Teriflunomide (TFN) limits relapses in relapsing–remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria.  Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease that represents one of the most common non-traumatic incapacitating neurological diseases in young adults . PatholoAlthough the exact disease pathogenesis remains unclear, it is assumed that an autoimmune attack mediated by autoreactive lymphocytes initiates the disease and leads to neurodegenerative processes ,5. In thTeriflunomide (TFN) is a disease-modifying drug (DMT) that has been recently approved as an oral therapy for patients with RRMS ,20. TFN Since TFN inhibits an enzyme that is closely associated with mitochondrial biology via the respiratory chain, we hypothesized that treatment of MS patients with TFN may also affect neuronal mitochondria. Although in MS, autoimmune attack is assumed to be one of the key players leading to neurodegenerative processes , numerou2O2) that induces oxidative stress in CNS cells.Recently, we showed that TFN could prevent alterations in mitochondrial morphology, motility, as well as oxidation potential in peripheral spinal root explants during oxidative stress . Conside2) levels in hippocampal slices at baseline and after treatments with H2O2 (200 µM) and H2O2 (200 µM) + TFN (50 µM). In vitro, changes in pO2 correlates inversely with neuronal oxygen consumption and cellular metabolism due to the constant supply with oxygen and glucose. To determine pO2, we performed a gradient descent of the oxygen electrode into the slice, which we refer to as ‘steps’ from the surface with the increment of 20 µm up to the core of the slice. A total of 10 slices were analyzed in three independent experiments. As depicted in 2 level no longer decreased (see methods). Thus, the depicted data covers only the descent to the core. Then, the oxygen electrode was raised to a 100 µm depth from the surface of the slice and 15 min of baseline activity was recorded. Immediately following the baseline recording, the H2O2 or H2O2 + TFN treatment in an artificial cerebrospinal fluid (aCSF) was applied for 30 min. Again, the steps were performed from the surface of the slice towards the core.To understand the effects of TFN on the metabolic activity in the nervous tissue, we investigated changes in oxygen partial pressure  relative the baseline). In contrast, the pO2 decreased towards the core when TFN treatment was added  relative to untreated), indicating an increase in the consumption of oxygen in the presence of TFN with respect to H2O2 treatment alone  or H2O2 (200 µM) + TFN (50 µM) were measured.Furthermore, we investigated the effect of oxidative stress and TFN application on synaptic transmission in acute hippocampal tissue. For that, stimulus-induced local field potential (LFP) signals were recorded in CA1 while stimulating the Schaffer collaterals using a paired-pulse facilitation protocol (see methods). The amplitudes of the population spike responses during baseline and treatment with H2O2-stressed brain slices treatment (0.88 ± 0.14-fold (mean ± SD) relative to the baseline), while the presence of TFN increased the PPR (H2O2 + TFN treatment (1.2 ± 0.62-fold (mean ± SD) relative to the baseline) (aseline) E.2O2 significantly decreased (0.64 ± 0.16-fold (mean ± SD)) with respect to untreated controls. The treatment with TFN along with H2O2 did not affect or restore the ATP levels (0.65 ± 0.03-fold (mean ± SD)). ). .2O2 or H2O2 + TFN. Then, the values of treated slices were normalized with their respective baseline images.We have shown previously that in peripheral nerves, mitochondria exposed to oxidative stress are affected even prior to the axonal damages ,30 and t2O2 reduced mitochondrial length (0.96 ± 0.37-fold (mean ± SD)) and that this effect was not abolished in the presence of TFN (0.97 ± 0.34-fold (mean ± SD))  (We observed that Hn ± SD)) C. Howeve47-fold) D  and after the treatment with H2O2-induced decrease in mitochondrial displacement was further decreased in the presence of TFN (data shown as mean ± SD: H2O2 treated: 1.06 ± 0.66-fold and H2O2 + TFN treated: 0.88 ± 0.59-fold)   A. Simila56-fold) B (Table 56-fold) .We know that mitochondria are dynamic organelles that can change their shape, ranging from punctuate structures to tubular networks depending on the cellular needs. Hence, we processed the images and segmented the fluorescent mitochondria into four different morphological categories: rod-shaped, puncta-shaped, network, and large mitochondria. Morphological parameters of mitochondria in treated slices were renormalized to their respective baseline images .2O2: 0.90 ± 0.41 length and 0.62 ± 0.39 area; and H2O2 + TFN: 0.88 ± 0.41 length and 0.64 ± 0.40 area)  .2O2- induced area reduction in puncta, network, and large (here only a trend) forms ; however, mitochondria became smaller  after exposure to H2O2. TFN- treatment prevented the reduction of mitochondrial size (0.96 ± 0.52). Similarly, TFN- treatment could prevent reduction in length and size of network-shaped mitochondria induced by H2O2 . In contrast, H2O2 induced the increase in length and size of large mitochondria . TFN prevented the oxidative stress-related increase in length but not the area of large mitochondria (data shown as mean ± SD: 0.95 ± 0.33 length and 0.91 ± 0.61 area)  forms E–H. Agai61 area) .2O2 + TFN: 0.42 ± 0.55 (rod), 1.01 ± 0.57 (puncta), 0.91 ± 0.60 (network), and 0.75 ± 1.11 (large)). Additionally, in case of rod-shaped and network mitochondria, it accentuated the effect of H2O2 (data shown as mean ± SD: 1.08 ± 0.68 (rod), 1.08 ± 0.60 (puncta), 1.78 ± 2.16 (network), and 1.02 ± 1.25 (large)) , 0.91 ± 0.37 (puncta), 0.94 ± 0.65 (network), 1.05 ± 1.03 (large); H2O2 + TFN: 0.56 ± 0.51 (rod), 1.07 ± 0.39 (puncta), 1.01 ± 0.76 (network), and 0.75 ± 0.91 (large)) ) . Furtherrphotype E–H (data(large)) .2O2, and the effects of TFN on H2O2-mediated neuronal and mitochondria changes were investigated. In the CNS slice model, TFN was used at a concentration of 50 µM. Using peripheral spinal root explants, we have already previously shown that TFN prevented oxidative stress-induced mitochondrial alterations at 1 µM concentrations [Mitochondrial alteration during inflammation is one of the major factors that contribute to neurodegeneration in inflammatory diseases such as MS. Thus, besides the importance of controlling inflammation, MS treatments should also aim at protecting mitochondria from damage . In thistrations . However2 and LFP revealed that TFN treatment could prevent the reduction of oxygen consumption and synaptic transmission upon the induced oxidative insult. An increment in pO2 with respect to the baseline recording during oxidative stress  that express cyan fluorescence protein (CFP) in neuronal mitochondria was conducted at the FEM under specific pathogen-free conditions. All experimental procedures were conducted in strict accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 and were approved by the Regional Animal Study Committee of Berlin, , approvals ID: G0101-14 and T0002-10.WT C57BL/6 mice were obtained from the Research Institute for Experimental Medicine—Forschungseinrichtungen für Experimentelle Medizin (FEM) at the Charité- Universitätsmedizin . Breeding of transgenic Tg(Thy1-CFP/COX8A)S2Lich/J mice  in HEPES artificial cerebrospinal fluid (HEPES aCSF) . The solutions were freshly prepared, and the pH adjusted between 7.3 and 7.4. The whole procedure was performed in temperatures between 34 and 37 °C.Brain slices were continuously supplied with carbogen (95% O2O2) dissolved in aCSF. In our previous studies, 200 µM H2O2 induced mitochondrial damage [2O2 induced only mitochondrial alterations [2O2 for 1 h [2O2 for incubation in submerged chamber during mitochondrial imaging experiments to ensure diffusion of H2O2 inside the 400 µm-thick brain slices. Since, H2O2 concentrations are known to quickly decrease after short period of time [2O2 was used in the interface chamber during the electrophysiology experiments to ensure adequate H2O2 availability to the slices in air-water interface, unlike in submerged chamber where the slices are inside the solution. The other treatment groups were DMSO (0.0005%), H2O2 (100 µM) +DMSO (0.0005%), and H2O2 (100 µM), along with TFN (50 µM) and TFN (50 µM) alone. DMSO was used as a vehicle control for TFN.To induce oxidative stress, acute slices were incubated for 30 min with hydrogen peroxide  for at least 90 min before the investigation (temperature ~34 °C). To induce oxidative stress, 200 µM H2O2 was added to the bath solution for 30 min.Hippocampal slices (400 µm thickness) were prepared using a Leica VT1200 S vibratome , as mentioned above. The slices were stored in an interface chamber and continuously supplied with freshly prepared and carbogenated aCSF , respectively. Both electrodes were placed in the stratum pyramidale of the CA1 area while stimulation  was performed with a stimulation electrode placed in the stratum radiatum between CA3 and CA1 using a Master 8 system . To measure changes in neuronal respiration, the pO2 electrode was advanced in 20 µm steps through the slices using a calibrated micromanipulator . In the in vitro condition, pO2 distribution depends on cellular O2-consumption since O2 diffusion and solubility are constant [2-gradient can be measured, from the surface till the core of the slice, the depth in which pO2 ceases to decrease. For electrophysiology measurements, baseline measurements were performed at 100 µm below the tissue surface for 15 min before and after 30 min treatment with H2O2 and H2O2 + TFN. Analog signals were digitalized using a Power1401 and recorded using Spike 2 software  for the recording. Offline analysis of the data was performed using Spike2.Stimulus-induced population spikes (PS) and pOconstant . Typicalg for 5 min. Then, the supernatant was collected and deproteinization with perchloric acid (PCA) was performed. ATP standards, in the range of 0 to 1 nmol, as well as samples, were then incubated with reaction-mix for 30 min. The fluorometric detection was performed at 535/587 nm in a plate reader . The calculation of the amount of the ATP in each slice was performed after the subtraction of the sample background and with the multiplication of the dilution factor, as described in the protocol by the manufacture.After the treatment of the slices, ATP assay was performed using the Abcam ATP assay kit  according to the manufacture’s protocol. Briefly, the tissue was homogenized in ATP assay buffer and centrifuged at 13,000× To image mitochondria in slices prepared from the mitoCFP transgenic mouse (Tg(Thy1-CFP/COX8A)S2Lich/J), an 830nm excitation wavelength and photomultiplier at 525 nm with 50 nm bandwidth was selected.At first, a region of interest (ROI) was selected and a time-lapse image stack of 30 steps with intervals of 3.6 s was obtained. Then, aCSF along with treatment was added without