Heart failure with preserved ejection fraction (HFpEF) remains a poorly characterized syndrome with many unknown aspects related to different patient profiles, various associated risk factors and a wide range of aetiologies. It comprises several pathophysiological pathways, such as endothelial dysfunction, myocardial fibrosis, extracellular matrix deposition and intense inflammatory system activation. Until now, HFpEF has only been described with regard to clinical features and its most commonly associated risk factors, disregarding all biological mechanisms responsible for cardiovascular deteriorations. Recently, innovations in laboratory and metabolomic findings have shown that HFpEF appears to be strictly related to specific cells and molecular mechanisms’ dysregulation. Indeed, some biomarkers are efficient in early identification of these processes, adding new insights into diagnosis and risk stratification. Moreover, recent advances in intermediate metabolites provide relevant information on intrinsic cellular and energetic substrate alterations. Therefore, a systematic combination of clinical imaging and laboratory findings may lead to a ‘precision medicine’ approach providing prognostic and therapeutic advantages. The current review reports traditional and emerging biomarkers in HFpEF and it purposes a new diagnostic approach based on integrative information achieved from risk factor burden, hemodynamic dysfunction and biomarkers’ signature partnership. Heart failure with preserved ejection fraction (HFpEF) is a heterogenous syndrome with specific molecular, genetic and metabolomic features, all of which reflect on vascular and myocardial cell adaptations . HFpEF eDespite recent improvements in treatment and diagnosis, HFpEF remains a poorly characterized syndrome with many unknown aspects related to different patient profiles, associated risk factors and pathophysiological pathways . Vast trNotably, recent machine learning analysis has attempted to cluster specific phenotypes by latent class study. In a post hoc analysis of TOPCAT, patients were classified into three categories according to vascular and cardiac remodeling: patients with mild LV hypertrophy and chronic pulmonary disease with normal vascular stiffness characterized by increased expression of metalloproteinase; older patients with multiple comorbidities, LV hypertrophy and reduced vascular compliance, characterized by an elevated tissue calcification biomarker; and obese subgroup with several metabolic alterations, increased renin–angiotensin system activity, lipidic profile derangement and increased inflammatory pattern . SimilarFinally, an analysis of SwedeHF and CHECK HF registries differentiated between five distinct phenotypes according to a combined approach including risk factors and associated comorbidities. The study confirmed that the cluster with CKD, coronary artery disease (CAD) and high diuretic amount revealed the worst outcomes  Figure .These data reveal some common features but also some discrepancies underlying the need for a more homogenous classification. Moreover, a detailed screening capable of identifying a specific cluster through the combination of structural cardiac abnormalities and the underlying molecular mechanisms responsible for the progression of this disease may lead to a better understanding of this syndrome and therefore to specific therapeutic target opportunities .Several HF risk prediction scores include biomarkers, mainly natriuretic peptides (NP), but important gaps exist regarding the knowledge of the underlying pathophysiological mechanisms, biological process and disease progression. Circulating biomarkers should reflect cardiac and extra cardiac disorders responsible for the HFpEF development and the related pathological pathways ,20. SincMyocardial Injury—High sensitivity troponin (HsTn) is universally considered a marker of myocardial damage in acute coronary syndrome (ACS). However, it has prognostic significance in HF and it implies myocardial damage apoptosis and progressive fiber loss independent of coronary vessel diseases. It could be the final outcome of microvascular dysfunction and subendocardial layer damage due to systemic oxygen reduction and therefore an altered supply–demand mismatch. Other features, such as increased wall tension, high left ventricle (LV) filling pressure and right ventricular dysfunction, are related with increased HsTn levels [n levels . In patin levels . Many ren levels ,24. Simin levels . These fn levels .Natriuretic peptides (NPs) are the hallmark biomarkers in HF and their measurement is accounted for in HF guidelines across the spectrum of the whole EF . The bioAdrenomedullin (ADM) is a regulatory peptide produced by endothelial and smooth muscle cells with antiproliferative, vasodilatatory and antiapoptotic effects. It is synthesized mainly by adrenal medulla but its receptors are expressed in many tissues such as lungs, heart and kidneys . It is cExtracellular Fibrosis—Collagen deposition and increased myocardial fibrosis are two relevant features in HFpEF. The more extensively analyzed biomarkers of this process are galectin-3 and soluble ST2. Galectin-3 is a glycoprotein involved in many inflammatory and profibrotic processes as a galactosidase family member, and it is synthetized by macrophage [crophage . It direcrophage . Galecticrophage ,44. Highcrophage . Moreovecrophage .Soluble ST2 is another marker reflecting myocardial fibrosis and it is overexpressed in HFrEF and HFpEF patients. It is primarily produced by myocardial cells, but also smooth muscle and endothelium cells, in relation to congestion or profibrotic stimuli . In HFpEMatrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) are two endopeptidases which induce extracellular collagen deposition; therefore, they are reasonably considered as two biomarkers of fibrosis in HFpEF . CollageAdditional collagen biomarkers such as procollagen type I (PIP) and procollagen type III N-terminal peptide (PIIINP) demonstrated a predictive role in high risk patients for HFpEF development. Both biomarkers reflect increased collagen deposition and turnover. They appear to be associated with the extent of collagen deposition in myocardial biopsies . HoweverInflammation—Systemic inflammation is a typical feature of HFpEF. It reflects the immune response to cardiac remodeling, systemic vascular injury and underlying triggers often associated with diseases, such as metabolic syndrome, diabetes, chronic lung disease and anemia [d anemia . Inflammd anemia . CRP andd anemia . CRP hasd anemia . HoweverGrow differentiation factor 15 (GDF-15) is a member of the cytokines family and it belongs to the transforming growth factor beta (TGFβ) family. It is highly expressed in inflammatory chronic diseases, and pulmonary, kidney, and cardiovascular diseases . Since iInterleukin-6 (IL6) and interleukin-1ß (ILß) are the most notorious members of the cytokines family. They are produced by activated macrophages and they are involved in several inflammatory and immunity processes . They coTumor necrosis factor α (TNFα) is another interleukin highly expressed in HFpEF. It correlates with adverse outcomes in a cross-sectional analysis . HoweverEndothelial dysfunction—Microcirculation and endothelial cells are two important features for HFpEF occurrence and microvascular dysfunction is one of the most common therapeutic targets. Dysfunctional endothelium increases the expression of adhesion molecules such as vascular cell adhesion molecules (VCAM), induced cell adhesion molecules (ICAM) and E-selectin that activates von Willebrand and other prothrombotic factors. Therefore, tissue growth factors (TGFs) and insulin growth factors (IGFs) are two other items of vascular alteration and increased proliferation [feration . The proferation . These pferation ,73. Unfoferation . ET1 levferation . Therefoferation .Plasminogen activator inhibitor (PAI-1) is the main inhibitor of tissue plasminogen activator and the intrinsic fibrinolytic system. It is increased in patients with HFpEF in association with D-dimer levels, suggesting an association with prothrombotic and procoagulant states in this setting . In the Insulin growth factor binding protein (IGFBP) is associated with inflammation, cell adhesion and senescence. It is increased according to left atrial dysfunction and dilatation reflecting diastolic dysfunction in HFpEF . In a maMetabolic dysfunction plays an important role in systemic processes which lead to HF. There is a complex interconnection between the myocardium and peripheral tissues and organs. Relevant studies show distinctive metabolic profiles that contribute to the severity of HF . HoweverThe most studied metabolites that are involved in the metabolic profile of HFpEF are serine, lysophosphatidylcholine (LPC), kynurenine and cystine, hydroxyproline, lactate, cGMP, symmetric dimethylarginine (SDMA), arginine, cAMP and acylcarnitine .In HFpEF mouse models, serine deficiency has been associated with inflammatory response and oxidative stress ,86. SeriThrough one-carbon metabolism in macrophages, serine is critical for the generation of phospholipid, biosynthesis of purine and thymidine, and production of methyl donor of S-adenosyl-methionine (SAM) and cellular glutathione. Serine is essential for the production of proinflammatory cytokines in M1 macrophages . In the In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS) through the conversion of its substrate, L-arginine, to L-citrulline. Arginine is known to act as a substrate for NO production by endothelial cells . Low levThe activation of cGMP precursors via the natriuretic peptide pathway increases the cGMP levels and a subsequent protein kinase G (PKG) . FurtherAnother finding was high levels of cystine, that is, an indirect index of inflammation . CystineKynurenine (Kyn) is a regulator of immune response, metabolized from tryptophan (Trp) during inflammatory conditions . The KyncAMP is produced via β-AR signaling and is inhibited via AMP-hydrolyzing enzyme phosphodiesterases, in which at least five families are expressed in the heart . Low cAMP levels in HFpEF suggest impaired cell signaling. Obese HFpEF patients showed an increased turnover of β-adrenergic r (β-AR) microdomains due to altered β-AR expression levels, blunted β-AR responsiveness and impaired β2-AR-coupled PDE activity . CurrentAdditionally, HFpEF patients displayed elevated concentrations of medium- and long-chain acylcarnitines . The acyFurthermore, inefficient β-oxidation leads to other metabolic pathways with an increased consumption of ketones and glutamate. The higher metabolite plasma concentrations, such as acetate and 3-hydroxybutyrate, are the markers of the worsening of heart function .However, the accumulation of long-chain acylcarnitines can have a toxic effect on the phospholipids sarcolemma. These acylcarnitines interact with different ion channels and produce cardiac arrhythmias ,106.Low levels of lysophosphatidylcholine suggest a dysregulated phospholipid metabolism . LysophoIn order to reduce the clinical variability of metabolomic results, the optimal approach could be sharing the data within different research groups, such as Biocrates, Metabolomics Society, Consortium of METabolomics Studies (COMETS) and Phenome and Metabolome aNalysis  .The non-coding genome which indicates small and long non-coding RNA is involved in gene regulation. Multi-microRNA is 21–22 nucleotide single-stranded RNAs that bind complementary messengers leading to degradation. They have been implicated in pathophysiologic processes that conduct HFpEF. Correlated with NT-proBNP, miRNAs are highly discriminatory and have improved specificity and accuracy in identifying non-acute HF . The subSince HFpEF is a heterogeneous syndrome characterized by multiple risk factors and several associated conditions, it could be very tricky distinguishing between the main pathophysiological driver by simply considering phenotypic classification. In this framework, a detailed laboratory screening may better elucidate the underlying mechanisms responsible for HFpEF appearance and evolution. Behind new and traditional biomarkers of inflammation, cardiovascular dysfunction and fibrosis, some emerging metabolites responsible for altered cell signals, energetic substrate and excessive immune response revealed additional diagnostic properties. The challenge of future research may systematically address the real value of clinical laboratory and metabolomic combination, to effectively initiate a precision medicine methodology.


