantly higher FT4 (9.3 ± 0.4 pmol/L), PSV (40.0 ± 2.6 cm/s), and IIEF-5 score (20.2 ± 3.6) compared to pre-treatment values. None of these endpoints showed significant change at T1 compared to T0 in patients of group B. Conclusions: Lt4 therapy is associated with an improvement of the erectile function at the vascular level, a decrease in MPV and total cholesterol. LT4 therapy should be considered in patients with arterial ED and SCH.Background: Arterial erectile dysfunction (ED) is an early sign of vascular damage. Rare evidence has been published so far as to whether subclinical hypothyroidism (SCH) affects arterial erectile function. Therefore, the objective of this study was to fill this gap. Methods: Patients with arterial ED and SCH were consecutive enrolled and randomly divided into Group A ( Erectile dysfunction (ED) is defined by the permanent or recurrent inability to achieve or maintain a penile erection adequate for sexual intercourse . Its gloErection is a complex mechanism, involving neurologic, hormonal, psychologic, and most of all, vascular factors. ED and cardiovascular disease (CVD) have been regarded as different manifestation of the same disorder and the evidence indicates arterial ED as an early marker of CVD ,4. AccorThyroid dysfunction has an impact on male sexual disorders. Particularly, most of the studies suggested a relation between ED and overt hypothyroidism . By contOn these premises, the aim of this study was to investigate the relationship between SCH and erectile function, by assessing the effect of levo-thyroxine (LT4) therapy on the penile blood flow, mean platelet volume (MPV), and total cholesterol in patients with SCH and arterial ED.n = 20) and Group B (n = 20). Group A was prescribed LT4 at the dose of 1 µg/kg/die for six months. Patients of the Group B did not receive any treatment. The following endpoints were assessed at enrollment (T0) and six months later (T1): body mass index (BMI), total testosterone (TT) TSH, free-thyroxine (FT4), MPV, total cholesterol, peak systolic velocity (PSV) values, and the International Index of Erectile Function 5-item version (IIEF-5) score.This is a randomized controlled study performed in patients referring to the Division of Andrology and Endocrinology, University of Catania, for ED. Forty Caucasian patients with arterial ED and SCH were consecutively enrolled. They did not have diabetes mellitus (DM), hypertension, dyslipidemia, and did not take any drug. Patients were randomly divided into Group A . Reference values were as follows: TSH0.34-4.2 µIU/mL, FT4 6.8–16 pmol/L, TT 47.8–980 ng/dL, total cholesterol 0–200 mg/dL, MPV7.2–11.1fL.The IIEF-5 provides a validated self-reported measure of ED. A score greater than 21 excludes ED. For scores ranging between 17–21, 12–16, 8–11, or 5–7, the ED is of low, moderate-low, moderate, or severe entity .Dynamic ultrasound of the penile arteries with pulsed Doppler analysis following intracavernous administration of 20 μg of alprostadil  was performed for the diagnosis of arterial ED. Ultrasound examination was undertaken with a GX MegasEsaote  device, equipped with linear, high-resolution, and high-frequency (7.5 to 14 MHz) probes dedicated to the study of soft body areas, with color Doppler for detecting slow flow and a scanning surface of at least 5 cm. Following injection, PSV was measured every 10 min for 20–30 min. A PSV <30 cm/s and a non-temporal peak systolic progression indicated the occurrence of an arterial disease.p value less than 0.05 was accepted as statistically significant.Results are shown as mean ± standard deviation (SD). The normality of the variables was evaluated with the Shapiro–Wilks test. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Duncan’s Multiple Range Test, using SPSS 22.0 for Windows . A This study was conducted at the Division of Andrology and Endocrinology of the teaching hospital “G. Rodolico”, University of Catania . The protocol was approved by the internal Institutional Review Board (n. 4/2019) and informed written consent was obtained from each participant after full explanation of the purpose and nature of all procedures used. The study has been conducted in accordance with the principles expressed in the Declaration of Helsinki.Group A and B did not differ for age, BMI, and serum TT levels in a statistically significant manner .At T0, Group A and B did not show any statistically significant difference for TSH A, FT4 FB, PSV FA, IIEF-5At T1, Group A showed significantly lower TSH A, MPV FA, and toTSH A, FT4 FB, PSV FA, IIEF-5Finally, Group A had lower TSH A, MPV FA, and toBy excluding patients with known causes of endothelial dysfunction such as DM, hypertension, dyslipidemia, this prospective study showed for the first time a positive effect of LT4 administration on penile blood flow and on factors related to vascular damage such as MPV and total cholesterol in eugonadal patients with arterial ED and SCH. Noteworthy, the benefit of LT4 administration on the erectile function is subjectively perceived from these patients, as derivable from the sharp amelioration of the IIEF-5 score.Previous studies have reported ED in patients with SCH . AccordiMPV is a prognostic marker in patients with CAD . Indeed,THRs are expressed in hematopoietic stem cells and thyroid hormones can modulate the production of blood cells, including platelets. Thyroid hormones are able to initiate platelet aggregation by the interaction with the VR and endothelium adhesion . This exLipids and endothelium are the main players in the pathogenesis of atherosclerosis. Lipids promote endothelium dysfunction and lipid-lowering regimens, by decreasing the amount of circulating inflammatory markers, are known to improve endothelial function and plaque burden . SimilarThe sharp improvement of the erectile function after treatment with LT4 in eugonadal patients with arterial ED and SCH opens new roads towards the implications of phosphodiesterase 5 inhibitors (PDE-5i) treatment. PDE-5i are well known to act by the increase of NO availability in the penile endothelium. As previously introduced, indirect evidence points to a role of TSH reduction in the increase of NO availability , which mIn conclusion, LT4 administration clinically improved the erectile function by restoring penile blood flow and decreased MPV and total cholesterol levels in patients with arterial ED and SCH. Euthyroidism prevents endothelial dysfunction with direct and indirect mechanisms in patients without other risk factors leading to vascular damage. LT4 administration should be considered in non-diabetic, dyslipidemic, hypertensive, and hypogonadic patients with ED of arterial nature and SCH.