moving the objective or the holding chamber. After 30 min, the same region was imaged again with same setting. This procedure was repeated with each single treatment group.Huygens deconvolution Scientific Volume Imaging software  was applied for the restoration of mitochondrial images obtained with two-photon microscopy. Images were opened in Huygens professional X11 and enabled automatic computation of point spread function (PSF). Then, the images were inspected with the logarithmic vertical mapping function. Once, the PSF was computed, the Classic Maximum Likelihood Estimation (CMLE) algorithm was applied to obtain effectively deconvolved images.Images were corrected for the background noise and image movement using Fiji Is Just ImageJ (Fiji) software ,50. The For morphology analysis, the corrected images were first segmented with Trainable Weka segmentation plugin in Image J software . A few mFor motility analysis, from the corrected images, five random ROIs within the image were selected. Then, each ROI was separately processed into Track mate plugin  for tract-test for the normal parametric data. Comparisons between two groups of non-parametric data were performed by Mann–Whitney U test. Comparison between more than two groups were analyzed with one-way analysis of variance and with Kruskal–Wallis test for non-parametric data followed by Dunn’s post hoc test. All the data are shown as mean ± SD. The data are shown as Tukey boxplot where the central line denotes the median; the lower and upper boundaries denote the 1st and 3rd quartile, and the whiskers denote the data except the outliers presented as individual dots. The outliers were not included in the analysis. p values ≤ 0.05 were considered significant. The significance of the data was further depicted as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.The data were analyzed with the Prism software . First, the Gaussian distribution of the variables was confirmed using all the three normality tests— KS, D’Agostino and Pearson omnibus, and Shapiro–Wilk. Non-normally distributed data were treated as non-parametric. Comparisons between the two groups were performed by Here, we conclude that TFN contributes to mitochondrial protection during oxidative stress. The drug might influence mitochondrial bioenergetics by reversibly limiting the reduction of flavin mononucleotide (FMN) via DHODH and thus, electron transport in the ETC . This in


Severe immune-related Adverse Events (irAEs) develop in 10–27% of patients treated with Immune-Oncology (IO) . The aim of our study was to evaluate efficacy and clinical outcome of metastatic renal cell carcinoma (mRCC) patients who stopped Immune Checkpoint Inhibitors (ICIs) due to early Grade (G) 3-G4 irAEs.We retrospectively collected data from 204 mRCC patients treated with ICIs in 6 Italian referral centers adhering to the Meet-Uro group, between February 2017 and January 2020. To properly weight the results, patients who did not report early G3–G4 toxicities have been included as control group.Primary endpoint was to evaluate 6 months Progression Free Survival (PFS) after early treatment interruption for Grade (G) 3–4 toxicities compared to the control group. Secondary endpoints were to evaluate Time to treatment failure (TTF) and overall survival (OS) in both groups. All statistical analyses were performed using SPSS software .18/204 (8.8%) patients had early treatment interruption for serious (G3-G4) irAEs. Early was defined as interruption of IO after only one or two administrations. Immune related nephritis and pancreatitis were the most common irAE that lead to treatment interruption. 6/18 patients received IO-IO combination whereas 12/18 patients antiPD1. In the study group, 12/18 (66.6%) were free from progression at 6 months since IO interruption, TTF was 1.6 months (95% CI 1.6–2.1), mPFS was 7.4 months (95% CI 3.16–11.6) and mOS was 15.5 months (5.1–25.8). In the control group 111/184 (60.3%) patients were free from progression at 6 months, TTF was 4.6 months (95% CI 3.5–5.6), mPFS was 4.6 months (95% CI 3.5–5.6) and mOS was 19.6 months (95% CI 15.1–24.0). In the overall population, mPFS was 5.0 months (95% CI 4.0–5.9) and mOS was 19.6 months (95% CI 15.1–24.0).ICIs seem to maintain efficacy even after early interruption due to severe irAE. Immune-Oncology (IO), alone or in combination, changed the paradigm of treatment of metastatic renal cell carcinoma (mRCC) showing an improvement in Overall Survival (OS) and Progression Free Survival (PFS) compared to standard of care –3. IO inSevere irAEs develop in 10–27% of patients treated with anti-Cytotoxic T-Lymphocyte Antigen (CTLA)-4, in amount 12–20% of patients treated with anti-programmed cell death (PD)-1 and 15–20% of patients treated with anti-programmed death-ligand (PD-L)-1  and 1.3–Fathal irAE have been reported in almost 0.36% of patients treated with anti PD-1/PDL-1, 1.08% in those treated with anti-CTLA-4 and 1.23% of patients treated with combination . IrAEs cAccording to recent reports, irAEs correlates with a better outcome . IrAE caTherefore, the aim of our study was to retrospectively evaluate the impact of early interruption of IO treatment, due to irAEs, on outcome of mRCC patients.We retrospectively collected data of mRCC patients treated with IO in 6 Italian referral centers adhering to the Meet-Uro group, between February 2017 and January 2020.Inclusion criteria were at least 18 years old at the time of enrollment, histological diagnosis of renal cell carcinoma and radiological diagnosis of metastatic disease.Patients treated with IO as single agent or in combination were considered eligible.Baseline characteristics were collected at the start of IO. Outcome data, including PFS, TTF, OS and toxicities, were collected too. Data included first line treatment, subsequent IO therapy and previous nephrectomy.The International Metastatic RCC Database Consortium (IMDC) prognostic risk group was computed at the index date based on the presence of six individual risk factors including time from diagnosis to systemic treatment < 1 year, hemoglobin < lower limit of normal, calcium > 10 mg/dL, platelet > upper limit of normal, neutrophil > upper limit of normal, Performance Status (PS) < 80% (Karnofsky).IrAEs were graded according to the National Cancer Institute Common Toxicity Criteria for Adverse Events .Primary endpoint was to evaluate 6 months PFS after treatment interruption for toxicities.Secondary endpoints were to evaluate OS and Time to Treatment Failure (TTF) in patients. TTF was evaluated as the interval from treatment initiation to premature discontinuation due to cancer progression, adverse events patients’ choice or death. PFS was defined as the time from treatment start to disease progression or death from any cause.Early interruption was defined as the interruption of ICIs after one or two administration, almost 2 months of treatment.Patients with no evidence of death were censored at the date of last tumor assessment.Real-world physician-assessed progression and response was based on clinical criteria or radiographic criteria using Response Evaluation Criteria in Solid Tumors (RECIST) guidelines, with imaging assessments occurring at clinically variable time points.Baseline demographic and clinical characteristics are described using frequencies and percentages for categorical variables.Descriptive analysis was made using median values and ranges. All statistical analyses were performed using SPSS software .All participating centers received local ethics approval for data collections. The study was conducted in accordance with good clinical practice and the Declaration of Helsinki.Data from 204 mRCC patients were retrospectively collected from 6 referral centers. To properly weight the results, patients who did not report early G3-G4 toxicities have been included as control group. Characteristics of patients are described in Table p < 0.0001)  patients were free from progression at 6 months from IO interruption whereas, in the control group, patients free from progression at 6 months, were 69/184 (37.5%) Immune related nephritis and pancreatitis were the most common irAEs that lead to early treatment interruption Table . AccordiIn patients who interrupted treatment due to early irAEs, TTF was 1.6 months (95% CI 1.6–2.1), mPFS was 7.4 months (95% CI 3.16–11.6) and mOS was 15.5 months (5.1–25.8). In the control group, TTF was 4.6 months (95% CI 3.5–5.6), mPFS was 4.6 months (95% CI 3.5–5.6) and mOS was 19.6 months (95% CI 15.1–24.0).In the overall population, median PFS was 5.0 months (95% CI 4.0–5.9) and median OS was 19.6 months (95% CI 15.1–24.0) whereas TTF was 4.1 months (95% CI 3.2–4.9). 125/204 patients were free from progression at 6 months.12/18 patients did not receive further treatment after IO due to clinical deterioration.IrAEs contribute to mortality and morbidity in mRCC and represent a relevant issue for healthcare system. Symptoms and signs are often insidious and similar to cancer related symptoms so that irAEs represent a clinical challenge for physicians.Biological biomarkers for irAEs are mostly unknown . BaselinIn clinical practice, pre-existing organ insufficiency is supposed to be associated to higher risk to develop irAEs as well as pre-exiting autoimmune disease (AID) . In patiIn our real-world analysis, the incidence of early G3-G4 irAEs was almost 10–15%.Outcomes here reported, demonstrate the efficacy of IO even after early interruption due to irAEs. Furthermore, after 6 months from treatment interruption, 66.6% of patients were free from progression demonstrating long-term benefit from IO. Indeed, G3-4 irAEs seem to be related to efficacy of IO even when treatment is early interrupted due to toxicities . DiffereThe hypothesis underling brilliant response after the onset of irAEs is that an exuberant activation of immune system against our self-tissues becomes an exuberant response against tumor tissue , 15. SevIt is reasonable to suppose that in patients who develop early G3-4 irAEs, ICIs promote adapted states of hyper-responsiveness in immune cells, which promote anti-tumor immunity. This subgroup of patients might have a “hyper-immune system” and early G3–G4 irAEs could represent a predictive parameter for persistent adaptations in the immune system, which can persist and display memory-like features. Limits of our study are the small sample size, the retrospective collection of data and the lack of central radiological review. A prospective validation is needed to straighten these findings.Pathogenesis and treatment of irAEs represent an interesting research field as it remains under debate. Interruption is often required after irAEs, with consequent doubts about treatment efficacy. Nevertheless, our study, with the limit of a retrospective collection, confirms that in mRCC patients, even after early interruption due to irAEs, IO maintain clinical and radiological efficacy.