Netherlands Heart Journal, Heckman and colleagues describe the feasibility and electrical properties of left bundle branch area pacing (LBBAP) [In this issue of the  (LBBAP) . In rece (LBBAP) . LBBAP i (LBBAP) . In cont (LBBAP) . Therefo (LBBAP) , 6. BothThe majority of publications on LBBAP are international cohort studies and show high acute success rates for implantation, a low threshold during follow-up and surprisingly few complications, in line with the study by Heckman et al. . In geneThe most widely used pacing electrode for LBBAP is the 3830 . This thin, lumen-less lead with a fixed helix design relies entirely on a guiding catheter for placement. The Medtronic C315 fixed curve workhorse sheath was primarily designed for HBP but can be easily employed for LBBAP as well. More recently, other medical device companies have introduced CSP tools . These catheters have a larger diameter than the C315 sheath to facilitate passage of a traditional, stylet-driven pacing lead. The combination of a stiffer guiding sheath and stylet-driven pacing lead may provide better stability and forward push to penetrate the septum, which can occasionally be cumbersome with the Medtronic 3830 in areas with extensive scarring or fibrosis. Emerging data suggest comparable success rates, electrical properties and safety aspects to those of the Medtronic 3830 . It shouThere is no doubt that LBBAP has great potential in the field of cardiac pacing. Nevertheless, many aspects need further refinement, and for LBBAP to reach prime time there is an urgent need for solid clinical evidence.


New approaches aimed at identifying patient-specific drug targets and addressing unmet clinical needs in the framework of precision medicine are a strong motivation for researchers worldwide. As scientists learn more about proteins that drive known diseases, they are better able to design promising therapeutic approaches to target those proteins. The field of nanotechnology has been extensively explored in the past years, and nanoparticles (NPs) have emerged as promising systems for target-specific delivery of drugs. Virus-like particles (VLPs) arise as auspicious NPs due to their intrinsic properties. The lack of viral genetic material and the inability to replicate, together with tropism conservation and antigenicity characteristic of the native virus prompted extensive interest in their use as vaccines or as delivery systems for therapeutic and/or imaging agents. Owing to its simplicity and non-complex structure, one of the viruses currently under study for the construction of VLPs is the human immunodeficiency virus type 1 (HIV-1). Typically, HIV-1-based VLPs are used for antibody discovery, vaccines, diagnostic reagent development and protein-based assays. This review will be centered on the use of HIV-1-based VLPs and their potential biomedical applications. Nanotechnology is defined as a fusion of state-of-the-art science and engineering where the production or assembly is aimed at the nanometer scale (1-100 nm) . Regardivia modification of the VLP surface  (® and Recombivax®) (®) , hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are typically employed for the production of VLPs in research . Severalrvarix™) , Hepatitmbivax®) , Hepatitax®) (®)  and malaax®) (®) . VLP-basax®) (®)  and SARSax®) (®)  are currHIV-1-based VLPs require just one polyprotein to self-assemble, making them particularly attractive nanoplatforms . This tyAlthough HIV-1-based VLPs have been mainly explored as vaccination nanoplatforms, they may be harnessed for other applications. The packaging of active molecules as well as the surface modification for targeting bestow VLPs with the ability to function as delivery platforms . VLPs caThis review compiles an updated set of HIV-1-based VLPs, addressing their current applications as viral vaccines, namely those with anticancer properties, as well as tools for targeted therapy. As biomedical application of VLPs is considered a hot topic, there are a few reviews on this subject . HoweverWithin the variety of viral particles under study, HIV-1-based VLPs have gained importance due to their recognized applicability as vaccines . HIV conHIV-1 is the most common subtype of HIV, a virus that derives from primates and targets the human immune system, giving rise to the acquired immunodeficiency syndrome (AIDS) . Given tgag (group-specific antigen), pol (polymerase) and env (envelope glycoprotein) , originating the proteins matrix (MA or p17), capsid (CA or p24), nucleocapsid (NC or p7) and p6 , and integrase (IN) (The primary proteins of replicative retroviruses are encoded in the HIV-1 RNA genome (9.7 kb) . Apart fprotein) . The gag) and p6 Figure 1) and p6 . The polase (IN) .Two envelope glycoproteins (Env), the surface Env glycoprotein gp120 and the transmembrane glycoprotein gp41 are formed upon processing of the polyprotein precursor gp160 by a cellular protease . gp41 isThe HIV-1 genome also encodes numerous regulatory and accessory proteins, namely Tat, which is necessary for transcription from the HIV-1 long terminal repeat, and Rev, which is involved in the transit of viral RNAs to the cytoplasm . Other pHIV-1-based VLPs are robust nanoplatforms of around 150 nm in size . The stagag gene, a Gag-VLP  Gag proteins  cells. This cell line is used in both research and industrial environments as it is easy to handle . Mammali93 cells . Strateg93 cells . It is t93 cells . High ma93 cells . This mi93 cells . Notwith93 cells . The pre to APCs . It is i to APCs . Moreove budding . Howeverassembly . These massembly .10 VLPs/ml was achieved, and the obtained VLPs exhibited a similar morphology to those produced in HEK-293  cell line is also employed in the manufacture of HIV-1-based VLPs . These c HEK-293 . Such fe HEK-293 . Albeit , Trichoplusia ni Pro™ (High Five) and Spodoptera frugiperda (Sf9)  . Higher nfection . The mainfection . Accordinfection . HIV-1 Vyprotein . This waell line . More re  against Gag and Pol epitopes . NeutralHIV-1-based VLPs are capable of inducing potent humoral and cellular immune responses, as reported by several studies . The potHIV-1-based VLPs are characterized by their simple self-assembly process, which requires just one polyprotein. In addition, as VLPs, they can elicit strong immune responses, encapsulate active molecules and display targeting moieties on the surface. These features make HIV-1-based VLPs attractive nanoplatforms for a wide range of medical applications, namely vaccination and drug delivery. These applications are depicted in The development of therapies such as antiretrovirals (ART) and pre-exposure prophylaxis (PrEP) have reduced the mortality associated with HIV-1 infection. However, these therapies are not able to end the epidemic since ART do not eliminate the virus completely and may lose efficacy due to drug resistance, and PrEP is a preventive strategy rather than a treatment. In addition, both have long-term side effects and are not accessible yet to all countries due to relatively high cost. Therefore, the need to develop a vaccine capable of improving the construction of HIV-1 antigens has been one of the priorities in HIV-1 treatment studies . Table The HIV-1-based VLP vaccines can be classified into five different groups , dependi®, which is a live attenuated vaccine composed of structural genes of four DENV strains combined with nonstructural genes of a yellow fever strain  and West Nile virus (WNV), are prevalent infectious agents in tropical and sub-tropical countries around the world transmitted mainly by mosquitoes, and effective vaccine candidates against them are required . The onlr strain . The poor strain . HIV-1 Vr strain . Firstlyan cells . After can cells . Both DIan cells . Lastly,an cells . FurtherThe influenza virus originates a highly prevalent respiratory disease that provokes annual epidemics as well as sporadic pandemics . The mosgag gene was fused to the gene that encodes GFP for quantification and characterization, and chemical additives were added to optimize VLP production (Foot and Mouth Disease (FMD), an infection that affects livestock, is caused by Foot-and-Mouth-Disease Virus (FMDV) and is mostly controlled through vaccination . The vacoduction . Lastly,oduction . It was oduction .9 VLPs/ml (The most recent pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had nefarious effects in public health and economy worldwide . This ou VLPs/ml . Finally VLPs/ml . This apThe uptake of VLPs occurs in several important immune cells, namely dendritic cells, macrophages, mast cells and B cells. The innate binding capability to their natural target cells is frequently lost following modification. This loss allows entry into the endogenous intracellular APCs and leads to the activation of CTLs. Such mechanism is called cross-priming and improves immune defense against viruses and tumors. Therefore, VLPs have been modified with the aim of presenting tumor-associated antigens .mut) into HIV-1 based VLPs with the aim of triggering a CD8+ T cell immune response, reported the cross-presentation of VLP-associated Nefmut in APCs and concluded that Nefmut VLPs efficiently penetrate B-Lymphoblastoid cell lines (B-LCLs). The most substantial benefit of utilizing the Nefmut VLP platform over other approaches is that Nefmut VLPs, apart from the Nefmut-based fusion protein, are comprised of only four viral components. Furthermore, distinct receptors can be used to pseudotype Nefmut VLPs, which can in turn overcome the antibody-dependent neutralization that occurs following numerous immunizations.Di Bonito et al.  studied in vivo electroporation of plasmids encoding HIV-1 Gag (pGag) could enhance immune responses. Firstly, the formation of particles following cell transfection with pGag was determined. Then, the induction of innate immunity was assessed in vivo. Lastly, the role of VLP assembly on the pGag immunomodulatory effects in vivo was evaluated. The obtained results show that the assembly of HIV-1 Gag VLPs does not influence the antigen-specific immunogenicity in vivo. On the other hand, electroporation of pGag or pGag* (pGag mutated version) alone delayed tumor growth concomitantly with therapeutic immunizations, which proves the Gag encoding plasmid can spur the innate immune response. Additionally, the HIV-1 Gag VLPs efficiently stimulate dendritic cell maturation, which are innate immune cells that penetrate tumors and are critical in the development of an anti-tumor T cell immunity  cultured in vitro via TLR-3 and -9 signaling  was also introduced into the 293SF-pacLV cells (Streptococcus pyogenes Cas9 with the C terminus of the Gag polyprotein and a plasmid containing the expression cassettes for both the mNeonGreen protein and a single guide RNA (sgRNA) were constructed  of trastuzumab, a monoclonal antibody against the human epidermal growth factor receptor type 2 (HER2), with the HIV protein gp41 .