Saccharophagus degradans produces an endo-β-mannanase of glycoside hydrolase family 5 subfamily 8 with three phylogenetically distinct family 10 CBMs located C-terminally from the catalytic domain (SdGH5_8-CBM10x3). However, the functional roles and cooperativity of these CBM domains in polysaccharide binding are not clear. To learn more, we studied the full-length enzyme, three stepwise CBM family 10 (CBM10) truncations, and GFP fusions of the individual CBM10s and all three domains together by pull-down assays, affinity gel electrophoresis, and activity assays. Only the C-terminal CBM10-3 was found to bind strongly to microcrystalline cellulose . CBM10-3 and CBM10-2 bound galactomannan with similar affinity (Kd = 0.2–0.4 mg/ml), but CBM10-1 had 20-fold lower affinity for this substrate. CBM10 truncations barely affected specific activity on carob galactomannan and konjac glucomannan. Full-length SdGH5_8-CBM10x3 was twofold more active on the highly galactose-decorated viscous guar gum galactomannan and crystalline ivory nut mannan at high enzyme concentrations, but the specific activity was fourfold to ninefold reduced at low enzyme and substrate concentrations compared with the enzyme lacking CBM10-2 and CBM10-3. Comparison of activity and binding data for the different enzyme forms indicates unproductive and productive polysaccharide binding to occur. We conclude that the C-terminal-most CBM10-3 secures firm binding, with contribution from CBM10-2, which with CBM10-1 also provides spatial flexibility.Carbohydrate active enzymes, such as those involved in plant cell wall and storage polysaccharide biosynthesis and deconstruction, often contain repeating noncatalytic carbohydrate-binding modules (CBMs) to compensate for low-affinity binding typical of protein–carbohydrate interactions. The bacterium  N-acetyllactosamine  is considered a key factor in dynamic systems, such as plant cell wall synthesis and degradation, microbe–host interplay, and synthesis and mobilization of storage polysaccharides. The carbohydrate active enzymes catalyzing these different reactions very often contain one or more noncatalytic carbohydrate-binding modules (CBMs) that facilitate the formation of enzyme–substrate complexes and can specifically bind with the substrate polysaccharides or with different polysaccharides located nearby, such as in the plant cell wall. It is common that polysaccharide hydrolases contain several CBMs, but it is difficult to understand the function of these individual CBMs because of their interplay with each other, substrate, other polysaccharides, and the catalytic domain (CD), and perhaps even more than one CD is present . Certaintosamine . There atosamine . Clearlytosamine , 3.Mannan polysaccharides  are founMannans are depolymerized by endo-β-mannanases found in glycoside hydrolase (GH) families GH5, GH26, GH113, and GH134 , some ofhttp://cazy.org)  . CBM famazy.org)   , 20, 21.azy.org) , 27, 28,azy.org) , 26, 29,azy.org) , 34, 35,azy.org) .Table 1SCellvibrio japonicus (CjGH10CBM10)  and phosphoric acid–swollen cellulose (PASC) with a dissociation constant (Kd) of 0.2 to 0.3 μM but not to hydroxyethyl cellulose (HEC) (CjGH10CBM10 is the only structure determined CBM10 (Protein Data Bank ID: 1E8R) (C. japonicus CBM10 (CjAA10CBM10) from LPMO  showed a similar polysaccharide-binding preference and bound with Kd = 7.5 to 17.3 μM to disordered (PASC) as well as to crystalline (Avicel and BMCC) cellulose (CjGH74CBM10) of the C. japonicus GH74 endo-xyloglucanase that also contains a CBM2, when analyzed as a GFP fusion did not bind to soluble tamarind seed xyloglucan and barley 1,3;1,4-β-glucan, both of which are substrates of the GH74 endo-xyloglucanase, or to HEC (Kd = 1.5 μM) to Avicel, which is not a substrate  , 37 (Tab10CBM10) . These rse (HEC) . It morese (HEC) . CjGH10Cellulose , which wellulose . Finallyr to HEC , but bouubstrate .Bifidobacterium animalis subsp. lactis Bl-04 (BaGH5CBM10) was able to bind to low-viscosity carob galactomannan (CGM-lv) with a Kd of 0.31 mg/ml but not to microcrystalline cellulose (Avicel), soluble cellulose (HEC), or insoluble ivory nut mannan (INM)  from the probiotic bacterium an (INM)   . Notablytomannan . The lactomannan  and alsotomannan .Clostridium cellulolyticum cellulase of GH9 adsorbed to PASC, Avicel, alkaline-pretreated sugarcane bagasse, and filter paper powder (Teredinibacter turnerae (GH5_2-CBM5-CBM10-GH6_4) and the marine bacterium Saccharophagus degradans (CBM2-CBM10-GH5_4) as well as the CjGH10CBM10 mentioned previosuly showed low affinity for Sigmacell cellulose type 20 (20 μm particles) and chitosan compared with the other type A CBMs  as well as its three CBM10 truncations  substitution of the first tyrosine (to a threonine) of the three aromatic residues critical for Avicel binding as demonstrated for C. japonicus CjGH10CBM10  laH10CBM10  and S1.FS. degradans having three C-terminal CBM10s   . These CBD79918) .SdGH5CBM10-1 linked to the CD of the S. degradans mannanase  and of the GH74 endoglucanase active on xyloglucan and mixed-linked 1,3;1,4-β-glucan (CjGH74CBM10)   Fig. 3)SdGH5CBM1SdGH5_8-CBM10x3, and its three stepwisely truncated forms lacking one, two, or all three CBM10s were recombinantly produced in Escherichia coli (C. japonicus (CjGH74CBM10)   was adop74CBM10) . All recSdGH5_8-CBM10x3 and its three truncated forms were active on all tested mannan polysaccharides but not on microcrystalline cellulose (Avicel) (SdGH5_8-CBM10x3 is similar to that of other characterized GH5_8 mannanases but with specific activities at the higher end (SdGH5CBM10-3) did not essentially affect the specific activity toward the good substrates, that is, CGM-lv and high-viscosity CGM (locust bean gum) and konjac glucomannan (KGM)  and the C-terminal (SdGH5CBM10-3) domains reduced activity by 36% to 40% for CGM-lv and high-viscosity CGMs and maintained activity for KGM compared with the full-length enzyme  showed twofold and sixfold higher activity than full-length enzyme. Overall, this suggests that the presence of only SdGH5CBM10-1 slightly hinders CGMs from interacting fully effectively with the CD. The presence of the C-terminal and middle CBM10 seems to prevent optimal productive interaction between the CD and the poor substrates INM and the highly substituted guar GG as reflected in the CD alone (SdGH5_8) and the enzyme form with only the first CBM10 (SdGH5_8-CBM10x1) having higher specific activities at low enzyme concentrations.The full-length (Avicel) . The subgher end , 18. Reman (KGM) ; Fig. 1.activity  on the ph enzyme . Remarkah enzyme . FinallySdGH5_8-CBM10x3 and truncated forms toward CGM-lv are in agreement with the findings on specific activity . Notably, the catalytic efficiency of CD alone is doubled and 1.4-fold higher than of the SdGH5_8-CBM10x1 and full-length enzyme, respectively. The Km of CD alone is low and similar to that of full-length SdGH5_8-CBM10x3, whereas the turnover number is 68% higher. The lowest turnover number is seen for full-length enzyme, whereas SdGH5_8-CBM10x1 has the highest turnover number . In the case of GG, the full-length enzyme showed the highest activity, whereas the CD alone in fact was the least active form (B). These results on the highly viscous and densely decorated guar GG indicated that the C-terminal CBM10-3 together with CBM10-1 play a role for activity at high-substrate concentrations. Thus, removal of CBM10-3 resulted in some reduction in activity, whereas removal of CBM10-2 as well did not make a difference, as the SdGH5_8-CBM10x1 and SdGH5_8-CBM10x2 showed the same activity. But the removal of CBM10-1 led to a further reduced activity. Although full substrate saturation was not possible, calculations applying the Michaelis–Menten equation on activity of the full-length SdGH5_8-CBM10x3 on INM indicated Km = 35 ± 14 mg/ml and kcat = 186 ± 53 s−1 (kcat/Km = 5.3 ml/[mg · s]). The Km is 1.3-fold higher as compared with a GH5_8 mannanase with one CBM10 from Streptomyces lividans (Km = 26.5 ± 11.5 mg/ml), whereas the kcat of that enzyme was 69 ± 1.2 s−1 resulting in a catalytic efficiency of 2.6 ml/(mg s) .The kinetics analysis on the insoluble crystalline INM showed the C-terminal CBM10-3 of full-length h enzyme A. In theive form B. These l/(mg s) ; hence, he CBM10 . This CBfindings A.Figure via the C-terminal CBM10-3, which deprives the CD access to susceptible substrate bonds. At the higher enzyme concentration used for the kinetics analysis (A), this is in part overcome. For the other poor substrate guar GG, the substrate is assumed to contain enzyme target bonds placed in different structural contexts and having different preferences for the productive enzyme attack to occur .To understand the reason behind the differences observed between the results of the enzyme kinetics on INM and guar GG  and the analysis A, this ito occur . Althougomannans , this isomannans B.SdGH5_8-CBM10x3 and the three CBM10s together (SdGH5CBM10x3) showed high affinity for CGM-lv  but was also clearly a combined effect of all the three CBM10s. The full-length enzyme and the three CBM10s together thus bound very strongly with Kd <0.13 mg/ml, whereas neither of the truncated enzyme forms lacking one, two, or all three CBM10 domains showed significant retardation in AGE. Both the C-terminal CBM10-3 and the middle CBM10-2 domains, however, bound reasonably well to galactomannan, the data suggesting that they together secures the full-length enzyme binding. This is opposed to the analysis of the innermost SdGH5CBM10-1_GFP, which showed ninefold to 18-fold reduced affinity compared with the other two CBM10s having Kd of 0.4 and 0.2 mg/ml, respectively (SdGH5CBM10-2_GFP), which has an insert of five amino acid residues and lacks a potentially stabilizing disulphide bridge similar to BaGH5CBM10 from the Bifidobacterium mannanase (Kd similar to that of BaGH5CBM10 (Kd = 0.3 g l−1)  but still has good affinity for insoluble cellulose; however, it does have the conserved tyrosine as the first of the three binding residues , and INM were assessed by retardation in AGE and pull-down assays, respectively. Neither the different enzyme forms nor the CBM10s individually or all three CBM10s together were able to bind HEC in AGE. By contrast, both full-length enzyme r CGM-lv . Althougectively . The secannanase  and S1, 3 g l−1) . But sur10-1_GFP . A possi residue ; Fig. 4.residues ; Fig. 4.SdGH5CBM10 and the combined three domain GFP fusions showed that SdGH5CBM10-3_GFP and SdGH5CBM10x3_GFP interacted strongly with INM, whereas the amount of protein bound in the case of both SdGH5CBM10-1_GFP and SdGH5CBM10-2_GFP was in the same range as the amount of GFP alone (A). Moreover, the qualitative pull down by Avicel showed only weak interaction with SdGH5CBM10-1_GFP and SdGH5CBM10-2_GFP, whereas the three CBM10s together (SdGH5CBM10x3_GFP) and the C-terminal domain SdGH5CBM10-3_GFP bound to Avicel  was too weak to obtain a Kd. Hence, SdGH5CBM10-3 is responsible for Avicel binding, in accordance with findings for other members of the same phylogenetic subgroup , whereas some bind soluble polysaccharide substrates with good affinity (e.g.SdGH5CBM10-2) or with low affinity (e.g.SdGH5CBM10-1). Members of the same phylogenetic subgroup as SdGH5CBM10-3-like CBM10s probably anchor full-length enzymes firmly to substrate or spatially adjacent polysaccharides, whereas CBM10s from phylogenetically different subgroups may still show binding to certain polysaccharides and enable the CD to flexibly survey the vicinity for susceptible glycosidic bonds while yet others may be protein domains serving as linkers or spacers not engaged in direct binding onto a crystalline cellulose via a classical CBM type A platform. In nature, multimodular enzymes characteristically participate in heterogenous catalysis on insoluble substrates to which firm binding is mandatory. However, binding is not the same as productive binding, and the density of functional and nonfunctional binding attack sites depends on the material and enzyme as illustrated in details for cellulose degradation by cellulases , galactomannan, and also insoluble seed storage mannan. However, the middle domain CBM10-2 shows also affinity with a 10-fold lower Kd than the Km for the good substrate galactomannan and may facilitate its interactions with the CD although the truncated enzyme lacking the C-terminal CBM10 lost ability to bind to the galactomannan. The CBM10-2 therefore may also serve as a linker that provides flexibility in polysaccharide interaction for the C-terminal domain and hence for the CD to interact productively with substrate. The role of the innermost CBM10-1 is more enigmatic, but at low enzyme and substrate concentrations, it was able to secure highest activity of the CD toward the two poor substrates (INM and guar GG) also relative to the activity of the CD alone or with two, respectively, three CBM10s. The CBM10-1 is in a small separate phylogenetic subgroup for which a possible hallmark and biological role, beyond this mentioned stabilization, remains to be identified.The multimodular endo-β-mannanase of family GH5_8 secreted by www.cazy.org;  (SdGH5CBM10s were made using HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred) (CjGH10CBM10 (Protein Data Bank ID: 1E8R) as template.CBM10 sequences were retrieved from the CAZy database  . A strucoscope/) . Homolog/hhpred)  with theCGM-lv, INM, and KGM were from Megazyme; high-viscosity CGM (locust bean gum), GG, microcrystalline cellulose (Avicel), and HEC were from Sigma–Aldrich. See S. degradans β-mannanase SdGH5_8-CBM10x3 (GenBank accession no.: ABD79918) without the predicted signal peptide (amino acids 1–21) and the three CBM10s SdGH5CBM10x3 fused with GFP, and GFP (AGT98536) were purchased , and cloned into the pET28a(+) vector using the XhoI and NheI restriction sites resulting in an N-terminal cleavable His tag. The three C-terminally truncated forms SdGH5_8-CBM10x2, SdGH5_8-CBM10x1, and SdGH5_8 , pET28a-SdGH5CBM10x3_GFP as template and mutagenesis primers. To get SdGH5CBM10-1_GFP, both SdGH5CBM10-2 and SdGH5CBM10-3 were deleted in one step. A one-step deletion of SdGH5CBM10-1 and SdGH5CBM10-2 was done to get SdGH5CBM10-3-GFP. Finally, SdGH5CBM10-2_GFP was obtained in two steps; first deleting CBM10-1 and then CBM10-3. The gene constructs were confirmed by sequencing.The genes encoding the  SdGH5_8  were con primers  designedE. coli. BL21, screened on LB agar including 50 μg/ml kanamycin, and starter cultures (10 ml) were made by inoculating LB medium, 50 μg/ml kanamycin with a single colony and incubated , which are used to inoculate 750 ml LB medium containing 10 mM glucose and 50 μg/ml kanamycin in shake flasks. Cultures were propagated  to an absorbance of 0.6 at 600 nm followed by decreasing the temperature to 16 °C and induction of expression by a final concentration of 0.1 mM IPTG. Cells were harvested  after approximately 20 h and stored at –20 °C until protein purification.All full-length and variant-encoding plasmids were transformed into g, 4 °C, 30 min). The filtered (0.45 μm) supernatant was loaded onto a 5 ml HisTrap HP column  pre-equilibrated with HisTrap equilibration buffer at a flow of 3 ml/min and eluted by a linear gradient from 2.5% to 100% HisTrap elution buffer  in 20 column volumes. Protein-containing fractions (based on absorbance at 280 nm) were pooled, concentrated to 4 ml  and further purified by gel filtration  at a flow rate of 0.5 ml/min. Fractions containing recombinant protein of correct size as based on SDS-PAGE were pooled and concentrated as above to 2 to 3 mg/ml. The absorbance at 280 nm was measured, and the protein concentration was determined using theoretical extinction coefficients.The proteins were purified in two steps. Cells were resuspended in HisTrap equilibration buffer , added two cOmplete Mini, EDTA-free, protease inhibitor tablets (Roche Diagnostics GmbH), lysed using a high-pressure homogenizer at 1 bar, added 3 μl Benzonase Nuclease (Sigma–Aldrich), and centrifuged  and enzyme  in 2000 μl and withdrawing 400 μl aliquots (200 μl in the case of INM) at 3, 6, 9, and 12 min. The kinetic assays were done in duplicates. In the case of the CGM-lv and INM data, the Michaelis–Menten model was fitted to the initial velocity data using GraphPad Prism 6 (GraphPad Software Inc).g, 10 min, 4 °C). Supernatants were transferred to fresh tubes and centrifuged , before 4 μl supernatant was heat treated in the presence of SDS-loading buffer and applied on the SDS-PAGE. The pellets from the pull-down assay were washed with 250 μl assay buffer and pelleted again as aforementioned, resuspended in 200 μl assay buffer, added 50 μl SDS-loading buffer, boiled for 10 min, and applied (4 μl) on the SDS-PAGE. For each protein, a sample of the 0.5 mg/ml protein stock used for the pull-down assay was treated as the supernatant sample and included on the gel.Qualitative screening of binding of the individual CBM10s separately and the triple CBM10 to microcrystalline cellulose (Avicel) and insoluble crystalline INM was done by a pull-down assay, where the samples were analyzed by SDS-PAGE: 10 mg polysaccharide (prewashed three times) was mixed with 200 μl 0.5 mg/ml protein in assay buffer . GFP was used as a control in the assay. The samples were incubated at 4 °C for 1 h with gentle agitation and then centrifuged  in 50 mM phosphate pH 7.0 and 0.1 mg/ml bovine serum albumin. GFP (10–150 μg/ml) served as a negative control. The mixtures were rotated end over end  and centrifuged . Supernatants were transferred to fresh tubes, centrifuged , and the concentration of unbound protein was determined by fluorescence  with excitation at 450 and emission at 510 nm using a standard curve made with each protein form (1–1250 nM) in assay buffer. The amount of bound protein was calculated by subtracting the unbound from the total protein, and the dissociation constant, Kd, was obtained by fitting Equation Quantitative pull-down assay was done with microcrystalline cellulose (Avicel) utilizing the presence of GFP for quantification . The totPC and FP are the bound and unbound protein concentrations, respectively. The pull-down assays were done in duplicates on two different days.Kd was determined from retardation distances according to the method  was done by AGE with 2.5 mg/ml CGM-lv or HEC in 12% acrylamide gels . Proteine method  using a e method  (EquatioRmi and Rmo are migration distances of sample relative to a reference protein in the presence and absence of polysaccharide, respectively; c is the polysaccharide concentration. The relative migration (Equation Equation  defines All the data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


SLCO1B1 gene is highly polymorphic and its function-impairing variants can predispose patients to adverse effects. The effects of rare genetic variants of SLCO1B1 are mainly unexplored. We examined the impact of eight naturally occurring rare variants and the well-known SLCO1B1 c.521C > T (V174A) variant on in vitro transport activity, cellular localization and abundance.Organic Anion Transporting Polypeptide 1B1 (OATP1B1) mediates hepatic influx and clearance of many drugs, including statins. The Transport of rosuvastatin and 2,7-dichlorofluorescein (DCF) in OATP1B1 expressing HEK293 cells was measured to assess changes in activity of the variants. Immunofluorescence and confocal microscopy determined the cellular localization of OATP1B1 and LC–MS/MS based quantitative targeted absolute proteomics analysis quantified the amount of OATP1B1 in crude membrane fractions.All studied variants, with the exception of P336R, reduced protein abundance to varying degree. V174A reduced protein abundance the most, over 90% compared to wild type. Transport function was lost in G76E, V174A, L193R and R580Q variants. R181C decreased activity significantly, while T345M and L543W retained most of wild type OATP1B1 activity. P336R showed increased activity and H575L decreased the transport of DCF significantly, but not of rosuvastatin. Decreased activity was interrelated with lower absolute protein abundance in the studied variants.Transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1. Four of the studied variants were identified as loss-of-function variants and as such could make the individual harboring these variants susceptible to altered pharmacokinetics and adverse effects of substrate drugs.The online version contains supplementary material available at 10.1007/s11095-021-03107-8. SLCO1B1), a sinusoidal hepatic uptake transporter, plays a critical role in the pharmacokinetics of many widely used drugs such as cholesterol-lowering statins  in the  statins . ResultsSLCO1B1 c.521 T > C variant is rather common: 32% of people with European ancestry are heterozygotes (TC genotype) and 4% are homozygotes (CC genotype) (in vitro—in vivo extrapolation (IVIVE) such physiologically based pharmacokinetic modelling (PBPK) incorporating in vitro data can help estimate the changes in in vivo pharmacokinetics and identify genotypes that could warrant caution when prescribing OATP1B1 substrate drugs (The enotype) . This haenotype) . To overte drugs .The three-dimensional molecular structure of OATP1B1 has yet to be established and knowledge on the important substrate-binding sites is limited. OATP1B1 is predicted to form twelve transmembrane helices, with cytoplasmic N- and C-terminals. Site-directed mutation studies have provided information on the effects of amino acid substitution on transporter function and identified transmembrane helices that are essential for transporter function . Based oSLCO1B1 c.521 T > C SNV served as a clinical reference variant. Our results provide new insight to amino acids critical to the transporter function and SNVs that may make their carriers susceptible to altered pharmacokinetics of OATP1B1 substrates.Here we characterized nine naturally occurring SNVs, where the amino acid substitution occurs in the putative transmembrane region of OATP1B1 Fig. . The varFetal bovine serum (FBS), Hank’s Balanced Salt Solution (HBSS), Dulbecco’s Modified Eagle Medium (DMEM), Coomassie Plus protein assay reagent and HyClone SfX Insect cell medium were from ThermoFisher Scientific . Q5® Site-Directed Mutagenesis Kit was from New England Biolabs . Oligomer  produced the mutagenesis primers. Toronto Research Chemicals  provided rosuvastatin hemicalcium and rosuvastatin-d6 sodium salt and DCF was from Santa Cruz Biotechnology . Dithiothreitol and iodoacetamide were purchased from Sigma-Aldrich . The peptide standards for MRM analysis were from JPT Peptide Technologies GmbH, Berlin, Germany. ProteaseMax surfactant, LyC endopeptidase and tosylphenylalanylchloromethyl ketone-treated trypsin were from Promega . All the other chemicals were purchased from Sigma-Aldrich .SLCO1B1 gene (Genebank™ accession number AJ132573.1) in pENTR221 entry vector using the primers described in Supplementary table SLCO1B1 genes along with previously cloned gene for enhanced yellow fluorescent protein  (Q5® Site-Directed Mutagenesis Kit and PCR introduced the single nucleotide variants (SNV) to the reference control)  were procontrol) .2. Twenty-four hours prior to transduction with baculoviruses, 0.5 *106 cells were seeded in each well of 48-well plates (either poly-D-lysine coated ThermoFisher Scientific™ Nunc™ or Corning™ CellBind™). Sodium butyrate added with the viruses at a final concentration of 5 mM  stimulated the expression of the proteins.HEK293 cells were cultured in DMEM, high-glucose, GlutaMax culture medium supplemented with 10% FBS at 37 °C, 5% COThe cellular uptake assay was conducted 48 h post-transduction on a heated (37 °C) orbital shaker plate. 