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Left bundle branch area pacing (LBBAP) induces delayed RV activation and is thought to be harmless, since the electrocardiographic signature is reminiscent to native RBBB. However, to what extent the delayed RV activation during LBBAP truly resembles that of native RBBB remains unexplored.This study included patients with incomplete RBBB (iRBBB), complete RBBB (cRBBB) and patients who underwent LBBAP. Global and right ventricular activation times were estimated by QRS duration and R wave peak time in lead V1 (V1RWPT) respectively. Delayed RV activation was further characterized by duration, amplitude and area of the terminal R wave in V1.n = 86), QRS duration [120 ms ] was longer compared to iRBBB patients (n = 422): 104 ms , p < 0.001, but shorter compared to cRBBB (n = 223): 138 ms , p < 0.001. V1RWPT during LBBAP [84 ms ] was longer compared to iRBBB , but shorter than cRBBB . LBBAP resulted in V1 R′ durations [42 ms ] comparable to iRBBB  but shorter than in cRBBB . During LBBAP, the amplitude and area of the V1 R′ wave were more comparable with iRBBB than cRBBB. V1RWPT during LBBAP was determined by baseline conduction disease, but not by LBBAP capture type.In patients with LBBAP (LBBAP-induced delayed RV activation electrocardiographically most closely mirrors the delayed RV activation as seen with incomplete rather than complete RBBB. Right ventricular (RV) apex pacing has been considered the standard pacing approach since its first attempt in 1958 –8. ConduThe study enrolled consecutive adult in- and outpatients diagnosed with either incomplete RBBB (iRBBB) or complete RBBB (cRBBB) on standard twelve-lead ECG between January 2015 and September 2018. LBBAP patients implanted between March 2020 and October 2021 were included in the LBBAP group.All patients were recruited at the Ghent University Hospital. The study was approved by the Ethics Committee of the Ghent University Hospital.Contemporary definitions of iRBBB and cRBBB were used to select RBBB patients. QRS duration cut-offs used for iRBBB and cRBBB were 110–119 and ≥120 ms respectively . PatientLBBAP implant was performed as previously described and both lumen-less and conventional stylet-driven pacing leads were used , 24. SucECG's were recorded at a paper speed of 25 mm/s and a calibration of 10 mm/mV with MAC 5500 ECG recording devices . To avoid any fusion with intrinsic rhythm during LBBAP, paced QRS morphologies were obtained during VVI pacing with a lower rate 20–30 beats higher then intrinsic heart rate. Global ventricular activation was measured as global QRS duration, in which the QRS was measured from its onset to the latest QRS offset in any lead , 27. TheAll electrocardiographic measurements were performed with digital calipers and adapted sweep speeds of 50 mm/s on the digitally stored ECG's. The delayed RV activation was further characterized by measuring the duration, amplitude and area of the delayed R wave in V1 using automated measurements provided by the 12SL algorithm  .Categorical variables are expressed as absolute number (percentage). Continuous variables are expressed as mean ± standard deviation in case of Gaussian distribution or median  if data follow a non-Gaussian distribution. Normality was tested using the Shapiro–Wilk test. To compare means and medians of continuous variables among groups the one-way ANOVA and Kruskall Wallis test was used. The Wilcoxon signed rank test was used for paired comparison of non-Gaussian distributed continuous variables. Multivariate analysis was performed to assess determinants of delayed RV activation using multiple regression analysis. Statistical significance was set at a two-tailed probability level of < 0.05. All statistical analyses were performed using SPSS software .Overall, the study included 731 patients with delayed RV activation: 422 patients with iRBBB, 223 patients with cRBBB and 86 patients with LBBAP. Baseline patient characteristics are summarized in In patients who underwent LBBAP, pacing indication was atrioventricular block in 62%, brady-tachy syndrome in 17%, sinus node disease in 16% and heart failure in 5%. In patients undergoing LBBAP, baseline QRS measured 112 ms , with 55% having narrow QRS, 14.5% left bundle branch block (LBBB), 14.5% RBBB and 17% non-specified intraventricular conduction delay (NIVCD). LBBAP pacing response was labeled as LBBP (non-selective and selective) in 62 (72%) patients, whereas LVSP was achieved in 24 (28%) patients.p < 0.001). V1RWPT during LBBAP was 84 ms  and longer compared to iRBBB patients , but shorter in comparison to cRBBB patients  , p < 0.001]. The V6V1 IPI for LBBAP patients was comparable to iRBBB patients . Compared to cRBBB, the V6V1 IPI was shorter for LBBAP patients .Representative examples of ventricular activation time measurements with iRBBB, cRBBB and LBBAP are shown in < 0.001] . V6RWPT p = 0.49], but shorter than in cRBBB patients  , p = 0.008]  and was comparable to V1 R′ duration in iRBBB patients  . Mean V1< 0.001] . R′ area= 0.008] .p < 0.001) in patients with LBBB, from 147 ms  to 136 ms  (p = 0.009) in RBBB patients and from 135 ms  to 128 ms  (p = 0.33) in patients with NIVCD. In patients with baseline narrow QRS (<120 ms), QRS duration increased from 94 ms  to 120 ms  (p < 0.001) with LBBAP. Ventricular activation times during LBBAP according to baseline conduction disease are summarized in p = 0.014). R′ duration in V1 was significantly shorter for patients with narrow QRS [39 ms ] undergoing LBBAP, compared to LBBAP patients with underlying RBBB [46 ms ], LBBB [48 ms ] and NIVCD [49 ms ], p = 0.04. Of interest, in LBBAP patients with presumed delay of the right bundle branch conduction (such as RBBB and NIVCD patients), the V1RWPT and V1 R′ duration were still shorter compared to cRBBB patients .With LBBAP, QRS duration shortened from 153 ms  to 116 ms  (n = 62) and LVSP (n = 24): 84 ms  vs. 80 ms , respectively (p = 0.43); and V1RWPT of both LBBP and LVSP patients resembled more V1RWPT of iRBBB patients compared to cRBBB patients.V1RWPT values were comparable for patients with LBBP  vs. 26 ms , respectively, p = 0.001].V6RWPT values were shorter in LBBP patients [42 ms ] compared to patients with LVSP capture type  were comparable to LVSP [45 ms , Due to differences in baseline characteristics between iRBBB, cRBBB and LBBAP patients , determiThis study is the first to compare the electrocardiographic pattern of LBBAP-induced delayed RV activation to the delayed RV activation observed in patients with conduction delay of the right bundle branch. Our results show that the delayed RV activation during LBBAP electrocardiographically mirrors more closely to native iRBBB than cRBBB, with activation times in between those of iRBBB and cRBBB patients. With LBBAP, the delayed RV activation seems to be determined by the underlying conduction disease rather than the type of LBBAP capture .LBBAP aims to capture the left bundle branch itself (LBBP) or the left-sided septal myocardium (LVSP) in the direct area of the left bundle branch. Several studies investigated the contraction and activation patterns of the left ventricle (LV) during LBBAP and revealed a fast and homogenous activation of the LV resulting in beneficial hemodynamic effects of LBBAP , 15. AltIn contrast to the LV activation patterns with LBBAP, data on delayed RV activation during LBBAP are scarce. As LBBAP aims to capture the LBB, the activation waveform needs to propagate from the left to the right ventricle. The exact mechanism of RV activation during LBBAP has not been elucidated, although retrograde invasion of the conduction system is suggested . IrrespeOf interest, we observed that the delayed RV activation during LBBAP is determined by baseline conduction delay and blocks, but not by the type of LBBAP capture. Indeed, both LBBP and LVSP resulted in similar electrocardiographic characteristics of delayed RV activation. This raises the hypothesis that RV activation with both LBBP and LVSP almost always occurs by activation of the right-sided conduction system capture, as pure myocardial conduction toward the RV would result in ECG characteristics of delayed RV activation resembling more to those seen with cRBBB. Moreover, even in patients with baseline cRBBB, LBBAP further shortens QRS duration, V1RWPT and V1 R′ duration, suggesting that RV activation occurs through the right-sided conduction system. Whether the pacing impulse during LBBAP systematically invades the right-sided conduction system and whether activation of the RV occurs through transseptal activation, or invading connection fibers between the left and right bundle branch or exclusively by retrograde invasion of the left bundle branch needs to be further elucidated .LBBAP is emerging as a popular pacing modality with growing worldwide adoption. This is mainly explained as LBBAP is characterized by excellent pacing characteristics (low pacing thresholds and high sensing amplitudes), overcoming the main limitations of HBP while still offering a near physiological pacing strategy. The first experience with LBBAP was published in 2017 and several questions remain unanswered regarding the long-term safety, lead performance, feasibility of lead extraction and most important, the long-term clinical outcome. Reports have shown preservation of left ventricular ejection fraction (LVEF) in patients with normal cardiac function undergoing LBBAP and significant improvements in LVEF when LBBAP is implanted in heart failure patients with reduced LVEF , 36. HowDuring ventricular pacing the normal sequence of electrical activation and electro-mechanical coupling is disrupted. It has been well established that with standard RV apical pacing the delayed activation of the LV can lead to deterioration of the LVEF, pacing-induced cardiomyopathy and adverse outcome including increased mortality –14, 17. Although both LBBAP and RBBB result in delayed RV activation, global ventricular activation patterns are unlikely to be identical. Indeed, with non-selective LBBP (the most frequently observed LBBAP pacing response during follow-up), direct myocardial capture of the basal septum occurs, which is different from septal activation during RBBB.To estimate the long-term effects of delayed RV activation by LBBAP, the prognostic outcome of patients with RBBB is sometimes extrapolated to patients with LBBAP. Our results show that the LBBAP-induced delayed RV activation is situated in between incomplete and complete RBBB, and mirrors more closely to iRBBB than to cRBBB. We believe that this observation may be relevant with regard to long-term outcome of LBBAP. First, it shows that with LBBAP the delayed RV activation still occurs by activation of the right-sided conduction system, resulting in only moderate conduction delay and probably a more physiological RV contraction than would be the case with purely myocardial conduction . Secondly, iRBBB has not been associated with adverse outcome in large population studies. As such, the delayed activation of the RV during LBBAP is unlikely to convey an adverse outcome.Although it is traditionally accepted that in patients without evidence of cardiac disease, cRBBB is not associated with increased risk of cardiac morbidity or mortality, conflicting data have emerged over the last years about the long-term prognostic significance of incidental cRBBB, especially when cRBBB is associated with heart failure . HoweverThe effects of pacing-induced delayed RV activation by LBBAP require careful follow-up and should be addressed in long-term follow-up studies. No assessments of mechanical contraction patterns or function of the RV were performed in this study, although we recognize that long-term follow-up studies with thorough evaluation of the myocardial contraction properties of the RV during LBBAP are needed. Non-invasive ECG imaging or ultra-high frequency ECG might better assess local activation times and depolarization characteristics of specific ventricular segments and could contribute to further insights into the exact mechanism of RV activation during LBBAP.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by University Hospital Ghent. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.EO and JD contributed to the conception and design of the article. EO, AD, SC, and JD contributed to the statistical analysis of the article. SK contributed to the literature research. EO wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Aristotle is often named as the first zoologist or biologist because of his writings on animals. Although Aristotle’s major intention in these books was to illustrate his ideas of how knowledge and understanding might advance, at least one modern biologist (C. Darwin) has recognized Aristotle's depth and breadth as being of surviving merit. Of greater surprise is the ongoing attention that his works continue to receive, including publications in contemporary scientific journals. This review identifies 38 peer-reviewed papers on various topics from Aristotle’s biological writings that have been published during the last 50 years. These papers are described according to content , specific creatures , publication outlet, distribution over the fifty year period surveyed, and visible trends in the topics studied. It is concluded that, in the highly-competitive field of peer-reviewed scientific publication and citation, Aristotle's biology continues to excite the interest of scientists and remains salient to modern science itself. Cuvier Gotthelf .1 When hs Faria, 010, workDarwin's comment about Aristotle was made in 1882. More recently, there has been a renewal of interest in what might (from a contemporary perspective) be called Aristotle’s biological writings in the last 50 years or so by two sets of authors with distinct motivations Tipton , 2014. FAlthough Tipton  claims tThis paper is concerned with only the first group of publications mentioned above  in order to ascertain how Aristotle’s biology is being made relevant to twenty-first-century biologists and to students of the history of biology.strictu sensu because the influence and intensity of discussion of this work on the side of the sciences is still greater than in classics and philosophy.While the influence that his work on biology had on Aristotle’s overall research program (when acknowledged) has, in previous decades, often been interpreted in a dichotomous pattern  focused upon the scientific relevance of Aristotle’s biology, (2) not concerned with philosophical aspects of his model of scientific inquiry, and (3) truly aimed at bringing the value of Aristotle’s biology to the attention of the modern reader in science and in the history of biology itself. First , a sear Shields  also proa or b (first or second columns), page numbers, and line numbers  for examples, using the Work title, Book number, and Bekkeri line notation, which denotes the columns as  Bekkeri .genetics; de Magalhaes and Costa . Föllinger  general problems like speciation and classification. They also link Aristotle’s biological works to the rest of his writing.specific topics in Aristotle’s biology. Several key areas in which such papers have been written are summarized below to provide a flavor of the kind of writing about Aristotle’s biology that has occurred during the last fifty years of relevant scientific literature.Although these papers focus upon more general aspects of Aristotle’s writing on biology, they are relevant to the papers on specific topics (described below) because they cover what might loosely be termed the epigenetics, first coined by Waddington and defined as “derived from the Greek word epigenesis, which Aristotle used for the theory that development is brought about through a series of causal interactions between the various parts” , then “a stockperson’s primary role is to promote the good of the animals in their care, ensuring that their needs, wants and desires are satisfied.”2224Perhaps due to his experiences as a child Shields ,20 AristAristotle asserted that, if a hen bird has mated with a cock and is pregnant, then if “she be trodden by another cock, the whole brood of chicks turn out like the second cock.”26Gross  describe29,32,Although it was published just before the search period (1970–2020), an earlier paper by Clarke and Stannard deserves mention because its authors present several arguments explaining why Aristotle reported that the brain occupied only the front of the head,30Aristotle is considered by many as “the founder of comparative anatomy,” although much of his work was based upon animals  because he did not perform dissections of humans Blits , p. 59.3material, form, origin of motion (or efficient), and end , and in doing so, provided a model of anatomy that went well beyond a simple description of parts, but also included the reasons for why those parts are as they are and how they might differ across species. This individual variety of animals’ form and function led Aristotle to state that “each and all will reveal to us something natural and something beautiful.”37Perhaps the strongest basis for the high opinion in which anatomists hold Aristotle is his focus upon function.Clinical Anatomy in 2007, Crivellato and Ribatti divert their discussion away from “many of his physiological concepts (that) turned out to be wrong” and instead focus on Aristotle’s development of a “precise topographical terminology” that cast the body into “a bipartite, symmetrical architecture,” including the front and back of the body, its top and what lay below, and its right and left sides (Crivellato and Ribatti organon) as an instrumental and functional part of the body , Tipton evaluates the accuracy of Aristotle’s descriptions of the habitat, diet, spawning, sexual dimorphism, and eggs of these fishes,Tipton describes Aristotle as a philosophical biologist for whom the two disciplines were intermingled Tipton , p. 370.Mullus surmuletus), a much-fancied eating fish in Ancient Greece and still today (“in antiquity a great delicacy … and continues to be a highly-prized fish today”)  appearing in Aristotle, Voutsiadou et al.’s work is comprehensive in its tabulation and description. They conclude that “All marine animal anhaima encountered in Aristotle’s zoological works are correlated with current marine invertebrate taxa”  focus on the sponges of the Mediterranean, confirming Aristotle’s comments about the diversity, nature, external morphology, symbiotic relationships, habitat, and value to humankind,, p. 1763The third relevant paper by Ganias et al.  replicatAristotle commented that “in Pharos and other places, the bite of the scorpion is not dangerous; elsewhere—in Scythia, for instance … the sting is fatal to man or beast.”History of Animals, including its appetite,Historia Animalium and other biological works, is not only extraordinarily detailed but in most cases accurate as well” . Similarly, classics journals have also published some of these papers on Aristotle’s observations of animals . However, there are twenty publications in scientific journals that do not purport to discuss historical issues . From a slightly different perspective, twenty-eight (or 73.7%) of the papers appear in science journals or journals devoted to the history of science, but only ten (or 26.3%) are published in history or classics journals. These data suggest that almost three-quarters of this selection of modern literature about Aristotle’s observation of animals was written for the specific consumption of scientists, while the remaining one-quarter could be intended to interest historians or classicists. On the basis that all these papers were peer-reviewed, it may be claimed that there is a reasonably active interest shown by scientists, historians, and classicists in Aristotle’s observations of animals.Second, the selection of publication outlets also provides an insight into the goals of these authors who comment on Aristotle’s relevance to modern science. As shown in Table Third, it is also of interest to note the distribution over time of the papers identified for review. Figure Finally, when the content and year are combined to detect any observable trends over time, as shown in Table While the relative status of Aristotle as a natural scientist vis-a-vis his traditional image as a philosopher has only relatively recently started to be debated in classical studies, his reception in modern science has steadily grown over the last five decades. Based on the papers reviewed here, plus the brief summary of their content, outlets, and timing of publication over a fifty-year period, it is argued that Aristotle’s observations of animals not only continue to excite interest in modern scientists but remain of relevance to modern biology and contribute to the general history of biology itself. Evidence of this claim is the extent to which highly-competitive publication space in mainstream historical, classics, and scientific journals is being given to papers that highlight Aristotle’s exact and voluminous output in what is now termed “biology,” his ability to demonstrate the principles of precise observation and hypothesis-formulation, and his fluid and informative communication with his audience. The fact that each of the 38 papers described herein was peer-reviewed by other researchers in the general fields of history, classics, and biology, and judged by them to be not only of relevance but also of sufficient scientific standing as indicators of an ancient but enormously comprehensive and still informative piece of work, further affirms the relevance of Aristotle’s work to modern science. As a statement about the ongoing history of biology itself, these papers also argue for the worth of an accumulative set of observations and deductions in the general field of biology, even from very long ago.