500 µl of transport buffer (HBSS with 4.17 mM NaHCO3 and 25 mM HEPES adjusted to pH 7.4 with NaOH) replaced the medium in the wells for a 3-min preincubation. After the removal of the buffer, uptake began with the addition of 125 µl of test solution containing test compound in transport buffer. Aspiration of the test solution stopped the uptake within the linear uptake phase for each substrate: 2 min for rosuvastatin and 15 min for 2,7-dichlorofluorescein (DCF). An immediate three-time wash with 500 µl ice-cold transport buffer followed. The cells were lysed with 125 µl of appropriate lysis buffer . DCF was quantified by fluorescence measurement of the cell lysates  with multimode microplate reader Varioskan LUX . The quantification of rosuvastatin is described in the following paragraph. The total protein was determined by mixing 10 µl of cell lysate with 300 µl Coomassie Plus reagent followed by absorbance analysis (595 nm) with Varioskan LUX.Uptake of test substrates was normalized to total protein amount. Uptake into eYFP expressing cells (representing passive influx) was subtracted from uptake into OATP1B1 expressing cells yielding active OATP1B1 mediated transport. This transport activity in the OATP1B1 variant cells was then normalized to the wild type OATP1B1 cells.Liquid chromatography (LC) mass spectrometry (MS) measurements of rosuvastatin were carried out with Waters ultra-high pressure LC–MS/MS instrument . Rosuvastatin D6 served as an internal standard (ISTD). Liquid chromatography coupled with a Waters UPLC HSS T3  column at 40 °C (1.5 µl injection volume) separated the analytes. Mobile phase consisted of 0.1% of formic acid  in ultrapure water (A) and 100% of LC–MS grade acetonitrile  (B). Gradient elution started with 20% of B at 0–0.2 min, continued with 20–95% B at 0.2–1.6 min and the complete run time was 5.5 min including column wash and equilibration with flow-rate as 0.3 mL/min.The MS measurements were carried out using Waters Xevo TQ-S triple quadrupole mass spectrometer coupled with an electrospray ionization (ESI) on a positive mode. Optimized ms-parameters were: capillary 3.5 kV, cone voltage 84 V for rosuvastatin and 62 V for ISTD, source temperature 150 °C and desolvation temperature 500 °C. Nitrogen was used as a desolvation gas (900 L h − 1) and a cone gas (150 L h − 1), argon as a collision gas. Multiple reaction monitoring quantified the analytes. The precursor and fragment ions for rosuvastatin were 482.19 > 258.12 (collision energy (CE) 32 V) and 482.19 > 300.19 (CE 34 V). For ISTD the ions were 488.17 > 264.17 (CE 30 V) and 488.17 > 306.24 (CE 38 V). The resulting data was analysed with Waters MassLynx V4.1 software.. . The cells were broken down with Dounce tissue homogenizer, resuspended in Tris-sucrose (TS) buffer  and held on ice. A 30-min centrifugation  followed. The supernatant was separated and a further centrifugation step  resulted in a pellet containing the crude membrane. The protein sample was suspended in TS buffer and quantified as described previously.HEK293 cells were cultured in T175 flasks for 24 h prior to addition of sodium butyrate  and baculoviruses containing either wild type or variant The absolute protein expression of OATP1B1 in the crude membrane preparations was quantified using LC–MS/MS-based QTAP approach. The protein sample preparation and quantitation with targeted LC–MS was based on the method described by Uchida et al. . BrieflyThe resulting peptides in the samples were analyzed with 6495 QQQ MS  with 1290 HPLC system (Agilent Technologies) and AdvanceBio peptide Map Column, 2.7 µm, 2.1 × 250 mm (Agilent Technologies) using HPLC method described earlier . MS cond+/K+-ATPase were selected according to the in silico peptide selection criteria published by Uchida et al., 2013 . Th. Th+/K+-Cellular uptake assays were conducted in triplicates in two to four separate experiments. The average of these technical triplicates is considered one data point and the data are presented as their mean ± SEM. The proteomics samples (presented as mean ± SEM) were prepared from four and immunofluorescence analysis from two separate batches of HEK293 cells.max) and Michaelis–Menten constant (Km) values of rosuvastatin transport for selected variants were calculated with non-linear regression from concentration‐dependent assay data and extra-sum-of-squares F-test assessed their statistical significance. P-values below 0.05 were considered significant in all analyses.The homogeneity of variance in the results was established with Levene’s test . Based on these results, the statistical significance of the differences in activity and expression levels was determined with either one-way analysis of variance (ANOVA) with the Dunnett’s post hoc test for multiple comparisons or Kruskal–Wallis one-way analysis of variance with Dunn’s post hoc test for multiple comparisons . Maximum velocity   approach to quantify OATP1B1. After normalization to Naype Fig. . The absWith the exception of P336R, all of the studied SNVs resulted in reduced expression of OATP1B1 in HEK293 cells Fig. . G76E, VImmunofluorescence and microscopy provided additional qualitative information on the localization of the OATP1B1 protein in control (non-transduced or eYFP expressing HEK293), wild type (WT) and variant OATP1B1 expressing HEK293 cells. Wild type OATP1B1 virus transduced cells showed typical concentrated band-like green color Figs.  and 8. NIn the G76E expressing cells, OATP1B1 was abundant yet it was not concentrated in similar band-like manner as in the wild type cells. In V174A, H575L, L193R and L543W variant cells the expression of OATP1B1 protein is appears lower than in the wild type Fig. . Insteadin vitro. G76E, R181C, T345M, H575L and R580Q were, to our knowledge, characterized for the first time. A trend (linear regression R2 0.76) was observed between protein expression levels and activity for the studied variants: all loss of function variants had levels below 50% of wild type OATP1B1  was not determined. Instead, the comparability of the variants was controlled by producing them at the same time with the wild type baculoviruses with standardized amount of DNA in each batch and the variants were only compared to the batch specific wild type virus. The amount of virus was tittered in in-house optimization experiments to achieve maximum transduction and the same ratio was used in all the experiments. The same protocol has previously been used by Sjöstedt et al. . We perf.  and 5, w. . While iSLCO1B1 c.211G > A, a variant that changes glycine to arginine at position 71, reduces OATP1B1 mediated methotrexate transport severely in vitro and is associated with reduced methotrexate clearance (in vitro transport of estrone-3- sulfate (E3S) significantly  genotype has resulted in increase in plasma AUC levels of e.g. rosuvastatin  was not altered in two studies  are both found in the putative transmembrane helix 7 Fig. . T345M dvastatin . In our max was reduced moderately. This in accordance with the first in vitro characterization, where this variant did not alter the uptake of E3S but reduced pravastatin Vmax (no change in Km)  . Cell-suH575L and R580Q, are found in transmembrane helix 11. Surprisingly, the H575L variant reduced transport activity in a substrate specific manner. The reduction in rosuvastatin transport was in line with reduction in protein abundance, yet DCF transport was more drastically reduced, indicating the possibility of additional changes in substrate recognition or translocation. R580Q impaired the transport of both tested substrates significantly and is likely caused by decreased protein expression. Immunofluorescence, however, suggested no drastic change in neither abundance nor cellular localization of OATP1B1. Previously, alanine substitution at position 580 resulted in over 50% reduction in E3S transport . In a WeOverall, the studied variants in transmembrane helices 2, 4 and 11 appear to be crucial for proper membrane localization and function of OATP1B1, while the studied variants in helices 7 and 10 retained much of the function of the wild type OATP1B1.SLCO1B1 c.521 T > C (V174A) genotype has prompted issuance of clinical prescribing guidelines  was significantly reduced, two (T345M and L543W) had similar activity and one (P336R) variant had enhanced transport activity compared to the wild type OATP1B1. Overall, our results support previous studies suggesting that transmembrane helices 2, 4 and 11 are important for proper membrane localization and function of OATP1B1. While there was a trend between protein amount and activity, protein amount does not fully explain the observed transport activities, indicating involvement in substrate recognition or translocation for some of these residues. G76E, L193R and R580Q caused loss of function and thus could predispose the individuals with these genotypes to adverse effects of OATP1B1 substrate drugs similarly to V174A.We have determined the transport activity and protein levels of nine variants of OATP1B1. To our knowledge, the G76E, R181C, T345M, H575L and R580Q variants were characterized for the first time. The We are grateful for the funding provided by Svenska Kulturfonden, Medicinska Understödsföreningen for Liv och Hälsa, DRA Consulting oy, Instrumentarium Science Foundation and Finnish Cultural Foundation. The UEF Metabolomics laboratory is supported by Biocenter Finland and Biocenter Kuopio and the Drug Discovery and Chemical Biology Network and the Light Microscopy Unit are funded by Biocenter Finland. Dr. Melina M. Malinen received funding from the European Union’s Horizon 2020 Research and Innovation program under the Marie Skłodowska-Curie grant agreement number 799510.Supplementary file1 (DOCX 20.3 KB)Below is the link to the electronic supplementary material.


The blood-brain barrier (BBB) prevents antibodies from penetrating the CNS and limits conventional antibody-based approaches to brain tumors. We now show that ENT2, a transporter that regulates nucleoside flux at the BBB, may offer an unexpected path to circumventing this barrier to allow targeting of brain tumors with an anti-DNA autoantibody. Deoxymab-1 (DX1) is a DNA-damaging autoantibody that localizes to tumors and is synthetically lethal to cancer cells with defects in the DNA damage response. We found that DX1 penetrated brain endothelial cells and crossed the BBB, and mechanistic studies identify ENT2 as the key transporter. In efficacy studies, DX1 crosses the BBB to suppress orthotopic glioblastoma and breast cancer brain metastases. ENT2-linked transport of autoantibodies across the BBB has potential to be exploited in brain tumor immunotherapy, and its discovery raises hypotheses on actionable mechanisms of CNS penetration by neurotoxic autoantibodies in CNS lupus.  Antibodies have revolutionized cancer therapy, but the blood-brain barrier (BBB) limits their use against brain tumors , 2. LocaSome autoantibodies reactive against host DNA penetrate live cells, and modified cell-penetrating autoantibodies are in development for use as single agents or delivery ligands in molecular therapy . Nucleos3E10 is not toxic to normal cells but is synthetically lethal to PTEN and BRCA-deficient cancer cells with defects in homologous recombination and repair of DNA double-strand breaks. Mechanistically, 3E10 inhibits base excision repair and homologous recombination and causes double-strand breaks to accumulate in and kill DNA repair-deficient cancer cells , 16–19. Necrosis is a defining feature of aggressive brain tumors such as glioblastoma multiforme (GBM) , and suchttps://doi.org/10.1172/jci.insight.145875DS1). ENT2 knockdown reduced penetration by DX1 to 0.36 ± 0.04 (P < 0.01) relative to cells treated with DX1 and control siRNA  relative to cells treated with DX1 in the absence of DP (Equilibrative nucleoside transporters are sensitive to inhibition by the pyrimido-pyrimidine derivative drug dipyridamole (DP) , 41. Thece of DP . These rP < 0.03) in BBB inserts relative to control blank inserts (P < 0.01 compared with DX1) (The ability of DX1 to cross an hCMEC/D3 transwell model of the BBB was tested using our previously described technique . Briefly inserts . In contith DX1) .P < 0.01) and 0.42 ± 0.11 (P < 0.01) at 15 and 30 minutes, respectively , i.v. DX1 (20 mg/kg) and i.p. control buffer (n = 4), or i.v. DX1 (20 mg/kg) and i.p. DP (70 mg/kg) (n = 4). DX1 was labeled with Alexa Fluor 750 (AF750) to facilitate detection in vivo. Twenty-four hours after treatment, brains were evaluated by IVIS to detect the labeled DX1, and radiance efficiencies were recorded. Control mice showed a mean background radiance efficiency in the brain of 28.1 ± 0.9 × 104. Mice treated with DX1 in the absence of DP exhibited a strong AF750 signal in the brain, correlating to the region of tumor formation, with mean radiance efficiency of 96.1 ± 3.2 × 104 (Tukey’s multiple-comparison test adjusted P < 0.0001 relative to control mice). Cotreatment with DP reduced the observed uptake of DX1 into the brain tumors, with mean radiance efficiencies decreased to 43.1 ± 1.0 × 104 (Tukey’s multiple-comparison test adjusted P < 0.0001 relative to mice treated with DX1 in the absence of DP). Representative images are shown in A panel of primary human GBM glioma stem-like cells (GSCs) derived from patient tumors is maintained at Yale School of Medicine under an IRB-approved protocol . GBM tumn = 7) or group 2 for treatment with i.v. DX1 (20 mg/kg) (n = 7) 3 times per week throughout the study. Consistent tumor growth between groups was confirmed by IVIS prior to treatment. One week after inoculation, mean radiance efficiencies (× 104) ± SEM in groups 1 and 2 were 0.5 ± 0.1 and 0.4 ± 0.1, respectively . At 2 weeks after inoculation, mean radiance efficiencies (× 104) ± SEM in groups 1 and 2 were 3.7 ± 0.9 and 3.8 ± 0.8, respectively . Treatment began after the week 2 IVIS measurements. At week 3 (1 week after the start of treatment), treatment with DX1 was observed to suppress tumor growth, with radiance efficiencies (× 104) in groups 1 and 2 of 42.6 ± 11.1 and 17.4 ± 4.1, respectively . In week 4, radiance efficiencies (× 104) in groups 1 and 2 were 358.1 ± 160.9 and 47.7 ± 17.7, respectively    . By week4 and 7)  and prolnk test) .n = 10) or DX1  3 times per week throughout the study. GBM model 2 GSCs have not been engineered to express luciferase, which necessitated examination of the effect of DX1 on tumor growth by harvesting brains at a preselected time point. At 9 weeks after tumor inoculation, 3 mice per group were euthanized, and brains were sectioned for analysis by H&E, Ki67, and TUNEL stains and immunostained for DX1. This time point was