In PNAS, Chu and Evans (1) argue that the rapidly rising number of publications in any given field actually hinders progress. The rationale is that, if too many papers are published, the really novel ideas have trouble finding traction, and more and more people tend to “go along with the majority.” Review papers are cited more and more instead of original research. We agree with Chu and Evans: Scientists simply cannot keep up. This is why we argue that we must bring the powers of artificial intelligence/machine learning (AI/ML) and open access to the forefront. AI/ML is a powerful tool and can be used to ingest and analyze large quantities of data in a short period of time. For example, some of us  have useAI/ML has become a powerful tool in science and engineering, used in a multitude of applications . In this letter, we are focusing on natural language processing (NLP). NLP, a branch of AI, is well described in many places . CommerOnly some simple steps need to be followed: identify the appropriate archive of published papers, train the NLP code using a subset of the archived papers, build “white” and “black” lists to help narrow the focus, and use the NLP to categorize and group papers by similarity and topic “closeness.” Network diagrams can help find linkages easily between papers where it may not have been obvious and, more to the point of CE , can easIn conclusion, we agree with CE  on the p


CD30 is overexpressed in several lymphoma types, including classic Hodgkin lymphoma (cHL), some peripheral T-cell lymphomas (PTCL), and some cutaneous T-cell lymphomas. The antibody–drug conjugate brentuximab vedotin targets CD30-positive cells and has been evaluated for the treatment of various lymphoma entities. This narrative review summarizes 10 years of experience with brentuximab vedotin for the treatment of CD30-positive lymphomas, discusses novel therapies targeting CD30 in development, and highlights remaining controversies relating to CD30-targeted therapy across lymphoma types. The collective body of evidence for brentuximab vedotin demonstrates that exploitation of CD30 can provide sustained benefits across a range of different CD30-positive lymphomas, in both clinical trials and real-world settings. Preliminary experience with brentuximab vedotin in combination with immune checkpoint inhibitors for relapsed/refractory cHL is encouraging, but further exploration is required. The optimal use of brentuximab vedotin for first-line therapy of PTCL remains to be determined. Further research is required on brentuximab vedotin treatment in high-risk patient populations, and in rare lymphoma subtypes, for which no standard of care exists. Novel therapies targeting CD30 include chimeric antigen receptor therapies and bispecific antibody T-cell engagers, which may be expected to further improve outcomes for patients with CD30-positive lymphomas in the coming years. Several antigens that have limited expression in normal tissue and are overexpressed by tumor cells are excellent candidates for the development of immunotherapeutics. CD30, a member of the tumor necrosis factor receptor family, is expressed on a small subset of activated T and B lymphocytes and is overexpressed in a variety of lymphoma subtypes . HighestDespite the viability of CD30 as a therapeutic target, initial clinical trials with “naked” anti-CD30 antibodies yielded disappointing clinical responses , 5. The In this review, we describe current evidence for anti-CD30 therapy in lymphoma, discuss ongoing controversies and data gaps, and provide perspectives on the direction of anti-CD30 therapy in the future.® (NCCN®) Clinical Practice Guidelines In Oncology (NCCN Guidelines®) recommend brentuximab vedotin in combination with doxorubicin, vinblastine, and dacarbazine (AVD) as a primary treatment option for stage III–IV disease, and as single-agent maintenance therapy following high-dose therapy and autologous stem cell transplantation (ASCT) for refractory disease with high risk of relapse [® for pediatric HL recommend brentuximab vedotin in combination with bendamustine, gemcitabine, or nivolumab as a reinduction therapy option for patients with relapsed/refractory cHL, and state that it may also be useful as maintenance post-transplant for some high-risk pediatric patients [The development of brentuximab vedotin has altered the treatment approach for patients with cHL. Treatment indications have been expanded since the initial approvals Table . Nationa relapse . Europea relapse . In olde relapse . The NCCpatients .Table 2Bp = 0.03) [p = 0.002) [p = 0.009; PFS: HR 0.68, 95% CI 0.53–0.86; p = 0.002) after a median of approximately 6 years follow-up has recently been reported [The recommendation for brentuximab vedotin in combination with AVD as primary treatment for cHL is based on data from the phase III ECHELON-1 study. In this trial, patients with previously untreated stage III–IV disease were randomized to receive brentuximab vedotin plus AVD (A + AVD) or bleomycin plus AVD (ABVD) . The pri = 0.03) . Seconda = 0.03) . The ben= 0.002) . Five-ye= 0.002) . Given t= 0.002) , these dreported . A + AVDreported . Of thosreported . Febrilereported , 15; howreported , 15, 16.p = 0.032; IPS 4–7, HR 0.588, 95% CI 0.386–0.894; p = 0.012) [Subgroup analyses from ECHELON-1 assessed the efficacy and safety of A + AVD as first-line treatment in older and high-risk patients , 18. In Other brentuximab vedotin combinations with chemotherapy have also been evaluated for first-line treatment of adults with cHL. In a phase II trial of patients with newly diagnosed advanced-stage cHL, brentuximab vedotin in combination with etoposide, cyclophosphamide, doxorubicin, and either procarbazine and prednisone (BrECAPP) or dacarbazine and dexamethasone (BrECADD) provided promising 3-year PFS rates of 90.2% and 89.7%, respectively . The BrEIn previously untreated patients for whom conventional chemotherapy is not an option due to age, frailty, or comorbidities, brentuximab vedotin monotherapy has been shown to be tolerable but with limited activity, highlighting a need for research into combination with other agents in this patient population . A phaseMost patients with limited-stage HL are cured with standard therapy, but some have reduced life-spans due to increased risk of late radiotherapy-related complications, such as malignancies   . After aIn an early phase II study that investigated brentuximab vedotin monotherapy in patients who had already relapsed or were refractory post-ASCT, brentuximab vedotin induced objective responses in 75% of patients . A recenA phase II trial of brentuximab vedotin as second-line monotherapy in patients with relapsed/refractory cHL reported an ORR of 75% and CR rate of 43%, representing an effective bridge to ASCT . BrentuxTreatment regimens combining brentuximab vedotin with immunotherapeutic agents have also been evaluated in the relapsed/refractory setting, although data are currently limited to early-phase studies. There is a strong rationale for such combinations, as these therapies have distinct mechanisms of action. Immune checkpoint inhibitors facilitate reactivation of T-cells that can target Hodgkin and Reed-Sternberg cells and may overcome the negative impact of anti-programmed cell death-1 (PD-1) pathway derangement caused by gene amplification . Brentuxp = 0.0013); however, no significant difference in OS was seen [The phase III AETHERA trial enrolled adults with unfavorable-risk cHL who had undergone HDT and ASCT and were at high risk of relapse or progression due to primary refractory HL, relapsed HL with remission duration of < 12 months, or extranodal involvement at the start of pre-transplantation salvage chemotherapy . PFS waswas seen . This bewas seen . Treatmewas seen , 52.Real-world data on brentuximab vedotin for cHL have demonstrated the utility and feasibility of CD30-targeted ADC therapy beyond the setting of clinical trials and specialized centers  reported good tolerability but limited clinical responses in 6 patients with relapsed or refractory cHL (of whom 5 had previously received brentuximab vedotin), with an ORR of 33% . EfficacBispecific T-cell engagers are another type of novel CD30-based therapy in clinical development. The CD30/CD16A-bispecific antibody AFM13 is a first-in-class innate cell engager that binds to CD16A on innate immune cells and CD30 on cHL cells, resulting in recruitment and activation of innate immune cells in close proximity to tumor cells, facilitating antibody-dependent cell-mediated cytotoxicity (ADCC) , 72. ActDespite the wealth of clinical evidence for brentuximab vedotin therapy in cHL, a number of questions remain. Firstly, the combination of brentuximab vedotin with immune checkpoint inhibitors in relapsed/refractory cHL has great potential, but it remains to be seen whether such combinations provide greater benefits than brentuximab vedotin plus chemotherapy or immune checkpoint inhibitors plus chemotherapy . Cost-eSecondly, for first-line treatment of advanced cHL, further data are required to determine the optimal brentuximab vedotin combination regimen. An ongoing phase III study comparing A + AVD versus nivolumab plus AVD in patients with advanced cHL will assess the value of first-line brentuximab vedotin and immune checkpoint inhibitor combination therapy .Thirdly, the use of PET status after cycle 2 (PET2) to guide potential switching of first-line therapy in patients with cHL remains unclear. Results from ECHELON-1 found that while PET2-positive and PET2-negative subgroups both benefitted from A + AVD compared with ABVD in terms of 5-year PFS per investigator, PET2-negative patients still fared better than PET2-positive patients; 5-year PFS rates for PET2-negative patients were 84.9% (95% CI 81.7–87.6) with A + AVD and 78.9% (95% CI 75.2–82.1) with ABVD, while in PET2-positive patients, PFS rates were 60.6% (95% CI 45.0–73.1) with A + AVD and 45.9% (95% CI 32.7–58.2) with ABVD . This suFinally, there is a scarcity of data from randomized controlled trials assessing the efficacy of brentuximab vedotin retreatment following first-line brentuximab vedotin in the relapsed/refractory cHL setting. Data from the ECHELON-2 study, which assessed the efficacy and safety of first-line brentuximab vedotin in patients with PTCL, suggest that retreatment of patients with cHL with brentuximab vedotin may be worth investigating .CD30 is consistently expressed by systemic ALCL (sALCL) cells and is an established component of sALCL diagnosis . As suchp = 0.0110; OS HR 0.66, 95% CI 0.46–0.95, p = 0.0244), with median PFS of 48 months versus 21 months after a median follow-up of 36 months [p = 0.0077; OS HR 0.72, 95% CI 0.53–0.99; p = 0.0424), which was most notable in the subgroup of patients with sALCL [An early clinical trial in which patients with CD30-positive PTCL received brentuximab vedotin administered sequentially with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or in combination with CHP and reported CR rates of 62% and 88% and 1-year PFS of 77% and 71%, respectively . Based o6 months . Patient6 months . The saf6 months . A subseth sALCL . Furtherth sALCL .In a phase II trial of 46 patients with newly diagnosed PTCL , the addition of etoposide to brentuximab vedotin plus CHP followed by brentuximab vedotin consolidation resulted in an ORR of 91% and a CR rate of 80%, with 18-month PFS and OS of 61% and 89%, respectively . AnotherBrentuximab vedotin has been evaluated for the treatment of pediatric patients with newly diagnosed ALK-positive CD30-positive ALCL . The addAside from ECHELON-2, clinical trial data on brentuximab vedotin therapy for PTCL are limited. However, several analyses have assessed brentuximab vedotin in patients with PTCL in real-life settings. In a single-center study of 27 patients with refractory/relapsed PTCL treated with brentuximab vedotin in a real-life practice, ORR was 63.0%, and after a median follow-up of 12.5 months, median PFS and OS were 5.2 and 12.5 months, respectively . ResponsCurrently, research into new anti-CD30 therapies for the treatment of PTCL appears to be limited. CD30 CAR-T cells have been shown to suppress tumor growth and increase tumor cell lysis in preclinical PTCL models , and ledAs the clinical efficacy of brentuximab vedotin is lower in PTCL than cHL, there are numerous outstanding questions. It is not clear whether brentuximab vedotin therapy is appropriate for use upfront in all PTCL cases, especially given the variability in CD30 expression across subtypes. Some studies have indicated that response to brentuximab vedotin can occur in patients who have low or undetectable expression of CD30 (< 10%) ; this isLike PTCL, CD30 expression in CTCL is variable by subtype . Brentuxp < 0.0001), a difference that was observable in both the MF and C-ALCL subgroups [Key evidence for the efficacy of brentuximab vedotin in CTCL comes from the multicenter phase III ALCANZA study, which enrolled adults with CD30-positive MF or C-ALCL who had received at least 1 previous therapy . Patientubgroups . Importaubgroups . Median ubgroups , and breubgroups . AE rateubgroups . PN was ubgroups . Final dubgroups .Real-world studies on brentuximab vedotin therapy for CTCL support the results of the ALCANZA trial, with good responses to treatment observed in patients with MF and C-ALCL –104 and Of the 3 types of lymphoma discussed so far, brentuximab vedotin therapy for CTCL is the least well studied. While the benefits of brentuximab vedotin in MF and C-ALCL are well-established, there remains a need for assessments of the value of combining it with chemotherapy for C-ALCL, as well as further studies on other subtypes. Additionally, as for PTCL, data on response to brentuximab vedotin in patients with low or no CD30 expression are also required to determine the optimal role of brentuximab vedotin across CTCL subtypes.In addition to cHL, PTCL, and CTCL, there are other tumor types that express CD30 and may benefit from CD30-targeted therapy. For example, CD30 is frequently expressed by Epstein-Barr virus (EBV) positive lymphomas. In a phase II trial of brentuximab vedotin in patients with relapsed/refractory EBV-positive CD30-positive mature T, NK, or B-cell lymphoma, ORR was 48%, and at a median of 20 months follow-up, median PFS and OS were 6.2 months and 15.7 months, respectively . AnotherSome CD30-positive B-cell specific lymphomas may also respond to brentuximab vedotin therapy. Although brentuximab vedotin has limited activity as a single agent in relapsed primary mediastinal B-cell lymphoma (PMBCL) , when usCD30 is variably expressed in DLBCL, at a frequency of up to 60% depending on cut-off value , 114, buThe last 10 years of experience with brentuximab vedotin have clearly demonstrated that CD30 is a highly valuable therapeutic target for lymphoma therapy. Exploitation of this target can improve outcomes for patients with a range of different CD30-positive lymphomas, and brentuximab vedotin is now an established standard of care in multiple treatment settings. Recent long-term data from phase III studies have demonstrated the durability of its benefit, and real-world data show that the benefits seen in clinical trials are translatable to clinical practice, including for tumor types not included in the trials. Several questions remain unanswered, however, including the importance of novel brentuximab vedotin combination regimens, such as those including checkpoint inhibitors. With respect to T-cell lymphoma, single-agent brentuximab vedotin is highly efficacious for the cutaneous lymphomas, ALCL and mycosis fungoides, even when they express low levels of CD30. The addition of chemotherapy to brentuximab vedotin in such groups requires further study. For patients with PTCL, the value of adding brentuximab vedotin to front-line chemotherapy for subtypes beyond systemic ALCL requires further study. In general, there remains a need for more data on brentuximab vedotin treatment of rare lymphoma subtypes, PTCL with low CD30 expression, and high-risk patient populations, at early disease stages, and in combination with checkpoint inhibitors.The novel anti-CD30 therapies that are in development may be expected to further improve outcomes for patients with CD30-positive lymphomas in the coming years. Bispecific antibodies and CAR-T cell therapies have shown considerable promise in early studies in cHL and, with continued success, may be expanded to other CD30-positive lymphoma types. Innovations in CAR-T cell design to improve antitumor activity, such as incorporation of variable fragments targeting membrane-proximal epitopes or with human antigen-recognition domains, or use of memory stem T-cells, may help to further improve the efficacy of this therapy for CD30-positive tumors , 118.Brentuximab vedotin has been a pioneer for the ADC approach to therapy. In the future, ADCs with novel antigen targets may also contribute to improved patient outcomes in lymphoma. Polatuzumab vedotin, an anti-CD79 ADC recently approved for the treatment of relapsed/refractory DLBCL, and camidanlumab tesirine, an anti-CD25 ADC in development for the treatment of various malignancies including cHL, have the potential to widen the range of treatment options for lymphoma , 120. Ad


It is known that the long noncoding RNAs (lncRNA) MALAT1 is associated with tumorigenesis and progression in various cancers; however, its functions and mechanisms in prostate cancer (PCa) initiation and progression are still unknown. In the present study, our findings revealed that MALAT1 plays a critical part in regulating PCa proliferation and glucose metabolism. Knockdown of MALAT1 affects the protein and mRNA levels of MYBL2. In addition, MALAT1 enhances the phosphorylation level of mTOR pathway by upregulating MYBL2. Knockdown of MALAT1 or MYBL2 in PCa cell lines significantly inhibits their proliferation capacity. Silencing MALAT1/MYBL2/mTOR axis in PCa cell lines affects their glycolysis and lactate levels, and we verified these findings in mice. Furthermore, we explored the underlying tumorigenesis functions of MYBL2 in PCa and found that high expression of MYBL2 was positively associated with TNM stage, Gleason score, PSA level, and poor survival rate in PCa patients. Taken together, our research suggests that MALAT1 controls cancer glucose metabolism and progression by upregulating MYBL2-mTOR axis. Prostate cancer (PCa) is the second most common malignant tumor in males of Western countries, but its five-year survival rate (98%) is much higher than that of renal cell carcinoma 75%) and bladder cancer (77%) . However% and blaArabidopsis [MYB proto-oncogene like 2 (MYBL2) belongs to the MYB transcription factor family and plays a critical role in regulating cell proliferation and apoptosis , 7. Varibidopsis  via the Moreover, the mTOR pathway plays a major role in the glucose metabolic program in cancers , and theIn the present study, we showed that MYBL2 is a downstream target of MALAT1. MALAT1 knockdown inhibits the expression of MYBL2 and reduced the phosphorylation level of mTOR pathway. Silencing MALAT1 and MYBL2 or using mTOR pathway inhibitor rapamycin significantly blocks PCa proliferation and decreases the Warburg effect. We thus reveal the MALAT1/MYBL2/mTOR axis and that MALAT1 and MYBL2 regulate PCa glucose metabolism via the mTOR pathway.2 at 37°C.The human PCa cell lines, PC-3 and C4-2, and the prostate epithelial cell line RWPE-1 were purchased from the American Type Culture Collection (ATCC). PC-3 and C4-2 cells were cultured in RPMI-1640 medium (Solarbio) containing 10% fetal bovine serum , while RWPE-1 was cultured in a keratinocyte serum-free medium (K-SFM) kit (Invitrogen). All cell lines were tested and authenticated by short tandem repeat (STR) profiling. The cells were grown in a humidified incubator under 5% COThis study was conducted on 98 archived paraffin-embedded clinical specimens  that were diagnosed both clinically and pathologically (Ethic Number: HProA150PG02). The clinical samples were provided by Shanghai Outdo Biotech Company and approved by the Institutional Research Ethics Committee in Shanghai of China for research purposes and the necessary information of patients is in the supplementary materials.μg of total RNA was isolated, and cDNA libraries were prepared using the Illumina TruSeq mRNA Stranded Library Prep Kit (Illumina) according to the manufacturer's protocol and analyzed by sequencing on an Illumina HiSeq 2000 sequencing platform (BerryGenomics). Reads per kilobase pair per million mapped (RPKM) reads value for each gene were assessed. A gene is considered significantly differentially expressed if the expression differs between any two samples with P value <0.05, as calculated by Cufflinks and compared to the potential mRNA of MALAT1 predicted by RNA RNAINTER (http://www.rnainter.org).A minimum of 3 Individual siRNAs against MALAT1 and MYBL2 are designed and provided by Genepharma Company, and the sequences of siRNAs are shown in Supplementary Table μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega) after DNase treatment (Promega). Subsequently, qPCR was performed on the cDNA using SYBR Green Mix (Roche) on CFX96 (Bio-Rad) real-time PCR machine. The primers are listed in Supplementary Table Total RNA was extracted using TRIzol (Thermo Fisher), and 0.5 μg total protein from each cell lysate was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking the membrane with 5% skimmed milk, the primary antibodies were MYBL2 , GAPDH , α-tubulin , mTOR , Phospho-mTOR , and PKM2 , and the secondary antibodies were goat antimouse and goat antirabbit .RIPA buffer was used for cell lysis. An equivalent of 15 μM EdU at 37°C for 2 h. After fixation with 4% paraformaldehyde for 30 min, the cells were exposed to 0.1% Triton X-100 for 10 min and washed with phosphate-buffered saline. Subsequently, the cells were treated with Click-iT EdU cocktail for 30 min and stained with 5 μg/mL of Hoechst 33342 for 30 min [Cell proliferation was estimated using the BeyoClick™ EdU cell proliferation kit (Beyotime). After transfection, .1.5 × 105 cells/well were seeded in six-well plates for 48 h and incubated with 10 r 30 min . The finBriefly, 1000 cells/well were seeded in six-well plates and cultivated for 9 days. Then, the cells were fixed with 2.5% glutaraldehyde and stained with methylene blue solution  at room temperature for 30 min. The plates were dried to capture the images .7 cells. The supernatants were collected by centrifuging for 20 minutes . Then, 800 μL precooled methanol/acetonitrile  was added; the mixture was sonicated in an ice bath for 20 min and incubated for −20°C 1 h for protein precipitation. Subsequently, the supernatant was collected by centrifugation of the mixture at 14,000g at 4°C for 20 min and analyzed on an Agilent 1290 Infinity LC system coupled with AB 5500 QTRAP mass spectrometer [We used wild-type and MYBL2 knockdown PC-3 cells at a density of 10trometer .