selected based on experience with this model in regard to time of onset of symptoms. Tumor area in each brain was evaluated by importing images of H&E- and Ki67-stained sections into ImageJ for contouring, and measurement of mean tumor areas on slices through the maximal thickness of the tumor. Representative H&E and Ki67 sections are shown in 2 versus 15.9 ± 1.5 mm2, P < 0.04, n = 3 mice per group)   . Both prr group) . In addir group) . DX1 wasr group) . The remnk test) . DX1 wasnk test) . These sn = 7) or group 2 for i.v. DX1 . The effects of DX1 delivered as a single cycle or as 4 consecutive cycles were tested in 2 independent studies, with 1 cycle defined as administration of control or DX1 3 times in 1 week. Mice were observed for behavior and weights, and brain radiance efficiency was monitored by weekly IVIS to track tumor growth. For the single cycle study, at week 1 prior to the start of treatment, mean radiance efficiencies (× 105) ± SEM in groups 1 and 2 were 1.2 ± 0.1 and 1.2 ± 0.1, respectively . Treatment with DX1 suppressed tumor growth, with radiance efficiencies (× 105) in groups 1 and 2 of: 2.4 ± 0.2 and 1.7 ± 0.1 in week 2 , 5.9 ± 0.6 and 2.9 ± 0.3 in week 3 , 21.6 ± 3.5 and 8.4 ± 1.5 in week 4 , 264.8 ± 72.0 and 71.9 ± 31.3 in week 5 , and 319.9 ± 122.5 and 161.8 ± 51.4 in week 6 , respectively (5) ± SEM in groups 1 and 2 were 1.2 ± 0.2 and 1.3 ± 0.2, respectively . Treatment with DX1 suppressed tumor growth, with radiance efficiencies (× 105) in groups 1 and 2 of: 2.5 ± 0.4 and 1.4 ± 0.3 in week 2 , 5.1 ± 0.7 and 2.0 ± 0.2 in week 3 , 20.2 ± 5 and 3.8 ± 0.8 in week 4 , 320 ± 66 and 20.2 ± 8.5 in week 5 , and 444 ± 49 and 35.3 ± 11 in week 6 , respectively (P = 0.42) (P < 0.02) (Breast cancer brain metastases are associated with increased incidence of PTEN and homologous recombination defects compared with primary breast tumors –33. The ectively . For theectively . Radiancectively . One cyc = 0.42) , similar = 0.42) . Four cy < 0.02) . DX1 was < 0.02) . These dAntibody-based immunotherapy has the potential to change paradigms in the management of CNS malignancies, but obstacles inherent to the neuroimmune interface must be overcome. The BBB excludes most antibodies and likely contributes to the suboptimal responses of primary and metastatic brain tumors to antibodies observed in clinical trials to date –49. The In previous work, cellular and nuclear penetration by 3E10 was demonstrated in multiple cell lines and shown dependent on the ability of 3E10 to bind DNA, the presence of extracellular DNA/nucleosides, and the expression of ENT2 in cells –52. MostThe pyrimido-pyrimidine derivative drug DP was used to facilitate consideration of the role of ENT2 in DX1 transport across a transwell BBB model and into brain tumors in vivo. DP blocked DX1 penetration into brain endothelial cells, transport across the transwell BBB model, and localization into orthotopic brain tumors. These findings further implicate ENT2 as mediator of BBB transport and brain tumor targeting by DX1. In regard to specificity of DP activity, DP exhibits greater inhibition of ENT2 as compared with the prototype nucleoside transport inhibitor nitrobenzyl mercaptopurine riboside (NBMPR). DP also has inhibitory effects on equilibrative nucleoside transporters ENT1 and ENT4 and on phosphodiesterases –56. BaseTaken together, our present findings support an ENT2-mediated mechanism of BBB penetration by DX1 and establish proof of concept for the use of a DNA-targeting lupus autoantibody against brain tumors. We also recognize DX1 as a potential delivery ligand for targeting linked cargo molecules to brain tumors and as a platform upon which additional antibodies with CNS bioavailability may be designed, including DX1-based bispecific antibodies. Furthermore, our findings raise the possibility that nucleoside salvage transporters are involved in the crossing of the BBB by neurotoxic autoantibodies in the syndrome of CNS lupus , 58 and DX1  was expressed in CHO cells and purified over a HiTrap Capto S column by FPLC as previously described . Purity DP (D9766) was obtained from MilliporeSigma. hCMEC/D3 BECs were cultured in 96-well plates and were then pretreated with control buffer (PBS + 10% FBS) or buffer containing 50 μM DP for 1 hour. Supernatants were then replaced with control buffer, buffer containing 10 μM DX1, or buffer containing 50 μM DP and 10 μM DX1. Thirty minutes later, cells were washed with PBS 3 times, fixed with chilled ethanol, and immunostained with the D5 anti-DX1 primary antibody , anti–rat AP-–conjugated secondary antibody , and finally with DX1 signal detected by color development as previously described . Cell st–ΔΔCt method was used to analyze the relative changes in gene expression between the tested samples according to the manufacturer’s protocol . After confirmation of ENT2 knockdown, efficiency of DX1 penetration into control or ENT2 siRNA–transfected cells was compared by protein L-based staining as previously described  or nontargeting control siRNA  were transfected into the cells using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The transfection was repeated the next day to ensure maximum knockdown. Successful ENT2 knockdown was confirmed by RT-PCR. RNA from ENT2 siRNA- and control siRNA–transfected knock down hCMEC/D3 cells was extracted using RNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol using 0.5 μg RNA in a total of 20 μL reaction. The mRNA level of ENT2 and β-actin was assessed using TaqMan Gene Expression real-time PCR assays . The results were expressed as the Ct. The relative quantification of the target transcripts normalized to the endogenous control β-actin was determined by the comparative Ct method (ΔCt), and the 2escribed .× 104 cells/insert), and hCMEC/D3 brain endothelial cells were adhered to the apical side (3.3 × 104 cells/insert).Cell culture inserts  were coated with 0.001% poly-L-lysine (PLL) solution  on the basolateral side and Fibronectin solution  on the apical side. NHA were adhered to the basolateral side of inserts  and blocked with 20% goat serum/0.5% Triton X-100 in PBS. The inserts were then transferred to wells containing 0.5% BSA/0.25% Triton X-100 in PBS (antibody incubation buffer [AIB]). The apical chamber contents were replaced with primary antibody against occludin (1:20) in AIB. Inserts were incubated at room temperature for 1 hour and then washed 3 times using PBS containing 0.1% Triton X-100 . Alexa 488–conjugated secondary antibody (1:200) in AIB was then added to apical chambers, and AIB to basolateral chambers, for 1 hour at room temperature. The inserts were subsequently washed with PBS-T as mentioned above. DAPI  counterstain was then completed by addition of DAPI in PBS to apical chambers for 10 minutes , followed by washes. All steps were conducted on inserts still attached to culture plate adaptors until final washes were completed. Inserts were then gently excised from the adaptors using a scalpel and mounted onto glass slides using Aqua Poly/Mount mounting medium to facilitate visualization of fluorescence by Evos FL microscope (Thermo Fisher Scientific). The slides were kept at 4°C overnight and imaged the next day.TEER of inserts with BBB (with both hCMEC/D3 and NHA layers of cells) and control blank inserts (with only PLL and Fibronectin) was serially measured by voltometer, and when the difference was more than 100, the BBB inserts were deemed ready to be used in experiments , 60. To × 104 GSCs in 5 μL PBS were injected into the right striatum (n = 10) 2 mm lateral and 0.5 mm posterior to the bregma and 3 mm below the dura, using a stereotactic apparatus with an UltraMicroPump (UMP3) (World Precision Instruments). Mice were then tracked by weekly IVIS , and once tumors were confirmed, mice were randomized to groups for treatment with tail vein and i.p. injection of control buffer  (n = 2), tail vein injection of DX1AF750  and i.p. injection of control buffer (n = 4), or tail vein injection of DX1AF750  and i.p. injection of DP  (n = 4). DX1 was labeled with equimolar AF750 by reaction with AF750 NHS Ester (Thermo Fisher Scientific) in PBS (pH 7.4). AF750-labeled DX1 was dialyzed  into PBS to remove excess AF750, and it was filter-sterilized prior to use. Twenty-four hours after treatment, mice were evaluated by IVIS to detect AF750 signal in the brain tumors.Studies were conducted under a Yale University IACUC–approved protocol. In the first model, luciferase-expressing GBM model 1 human GSCs were used to establish orthotopic GBM tumors in the brains of nude mice. Female athymic NCr-nu/nu mice  maintained in a pathogen-free environment were anesthetized by i.p. injection of ketamine and xylazine (Covetrus). In total, 6 Volumes of GBM spheroids treated with control media or media containing DX1 were monitored over 7 days. An Evos FL microscope (Thermo Fisher Scientific) was used to capture representative bright-field images over multiple fields of view. Analysis of GBM spheroid volume was performed using ImageJ. Sphere images were imported, and the scale was thresholded to calibrate for pixel size. Sphere diameters were determined and used to determine the equivalent sphere volume for each spheroid. Relative volumes were calculated as described in each experiment.The impact of control media or media containing DX1 on the clonogenic survival of 231-BR cells was evaluated by colony formation assay as previously described .× 104 to 6 × 104 GSCs in 5 μL PBS were injected into the right striatum , 2 mm lateral and 0.5 mm posterior to the bregma and 3 mm below the dura, using a stereotactic apparatus with an UltraMicroPump (UMP3) on day zero. Treatment was initiated 2 weeks after inoculation. Mice were treated with control buffer  or DX1  by tail vein injection 3 times per week. Mice were monitored daily for general health, body weight, grooming, behavioral changes and any signs of distress. Mice exhibiting neurological symptoms or significant weight loss were humanely euthanized. In the GBM model 1 study, tumor sizes were tracked by weekly IVIS as previously described  were used to establish orthotopic GBM tumors in the brains of nude mice. PTEN-deficient status of tumors was confirmed by anti-PTEN immunostain on sections of established tumors as previously described . Female escribed . For theescribed . The rem× 105 231-BR cells in 100 μL PBS. Successful establishment of brain metastases was confirmed by IVIS 1 week after injection. Mice were randomized into groups with equivalent mean radiance efficiencies in the brain, and they were treated with control vehicle  or DX1  by tail vein injection 3 times per week for 1 or 4 cycles. Mice were monitored daily for general health, body weight, grooming, behavioral changes, and any signs of distress. Mice exhibiting neurological symptoms or significant weight loss were humanely euthanized. Tumor burden was monitored by weekly IVIS. Kaplan-Meier plots of survival were generated in GraphPad Prism (version 7).Studies were conducted under a Yale University IACUC–approved protocol. The 231-BR brain-seeking subclone of the MDA-MB-231 cell line, engineered to express luciferase to allow detection by IVIS, was used to establish brain metastases in nude mice. Two independent studies with this model were conducted in order to test the effects of a single cycle or 4 cycles of DX1. In each study, on day zero, 14 female athymic NCr-nu/nu mice  maintained in a pathogen-free environment were anesthetized by i.p. injection of ketamine and xylazine, and they were subjected to intracardiac injection of 1.75 t test was used to evaluate the statistical significance of data. P ≤ 0.05 was considered significant. Data are presented as mean ± SEM in figure parts in which error bars are shown. Mice were randomized to respective treatment groups. Sample sizes were selected based on power analysis informed by previous results with the parental 3E10 antibody and fragments. Endpoints were prospectively selected. Number of replicates for each experiment are indicated in the respective figures. No investigator blinding was used.Log-rank test was used to determine the statistical significance of in vivo survival data. Tukey’s multiple-comparison test was used to determine the statistical significance of radiance efficiency data in Studies were conducted under a Yale University IACUC–approved protocol.Study conception and design were contributed by JZ and JEH. Performance of experiments and analysis of data were contributed by ZR, GD, SZ, AS, CKM, XC, BJC, PZ, JL, NJWR, JZ, and JEH. Manuscript preparation and/or review were contributed by ZR, GD, SZ, AS, CKM, XC, BJC, JL, PZ, NJWR, CHJ, VD, JAC, AH, JMB, JZ, and JEH. The order of the co–first authors  was based on the timing of each author’s initiation of their work on the project.