μM), 2-deoxy-glucose (100 mM), and glucose (10 mM) to the plate. The final ECAR values were normalized to the cell numbers [The capacity for glycolysis was assessed using the Seahorse XF cell Mito stress test kit or glycolysis stress test kit from Seahorse Bioscience on a Seahorse Bioscience XF96 extracellular flux analyzer . Cells (1.5 × 104 cells/well) were cultured in microplates XF96 (Seahorse Bioscience) the day before estimation. Finally, the ECAR values were measured by adding oligomycin  were purchased from the Shanghai CQ and raised in a barrier facility under a 12-h light/dark cycle. Mice were randomly divided into NC or siMALAT1 group with 6 mice in each group. Then, vector control or MALAT1 knockdown PC-3 cells were injected subcutaneously into the flanks of mice NC or siMALAT1 group. Next, we recorded tumor volume once a week for a total of 6 weeks. Six weeks later, mice were sacrificed, and tumors of each mice were excised, imaged, and weighed. Ki67, MYBL2, Phospho-mTOR, mTOR, and PKM2 protein levels were measured by immunohistochemistry.All animal experiments were approved by the Institutional Animal Care and Use Ethics Committee of Shanghai CQ and performed in the Department of Laboratory Animals of Shanghai CQ under the institutional guidelines. Adult male nude mice , and the sections were blocked using 3% H2O2. Subsequently, the slides were incubated with primary antibody Ki67 (Affinity), MYBL2 (Affinity), mTOR (Proteintech), phospho-mTOR (Proteintech), and PKM2 (Affinity), and secondary antibodies antirabbit and antimouse (Proteintech). Finally, the slides were incubated with the horseradish peroxidase substrate diaminobenzidine (Thermo Scientific) and counterstained with hematoxylin, and we used IHC score to quantify IHC results.Tumor tissues were isolated from mice and PCa patients and sliced into 5-t test. P values displayed as ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 indicated statistical significance.Statistical analyses were conducted using SPSS 21.0 or GraphPad Prism 7 software. The error bars represent the standard deviations from the mean. All experiments were repeated at least three times. The difference between groups was analyzed by Student unpaired To verify the role of MALAT1 in PCa, we first analyzed the MALAT1 expression profile in primary organ tissues and PCa using the Cancer Genome Atlas (TCGA) database and found that MALAT1 was highly expressed in the prostate than other tissues, and the expression was even higher in PCa Figures . In the Next, we tried to verify the regulatory correlation between MALAT1 and MYBL2 and found that MALAT1 knockdown decreased the expression of MYBL2 at both RNA and protein levels Figures . In contArabidopsis [mTOR pathway plays a critical role in tumorigenesis and cancer progression , and a pbidopsis . In ordeThe increased understanding of cancer has identified glucose metabolism as one of the first hallmarks of cancer . The mTOThe MALAT1/MYBL2/mTOR axis was then evaluated in the mouse model. In the in vitro experiment, silencing the MALAT1/MYBL2/mTOR axis significantly reduced colony number and glycolysis. Next, we injected MALAT1 knockdown PC-3 cells (siMALAT1) and vector control PC-3 cells (NC) in the mouse model, and the tumorigenic ability in the siMALAT1 group was also inhibited : only 3/P = 0.015) and lower progression-free survival (P < 0.001) (Figures The clinical relevance of MYBL2, a potential carcinogen, was evaluated in human PCa. First, we analyzed TCGA data and found that MYBL2 mRNA levels were upregulated in T3-4 stage than in T2, in N1 PCa compared to N0, and had high PSA level and Gleason score Figures . Moreove Figures . We also Figures . These r Figures .LncRNA MALAT1 is a classical carcinogen in various tumors, controlling several aspects of cancer progression . In PCa,As a potential oncogene and biomarker in aggressive cancers , 31, MYBArabidopsis [mTOR signaling pathway affects tumor proliferation, metastasis, and drug resistance , 36, andbidopsis , and theGlucose metabolism is an intrinsic characteristic of cancer cells during carcinogenesis and tumor progression, proving advantageous to tumor proliferation . In PCa,Aerobic glycolysis, or the “Warburg effect,” is a unique tumor metabolism promoting cancer progression . HoweverWe also verified our findings in a nude mouse model. Our study shows that the tumorigenic capacity of mice injected MALAT1 knockdown PC-3 cells was weaker than that of the control group, and only 3/6 mice progressed to tumorigenesis. This result indicated low tumorigenic capacity due to MALAT1 reduction, which was supported by the final weight and volume of the tumor growth. Overall, the results of animal experiments are consistent with the above in vitro findings.Arabidopsis, MYBL2 is a downstream target of the mTOR pathway but an upstream gene in mammals; these differences necessitate the exploration of the connection between MYBL2 and all the known upstream targets of mTOR and the underlying mechanisms. Besides, our founding reveals that MYBL2, as a highly potential tumorigene, provides a new character of MALAT1in PCa. Interestingly, more and more researches using Chinese medicine to treat cancer [In the present study, we revealed the metabolism function of the MALAT1/MYBL2/mTOR axis in PCa. However, since it is a novel axis, the mechanisms of its abilities require further examination. mTOR pathway is also a mediator for other metabolisms in various cancers , and fatt cancer , 51 giveTaken together, the current study demonstrated that MALAT1 regulates MYBL2 and controls the mTOR pathway in PCa cells. We revealed MALAT1/MYBL2/mTOR axis for the first time and suggested that this novel axis mediates glycolysis and the Warburg effect in PCa cells. These results pointed out that silencing MALAT/MYBL2/mTOR axis might be considered as potential new therapy based on glucose metabolism for PCa .


With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF  It has  T cells .via a non-canonical NF-κB pathway and activation of TNFR2 is important for proliferation, survival, and lineage stability of Treg cells, as well as the development of thymic Treg cells from Treg precursor cells  and mitogen activated protein kinase (MAP kinase) pathway to deliver a proinflammatory signal . In contor cells  and is aAt physiological concentrations soluble TNF activates TNFR1 but not TNFR2, whereas the membrane bound form of TNF can activate both receptors . UncontrTNF blockade by biologics has been a clinical success story . These aRA is a common chronic inflammatory disease with a global prevalence of 0.51% . It is aThe use of anti-TNF biologics such as infliximab, adalimumab, etanercept and certolizumab pegol have revolutionized the treatment of RA. Despite this success, the rate of disease remission in RA is still rather low with only 25% of patients reaching remission. In addition, it has also been reported that only 25% of patients achieve low disease activity (LDA) within 6 months  of anti-In silico tools were also used to identify plant-derived natural products that prevent the formation of TNF trimers  . Crystalin vivo efficacy was assessed in an animal model of arthritis. This data, along with predictions from Quantitative Systems Pharmacology (QSP) and the molecule’s ADME and PK properties, were used to determine suitable doses for human clinical trials. Additionally, in order to provide a biomarker of TNF occupancy in humans, we developed an ELISA using an antibody described by SAR441566, is a novel small molecule arising directly from the molecules first described by O’Connell et al., in 2019. The potency of SAR441566 was determined by protein and cell binding and activity against endogenous TNF in whole blood, while Here we discuss the preclinical development of SAR441566, which has recently become the first anti-TNF small molecule to enter phase 1 clinical trials.via amine coupling chemistry to a level of ∼1,500 Response Units (RU). HBS-P  was used as the running buffer with a flow rate of 10 μl/min. A reference surface was prepared by activating and deactivating an appropriate flow cell. SAR441566 was flowed over human TNF at five concentrations of doubling dilutions from 10 to 0.625 µM, plus a blank. The assay was run at 25°C with a flow rate of 100 μl/min in HBS-P supplemented with 1% dimethyl sulphoxide (DMSO). Double referenced background-subtracted binding curves were analyzed using the T200 BiaEvaluation software (version 1.0) following standard procedures. Kinetic constants for the on-rate (ka) and off-rate (kd) were determined for SAR441566 binding to human TNF from the fitting algorithm and the affinity constant determined (KD). The data were analyzed in BiaEvaluation software then presented in GraphPad Prism 7.SAR441566 binding to human TNF was demonstrated by surface plasmon resonance (SPR) using a Biacore T200 . Here, the ligand TNF, was captured on the immobilized surface while the analyte SAR441566, was flowed over the captured surface. The sensor detects the change in mass on the sensor surface as the analyte binds to the ligand to form a complex on the surface. This corresponds to the association process. The dissociation process is monitored when the analyte is replaced by buffer. Human TNF (VCID7359) was produced by Beryllium (now UCB) in a stock concentration of 4.98 mg/ml at pH 7.4 and stored at −80°C. A solution of human TNF for the immobilization was prepared by dilution of the stock solution at concentration of 4.98 mg/ml to 10 μg/ml using immobilization buffer (Na acetate 10 mM pH 5.5). Human TNF was immobilized on a CM5 Series S Sensor Chip in vitro potency of SAR441566 was determined using an assay that measured zymosan-activated CD11b expression on granulocytes in human whole blood. Zymosan works by binding to toll-like receptor 2 (TLR2) and other receptors, including Dectin-2, , the assay stimulus, was added  with thorough mixing and incubated for 3 h at 37°C/5% CO2. The reactants were labelled by transferring 10 μl to a fresh assay plate containing 10 μl of anti-human CD45 antibody (Pacific Blue-labeled) and anti-human CD11b antibody (Phycoerythrin-labeled) for 1 h at 4°C. The blood was fixed/lysed overnight (4°C) using FACS/lysing solution  and analyzed with a FACS CANTO II (BD Biosciences). The number of single cells  positive for CD45 and expressing CD11b was determined using FlowJo software (BD Biosciences). Maximum inhibition of CD11b activation used for inhibition curve calculation was determined with an excess of an anti-TNF blocking antibody (in house anti-TNF antibody CB6). Activity Base (IBDS) was used to fit (4 PL) a dose dependent inhibition curve and calculate IC50 and Emax.The ectin-2, , and hasectin-2, . In turnectin-2,  which ca2. Zymosan, was added  with thorough mixing and incubated for 3 h at 37°C/5% CO2, as described for potency measurement. Plasma samples were collected by centrifugation at 2250g for 10 min and assayed for total TNF and TNF bound to SAR441566. The SAR441566 bound TNF was determined by ELISA using a capture antibody specific to compound-bound TNF (CA 1974), previously described in 50) and 90% occupancy (OCC90) of TNF using IDBS XLfit software.An enzyme-linked immunosorbent assay (ELISA) was used to determine the potency at which SAR441566 occupies the TNF in zymosan activated human whole blood. For the TNF occupancy assay a serial dilution of SAR441566 was pre-incubated in 384 well plates with fresh human whole blood  from UCB donors for 1 h at 37°C/5% COBlood samples obtained from healthy donors at UCB Celltech were taken with informed consent under UCB Celltech United Kingdom HTA license number 12504. All human blood and blood products were then used, stored and disposed of in accordance with UCB’s internal procedures and the United Kingdom Human Tissue Authority (HTA) Act guidelines, Code E.