Trilaciclib is a first-in-class CDK4/6 inhibitor that transiently arrests hematopoietic stem and progenitor cells (HSPCs) in the G1 phase of the cell cycle to preserve them from chemotherapy-induced damage (myelopreservation). We report integrated analyses of preclinical and clinical data that informed selection of the recommended Phase II dose (RP2D) used in trilaciclib trials in extensive-stage small cell lung cancer (ES-SCLC).A semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model developed from preclinical data guided selection of an optimal dose for G1 bone marrow arrest in a first-in-human Phase I study (G1T28-1-01). PK, PD, safety, and efficacy data from G1T28-1-01 and two Phase Ib/IIa studies (G1T28-02/-03) in ES-SCLC were analyzed to support RP2D selection.2 dose would induce a 40–50% decrease in total bone marrow proliferation in humans and almost 100% cell cycle arrest of cycling HSPCs. Consistent with this model, analysis of bone marrow aspirates in healthy volunteers after trilaciclib 192 mg/m2 administration demonstrated almost 100% G1 arrest in HSPCs and 40% decrease in total bone marrow proliferation, with minimal toxicity. G1T28-02/-03 reported similar PK parameters with trilaciclib 200 mg/m2 but slightly lower exposures than expected compared with healthy volunteers; consequently, 240 and 280 mg/m2 doses were also tested to match healthy volunteer exposures. Based on PK and relevant safety data, 240 mg/m2 was selected as the RP2D, which was also favored by myelopreservation endpoints in G1T28-02/-03.Model simulation of bone marrow arrest based on preclinical data predicted that a ≥ 192 mg/m2 as the RP2D for trilaciclib.Integrated PK/PD, safety, and efficacy data support 240 mg/mNCT02243150; NCT02499770; NCT02514447.The online version contains supplementary material available at 10.1007/s00280-021-04239-9. Damage to hematopoietic stem and progenitor cells (HSPCs) by cytotoxic chemotherapy causes multilineage myelosuppression, which can manifest as neutropenia, anemia, thrombocytopenia, and/or lymphopenia –3. ChemoTrilaciclib is a first-in-class, highly potent, and selective intravenously administered cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, developed to preserve multiple hematopoietic cell lineages from chemotherapy-induced damage. When administered prior to chemotherapy, trilaciclib transiently arrests CDK4/6-dependent proliferating cells in the G1 phase of the cell cycle to prevent CIM , 10, 11.There was a theoretical concern that during dose escalation with chemotherapy, low doses of trilaciclib could produce an insufficient G1 arrest, thereby releasing HSPCs from G1 arrest in a synchronous manner while therapeutic levels of chemotherapy were present, and potentially resulting in increased CIM. Therefore, it was important to ensure that a biologically effective dose (BED) of trilaciclib was identified that was sufficient to maintain G1 cell cycle arrest of HSPCs throughout the duration of clinically relevant chemotherapy exposure. Because trilaciclib is a first-in-class molecule and no prior clinical evidence was available from similar molecules to inform dose selection, pharmacokinetic (PK) and pharmacodynamic (PD) modeling of available preclinical trilaciclib data , 13 was 2 as the dose for the Phase II randomized trials of trilaciclib in patients with ES-SCLC.Here, we describe these integrated analyses of preclinical and clinical PK, PD, safety, and efficacy data that formed the basis for selecting 240 mg/mOwing to the invasive nature of the PD marker collection (i.e. bone marrow aspiration), a semi-mechanistic PK/PD model based on animal PK/PD data and human PK data was developed to guide the selection of an optimal dose level for bone marrow aspiration in humans during dose escalation in the Phase I first-in-human study. The model was constructed using PK data from four species  and PD data from three species .The PK/multi-compartment PD model was based on a myelosuppression model previously published by Friberg et al. , with moThe model was constructed in two stages. First, preclinical PK data  were compiled and modeled by allometric scaling. A sequential PK/PD modeling approach (population PK parameters and data approach ) was theEmax) in plasma on the transition rate constant from G1 to cycling for HSPCs. The effect of 5-fluorouracil (5-FU) was modeled as removing (killing) cells while HSPCs are cycling. Owing to model complexity, only a minority of parameters could be estimated. The remaining parameters were either fixed using literature-based values or reliance on the model to find the best fit.A combined systems biology and empiric modeling approach was taken, largely based on an understanding of the biology of hematopoiesis and the mechanism of action of trilaciclib, with literature values used when needed, and domain expert estimates used when literature values were not available. The multi-compartment PD model Fig. a was bui2. The PK model for trilaciclib was fixed to the final model from the PK analysis, and an additional 5-FU model was fixed to the literature values  5 mg min/mL) on Day 1 and etoposide (100 mg/m2) on Days 1–3, and IV trilaciclib (or placebo in Part 2) was administered once daily before chemotherapy. The primary objective of the study was to define the trilaciclib RP2D (Part 1) and to assess the safety and tolerability of trilaciclib in combination with E/P (Parts 1 and 2). Secondary objectives included analysis of the PK profiles of trilaciclib and of E/P (Part 1), and assessment of trilaciclib efficacy (Parts 1 and 2), including thorough evaluation of common myelosuppression endpoints. In the dose-finding Part 1 portion, blood samples for PK analysis were collected at prespecified time points on Cycle 1 Day 1 and Cycle 1 Day 3 .G1T28-02 (NCT02499770) was a multicentre Phase Ib/IIa study in patients with newly diagnosed ES-SCLC . Eligibl. Part 1 was a Phase Ib, open-label, dose-finding portion. Part 2 was a Phase IIa, randomized, double-blind, placebo-controlled portion. In Part 1, trilaciclib  was administered once daily prior to chemotherapy on Days 1–5 of each 21-day topotecan cycle . In Part 2a, patients were randomized 2:1 to receive trilaciclib 240 mg/m2 prior to topotecan 0.75 mg/m2, or placebo prior to topotecan 1.5 mg/m2 using the same schedule as that in Part 1. Part 2b evaluated trilaciclib 240 mg/m2 prior to topotecan 1.5 mg/m2 using the same schedule as that in Part 1. Study objectives included assessment of dose-limiting toxicities , safety and tolerability (Parts 1 and 2), PK (mainly Part 1), and myelosuppression efficacy (Parts 1 and 2) of trilaciclib in combination with topotecan. In Part 1 of the study, blood samples for PK analysis were collected at prespecified time points on Cycle 1 Day 1 and Cycle 1 Day 4 for all patients.G1T28-03 NCT02514447) was a multicentre Phase Ib/IIa study in patients with previously treated ES-SCLC  increased dose dependently following a single 30-min IV infusion of trilaciclib over the dose ranges tested (0–192 mg/m2), with AUC0-inf increasing slightly more than dose proportionally. Mean (range) AUC0-inf for both 192 mg/m2 groups combined (n = 18) was 3029 (2379–3869) ng h/mL (Online Resource 6).Of the 33 subjects enrolled in the SAD part of the study, 24 received trilaciclib  with a maximum mean inhibition of 37.2% and 60%, respectively, at 4 h after end of infusion . Baseline demographics and disease characteristics were generally comparable across the treatment groups in Part 1  than expected on the basis of exposure at 192 mg/m2 in Study G1T28-1-01; this prompted the safety monitoring committee to increase the dose to 240 mg/m2, which almost achieved the target AUC of 3100 ng h/mL . A dose of 280 mg/m2 was also tested, but exposures were significantly higher and resulted in drug accumulation (gMean AUC: 3490 h ng/mL on Day 1 and 4520 h ng/mL on Day 4) that had not been observed at lower dose levels. The safety and efficacy of three dose levels of trilaciclib: 200, 240, and 280 mg/m2 were evaluated when trilaciclib was administered prior to 0.75 mg/m2 of topotecan. No DLT (0 of 10 patients) was reported for the 240 mg/m2 trilaciclib cohorts. By contrast, 2 of 4 and 2 of 7 patients reported DLTs in the 200 mg/m2 and 280 mg/m2 trilaciclib cohorts, respectively. From laboratory abnormalities, the 240 mg/m2 dose showed fewer Grade 3/4 neutropenia and thrombocytopenia events than did the 200 and 280 mg/m2 doses . The efficacy/safety comparisons for trilaciclib dose levels were based on a topotecan dose of 0.75 mg/m2, which is half of the recommended dose (1.5 mg/m2). However, the PD effect of trilaciclib is assumed to be related to host cells, hence is independent of the chemotherapy dose.Similar to Study G1T28-02, PK analysis n = 1 showed 2 would result in a decrease in total bone marrow cycling cells of slightly less than 50%; therefore, this dose was selected as the BED, to be evaluated further in the first-in-human Phase I study (G1T28-1-01).To rationally design a schedule for administering trilaciclib prior to chemotherapy, it was critical to understand the magnitude and duration of G1 cell cycle arrest of HSPCs at a given dose level of trilaciclib. Therefore, in addition to obtaining first-in-human safety experience, a key goal of the Phase I study was to demonstrate the biological activity of trilaciclib, including the maximal level of inhibition and the duration of response in HSPCs, and to define the starting dose level of trilaciclib for use in combination with chemotherapy in patients with ES-SCLC. Owing to the difficulty of sampling human bone marrow for HSPCs and other cell types, dose selection was started from an evaluation of available preclinical data from mice and dogs , 13, whi2 and 192 mg/m2, with a dose-dependent decrease in PHA-stimulated lymphocyte proliferation, providing proof of concept that there was a dose-dependent decrease in proliferating CDK4/6-dependent cells in the blood due to CDK4/6 inhibition. In addition, the significantly higher level of inhibition for lymphocyte proliferation at 192 mg/m2 aligned with the simulation results that supported 192 mg/m2 as the BED for assessing bone marrow proliferation. Data from bone marrow HSPCs were more definitive, providing information for the target organ and cell populations. These data demonstrated that administration of trilaciclib at the BED of 192 mg/m2 resulted in almost 100% G1 arrest in most bone marrow stem and progenitor subsets, with the exception of erythrocyte and megakaryocyte lineages, which persisted for 32 h.Lymphocytes are known to be sensitive to CDK4/6 inhibition and were evaluated as a surrogate PD marker of trilaciclib activity as well as a surrogate for bone marrow aspirates. In healthy volunteers, a robust PD effect was demonstrated with trilaciclib 96 mg/mThe total bone marrow data were consistent with the model-predicted results, showing an approximate 40% decrease in total bone marrow cells in the S phase with some recovery by 32 h. Trilaciclib was well tolerated at all test doses. The most commonly reported AEs were headache and nausea. No severe AEs were reported, and all moderate AEs spontaneously resolved within 24 h .2 was pharmacologically active with minimal toxicity in healthy volunteers, a dose of trilaciclib 200 mg/m2 (rounded up from 192 mg/m2) was selected as the starting dose for the subsequent Phase Ib/IIa ES-SCLC studies. Subsequent PK assessments across Part 1 of Studies G1T28-02 and G1T28-03 revealed that the PK parameters of trilaciclib at 200 mg/m2 were similar in each study, but that exposures (AUC) were slightly lower than the target AUC established with 192 mg/m2 in Study G1T28-1-01. On the basis of these observations and relevant safety data, the dose of trilaciclib was increased to 240 mg/m2. Trilaciclib was also tested at a dose of 280 mg/m2 in Study G1T28-03; however, owing to the level of accumulation and accompanying safety data, the 280 mg/m2 dose was not selected as the RP2D. The poorer efficacy observed in myelosuppression endpoints with the 280 mg/m2 dose may be due to the longer-than-necessary arrest of HSPCs in the G1 phase. This likely resembles an inverted U-shaped dose–response curve, whereby prolonged inhibition could cause myelosuppression.On the basis of PK/PD data from Study G1T28-1-01, which indicated that trilaciclib 192 mg/m2 as the Phase IIa dose. DLT assessments showed reduced DLT with trilaciclib 240 mg/m2 versus 200 mg/m2 (G1T28-02 and G1T28-03) and 280 mg/m2 (G1T28-03). Additionally, there was a reduction in hematologic Grade 3/4 laboratory abnormalities with the 240 mg/m2 dose compared with the 200 mg/m2 (G1T28-02 and G1T28-03) and 280 mg/m2 (G1T28-03) doses.Evaluation of safety data in Part 1 of both the G1T28-02 and G1T28-03 studies supported the selection of trilaciclib 240 mg/m2 reduced the risk of CIM in patients with ES-SCLC undergoing chemotherapy, and that it had a favorable outcome over the 200 mg/m2 dose. These findings are consistent with the subsequent Phase IIa efficacy results of the G1T28-02 and G1T28-03 studies, which demonstrated a clinically and statistically significant reduction in the DSN in Cycle 1 and occurrence of SN across the treatment period when trilaciclib was administered prior to chemotherapy [90 ~ 50 ng/mL), cells in the S, G2, and M phases would be fully depleted  in approximately 1.5–2 h. Even shorter periods of trilaciclib administration prior to chemotherapy will still result in substantial protection, particularly for stem cells, as only a small fraction of stem cells are cycling at any one time [Efficacy data from Part 1 of both the G1T28-02 and G1T28-03 trials clearly showed that trilaciclib 240 mg/motherapy , 14. In one time , 29. Mor2 was identified as the BED and was selected for further safety and efficacy evaluation. The prediction for the PD effect estimated from preclinical data was confirmed by analyses of bone marrow aspiration from the first-in-human study. Subsequent evaluation of trilaciclib administered prior to chemotherapy (E/P or topotecan) in patients with ES-SCLC led to a PK- and efficacy-guided dose adjustment with the selection of trilaciclib 240 mg/m2 as the RP2D for Part 2 of Studies G1T28-02 and G1T28-03. In Parts 1 and 2 of both studies, trilaciclib 240 mg/m2 was well tolerated, with few trilaciclib-related AEs, and demonstrated a decrease in laboratory abnormalities and additional myelosuppression endpoints compared with placebo. Improvements were consistently more favorable in patients treated with trilaciclib 240 mg/m2 compared with those receiving trilaciclib 200 mg/m2 or 280 mg/m2, supporting trilaciclib 240 mg/m2 as the RP2D.In summary, a PK/PD model was constructed to predict human PD of trilaciclib for the first-in-human study to guide selection of the optimal dose at which to evaluate bone marrow aspirates, which is an invasive procedure. Using model simulations and available PK and PD data from Study G1T28-1-01, a trilaciclib dose of 192 mg/mSupplementary file1 (DOCX 330 KB)Below is the link to the electronic supplementary material.


The resulting sensor was successfully applied for the sensitive detection of paroxetine·HCl (PRX), a selective serotonin (5-HT) reuptake inhibitor (SSRIs), in its pharmaceutical formulations. The sensor was characterized morphologically using scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) and electrochemical techniques such as differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The proposed sensor, poly (dl-met)/AuNPs-GCE, exhibited a linear response range from 5 × 10−11 to 5 × 10−8 M and from 5 × 10−8 to 1 × 10−4 M using DPV with lowest limit of detection (LOD = 1 × 10−11 M) based on (S/N = 3). The poly (dl-met)/AuNPs-GCE sensor was successfully applied for PRX determination in three different pharmaceutical formulations with percent recoveries between 96.29% and 103.40% ± SD .This work demonstrates a facile electropolymerization of a  S,4R)-4-(4-fluorophenyl)-3-piperidine) is known as a selective serotonin (5-HT) reuptake inhibitor (SSRIs) antidepressant drug, used to treat depression, panic disorders, generalized and social anxiety disorders, phobias, posttraumatic stress disorders and obsessive compulsive disorders , a working electrode [CHI-104 Glassy carbon electrode (GCE) 3 mm in diameter] , and 1 mm platinum wire as an auxiliary electrode. pH measurements were performed using a digital Jenway pH meter, model 3510 . Surface topographic characterization was performed using a 5600 Ls atomic force microscopy (AFM) system,  in the contact mode, and field emission electron microscopy .Stock solution of 1 mM of paroxetine was prepared in ethanol, and then diluted in PBs pH 7.5 to the required concentrations. Phosphate buffer solution (PBs) was prepared by dissolving appropriate amounts of potassium dihydrogen phosphate anhydrous and dipotassium hydrogen orthophosphate in ultra-pure water to yield 0.1 M PBs pH 7.5.For sampling of pharmaceutical formulations, ten tablets of each formulation were ground in a mortar and weighed, then equivalent weights of one tablet were dissolved in 0.1 M HCl: ethanol mixture (1:5), afterwards the solutions were centrifuged at 5000 rpm for 10 min, filtered using (ultra-pure) PTFE 0.45 μm and diluted in 0.1 M PBs pH 7.5 to prepare the required concentrations.2SO4 from 0 V to 1.5 V at a scan rate of 0.1 V/s until a stable signal was obtained. The cleaned GCE was then immersed in 0.5 M H2SO4 solution containing 1 mg/mL of HAuCl4 at constant potential of −0.2 V for different deposition times using chronoamperommetry (i-t) as previously reported for AuNPs deposition 3/4− prepared in 0.1 M KCl as a supporting electrolyte. All measurements were performed at room temperature.The electrochemical measurements were performed on the surface of poly , pH of the analyte detection media, and electrochemical parameters  should be investigated.dl-met, the surface of GCE was modified at different deposition time intervals with AuNPs, and the IPRX of 10 µM PRX on the AuNPs-GCE were recorded and compared with the response of the bare GCE. IPRX was at 80 s deposition of AuNPs-GCE. AuNPs increases the IPRX by ~10-fold of the bare GCE, this may be due to AuNPs enhanced the electron transfer and increased surface area of the GCE thus increasing its sensitivity 3−/4− prepared in 0.1 M KCl. 6]3−/4− on the bare GCE (curve a), which showed a weak redox peak compared to the AuNPs-modified GCE (curve b). The observed increase in the redox peak can be attributed to the electroactivity of AuNPs. A further increase in the redox peak was noticed on the surface of poly (dl-met)/AuNPs-GCE (curve c) which gave the highest redox peak, indicating the formation of a layer of a conducting polymer.The stepwise modification of the sensor was investigated electrochemically using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in 1 mM 3−/4− and the square roots of the scan rate shown in dl-met)/AuNPs-GCE was under diffusion control, respectively 3−/4− (1 × 10−6 mol/cm3) and A is the surface area of the electrode [dl-met)/AuNPs-GCE were calculated to be 0.0081, 0.0100 and 0.012 cm2, respectively, confirming the enhancement of conductivity of poly (dl-met)/AuNPs-GCE due to the increase of the active sensor surface.The working surface area of the sensor was calculated from the Randles-Sevcik Equation (Equation (3)):lectrode ,39. The IPRX of 10 μM PRX in a solution containing equimolar, 10-fold, and 100-fold of these compounds under optimum sensor conditions. Dopamine, urea, ascorbic acid, glucose and lactose in their equimolar ratio with PRX were found to positively affect the response by 2.06%, 2.02%, 0.80%, 5.00% and 2.80%, in 10-fold ratio 0.62%, 1.32%, 4.82%, 2.72% and 1.47% and in 100-fold ratio 1.50%, 2.20%, 0.90%, 2.47% and 4.7%, respectively, which revealed that poly (dl-met)/AuNPs-GCE did not show any significant interference due to these interferents within the studied concentration ranges.Certain compounds that are frequently found as components of tablets or biological media and various species with potential interference capabilities in the PRX measurement such as dopamine, urea, ascorbic acid, glucose, and lactose were tested. The interference effect of these compounds was examined by determining the −11 to 1 × 10−4 M in 0.1 M PBs pH 7.5 under the optimized experimental conditions. dl-met)/AuNPs-GCE from 5 × 10−11 to 1 × 10−4 M, the peak current was found to increase with increasing PRX concentration. Two linear ranges were noticed the first a linear range, shown in −11 to 5 × 10−9 M, R2 = 0.9912, represented by regression Equation (4) and the second range from 5 × 10−9 to 1 × 10−4 M, R2 = 0.9950 represented by regression Equation (5) as shown in in −11 M which indicated the sensitivity and lower LOD of the presented sensor compared to other previously reported methods given in The standard calibration curve was constructed by measuring different concentrations of PRX, ranging from 5 × 10dl-met)/AuNPs-GCE sensor was investigated by recording IPRX in 10 µM PRX at pH 7.5 five times within the same day on the same sensor prepared under the optimized conditions. The results revealed a good repeatability expressed as RSD% of n = 5 was 3.47%. The reproducibility of the poly (dl-met)/AuNPs-GCE was also tested by preparing three different sensors on three different days upon the same optimum conditions. The results showed that RSD% of n = 3 were in range of 3.37%–4.17% revealing the high reproducibility of the method. The stability of the sensor was tested by measuring 10 µM PRX at pH 7.5 at different time intervals over one month and the sensor was found to have a very stable response and retained up to 97% of its response.The repeatability of the poly (dl-met)/AuNPs-GCE sensor for PRX was determined in three different pharmaceutical formulations using the standard additions method at different concentrations [® 12.5 mg/tab are varying from , Paroxetine CR® 25 mg/tab  and Paroxetine CR® 37.5 mg/tab  indicating that the developed poly (dl-met)/AuNPs-GCE sensor is suitable for PRX detection in its commercial dosage forms without interferences from any pharmaceutical additives or excipients.The accuracy and applicability of poly (trations . Table 2dl-met from −0.8 V to 1.5 V for five cycles at a scan rate of 0.1 V s−1. The poly (dl-met)/AuNPs-GCE sensor showed a better conductivity and high surface area to detect 10 µM PRX at pH 7.5, step potential of 0.005 V, pulse amplitude of 0.1 V over bare GCE and AuNPs-GCE. The sensor showed high selectivity towards PRX in the presence of some common biological interferents such as glucose, lactose, urea, dopamine, and ascorbic acid. poly (dl-Met)/AuNPs-GCE showed linearity from 5 × 10−11 to 5 × 10−8 M and 5 × 10−8 to 5 × 10−4 M with detection limit of 1 × 10−11 M (S/N = 3) using DPV. The proposed sensor has the lowest LOD, good reproducibility and applicability in pharmaceutical detection of PRX compared to other previously reported electrochemical sensors, which represents a simple and cost-effective approach for PRX detection in regulator and quality control units in pharmaceutical companies.A new electrochemical sensor for selective and sensitive detection of PRX was developed. The sensor was fabricated using a GCE modified with electrodeposited AuNPs at constant potential of −0.2 V for 80 s and electropolymerized