® 488 (AF488) using a Protein Labelling Kit  according to the manufacturers’ instructions and added to the fresh NSO-mTNF cells or fixed and permeabilized cells at a final concentration of 4 ng/ml . Cells were plated into 96-well dark walled imaging plates . Images were acquired using a Cellomics Arrayscan (Life Technologies) with a ×20 objective, ORCA-ER camera (Hamamatsu). Permeabilization of the cells allows CA1974 to enter the cell and bind intracellularly to the SAR441566 bound TNF complex while the use of live cells only captures SAR441566 bound to TNF on the cell surface.The binding of SAR441566 to human TNF on the cell surface and intracellularly was assessed using an in-house non-secreting murine melanoma cell line (NSO) engineered to express a non-cleavable form of human membrane TNF (NSO-mTNFΔ1-12)  were suspended in 0.9 ml DMEM  containing 10% FBS  and 1x NEAA  in 5 ml polystyrene round bottomed tubes . The final assay concentration of SAR441566 was 0.1 μM. This was formulated in identical assay media at ×10 final assay concentration (1 μM). To assess the binding kinetics of SAR441566, 100 μl of media containing 1 μM SAR441566 was added to the NSO-mTNF cells in 0.9 ml of media to give a final assay concentration of 0.1 μM SAR441566 in 1 × 106 of NSO-mTNF cells. Importantly, SAR441566 and CA 1974 (4 ng/ml) were added at the same time (Time 0). The increase in AF488 labelling of the cells reflects the increase in the changed conformation of TNF bound to SAR441566. Cells were analyzed and recordings taken at baseline and multiple time points up to 90 min by flow cytometry , gated by FSC, SSC, and AF488 staining. Mean fluorescence intensity increased over time and was measured and used to calculate the kinetics of binding and populate the QSP model predictions  model in male DBA/1 mice, across three independent studies. All in vivo studies were reviewed by an internal Ethical Review Body and conducted in accordance with the Animals (Scientific Procedures) Act 1986, EU Directive 2010/63/EU. Chicken type II collagen  was dissolved in 0.1 M acetic acid at a concentration of 2 mg/ml and rolled overnight at 4°C. The next day (Day 0) the CII solution was emulsified in an equal volume of complete Freund’s adjuvant (Sigma Aldrich) to give a final concentration of 1 mg/ml. Mice aged 10–15 weeks old were sensitized with 0.1 ml (100 µg CII) of emulsion injected intradermally on the flank under anesthesia. Fourteen days post-sensitization mice received a booster intradermal injection of CII emulsified in incomplete Freund’s adjuvant (Sigma Aldrich) on the opposite flank. Mice were assessed for signs of arthritis from Day 14 onwards. When signs of arthritis were seen, mice were weighed, and disease scored. Disease score was graded on a 0 to 3 scale as follows: grade 0, no swelling; grade 1, wrist or ankle swollen; grade 2, wrist/ankle and pad swollen; grade 3, wrist/ankle, pad and digits swollen. Disease score was the sum of all 4 paws (maximum score per mouse was 12). Mice were dosed therapeutically for 20 days with either vehicle or SAR441566 ) starting from the first day they showed signs of arthritis. The efficacy of SAR441566 was assessed over a dose range of 1, 3, 10 and 30 mg/kg. The area under the curve (AUC) of the disease score was calculated from baseline for individual mice in each treatment group. Blood samples were taken via the tail vein from a subset of mice in each group at 1 and 14 h post dose of SAR441566 on day 1 to obtain exposure levels.In one of the CIA studies an in-house anti-mouse TNF antibody (Ab501), that has previously been shown to be efficacious in an arthritis model , was use4)) and assuming a normal distribution with a vague prior for the variance between observations ) (The mean area under the curve (AUC) change from baseline data was analyzed using a linear model suitable for the randomized design. The statistical analysis was performed in a Bayesian framework  fitting 3,1e−3)) . The posX-ray micro-computed tomography (micro-CT) imaging of mouse limbs originating from the CIA model was performed using a SkyScan 1176 scanner . Dissected mouse limbs of each animal were fixed in 10% neutral buffered formalin. One day before micro-CT imaging, limb bone samples were carefully washed in phosphate-buffered saline (PBS) and then installed in polypropylene syringes, one for each with labels, at 4°C with 35% ethanol.3 calcium hydroxyapatite phantoms. Scanning settings of 50 kV, 500 μA, 8.6 µm pixel resolution, 0.15° or 0.3° rotation steps, and 4-frame imaging average with a 0.5-mm Al filter were applied.Prior to micro-CT scanning, samples were warmed to room temperature. A batch scanning approach  with impThe acquired X-ray projection images were reconstructed using NRecon software  with beam hardening correction and post-alignment. Three-dimensional (3D) bone images of limbs were segmented and recorded individually with their labels for analysis.For 3D micro-CT analysis of the tibia trabecular, cortical, and whole bones, MATLAB  protocols were developed to automatically select volume of interest (VOI) for each limb bones. CT-Analyzer  was then used to segment and analyze the limb bones. After all, VOIs of micro-CT images were obtained, automatic batch processing was developed and applied for two-dimensional (2D) section analysis and 3D volumetric analysis, including bone mineral density (BMD) calculation. This analysis was applied to assess limb paws with 400-slice VOIs. The trabecular bone analysis methods and parameters  were alsIn vitro metabolic clearance was determined in cryopreserved primary human hepatocytes (20-donor mixed gender pool obtained from BioIVT). The incubation was performed using suspended cells as described in In vivo PK studies were performed after intravenous (i.v.) and oral (p.o.) administration in rat, dog and cynomolgus monkeys at various doses using recommended application volumes for the different species: Rat 1 ml/kg for IV, 2 or 10 ml/kg for PO, dog 0.3 ml/kg IV, 5 ml/kg PO, Cynomolgus Macaque 2 ml/kg IV and 5 ml/kg PO. Studies were carried out according to internal guidelines and governmental requirements. SAR441566 was quantified from EDTA plasma and tissue homogenates. Tissue samples were weighed, homogenized in water and diluted in rat plasma with an exploratory liquid chromatography-mass spectrometry (LC-MS/MS) method after protein precipitation, with a quantification limit between 1 and 100 ng/ml. The pharmacokinetic parameters were calculated by the program Phoenix WinNonlin 6.4 using a non-compartmental model and linear trapezoidal interpolation calculation.The permeability of SAR441566 was determined at pH 7.4 in trans-well experiments with Caco-2 TC7 cells, internally validated for BCS classification (ICH-M9) with the probe substrates listed in the guideline. Every week cells are controlled for monolayer integrity using mannitol and low and high permeable compounds (metoprolol and propranolol) with acceptance criteria. In addition, digoxin is used for P-gp positive control in the absence and presence of cyclosporine A. Bi-directional flux/efflux experiments with increasing concentrations of SAR441566 and specific inhibitors for P-gp and BCRP  were used to characterize efflux transporter substrate properties of SAR441566. Fraction unbound of SAR441566 across preclinical species and human was determined by equilibrium dialysis with [3H]-labelled SAR441566 at six concentrations between 0.01 and 50 μg/ml at 37°C for 2 h, where equilibrium between plasma and buffer was reached. ® software (using MATLAB® R2020b). Changes to nominal parameters to this model are within the bounds provided by the source. Only intracellular TNF production, binding by SAR441566 and trafficking to the cell membrane was added to the model, which is based on the TNF residence time of 2–3 h measured in the membrane binding assay  biology  to the cng assay . To modein vitro data. Total clearance was assumed to be the sum of hepatic and renal clearance, with renal clearance being negligible as only traces of unchanged compound could be recovered in a mass balance study in rats (data not shown). Scaling hepatic clearance from in vitro to in vivo was performed using a value of 8 μl/min/106 cells for in vitro intrinsic clearance as determined in suspended human hepatocytes (® Simulator V16R1 (Method 2)  by simple three-species allometry with correction for interspecies differences in fraction unbound in plasma . In addiatocytes  using thatocytes . Vss wasatocytes  using thethod 2) .D]) value of 15.1 nM (n = 4) (−1s−1) and 2.19e−5 (s−1), respectively. The average t1/2 of SAR441566 for human TNF was determined to be 9 h:The affinity of SAR441566 for human TNF was determined using SPR technology on a Biacore T200 . The stu (n = 4) . The aveThe potency of TNF inhibition by SAR441566 was assessed in a human whole blood assay. Endogenous TNF expression is driven by Zymosan and this in turn produces CD11b on the cell surface.Briefly, the potency of SAR441566 to inhibit TNF in human whole blood was assessed by the addition of serial dilutions of the compound to blood prior to the addition of zymosan and, following an incubation, detection of CD11b expression on granulocytes by flow cytometry.50 of 35 nM  and an IC90 of 163 nM  (SAR441566 inhibited the expression of CD11b in zymosan-stimulated human whole blood with an IC–191 nM) . This va50) and 90% occupancy (OCC90)  to detect compound bound TNF. TNF occupancy increased proportionally with SAR441566 concentration and concentration-response curves were generated to determine the concentrations of SAR441566 required to achieve 50% occupancy (OCC (OCC90) .50 for total compound (includes protein bound) of 14.3 nM; , and with an OCC90 of 64.3 nM . This equates to a free SAR441566 OCC90 concentration of 16 nM.SAR441566 occupied human TNF with an OCCSAR441566 is a small molecule drug with a molecular weight of 502.5 Da, a moderate hydrophobicity and a partially charged tertiary amine with a pKa of 8.6. The molecule does not violate any of the 5 rules of Lipinski . SAR4415In Caco-2 TC7 cells, absorption was pH- and concentration-dependent with a higher absorption at a neutral pH of 7.4 compared to pH 6.5. At saturating concentrations SAR441566 was a high permeable compound. Bi-directional Flux/Efflux experiments identified SAR441566 as a substrate of efflux transporters and further analysis with specific inhibitors PSC833 (P-gp inhibitor) and Ko-143 (BCRP inhibitor) revealed SAR441566 as a P-gp substrate .In rat, a direct elimination of parent SAR441566 into the gut was observed in addition to oxidative metabolism in a mass balance study (data not shown).As a highly permeable and partially cationic compound, SAR441566 showed the expected high tissue distribution in rat, with tissue to plasma ratios up to 53 with highest accumulation in kidney, lung and liver, and very low distribution into the brain , 5.® 488 labelled IgG antibody (CA 1974) to detect TNF bound to SAR441566. Experiments allowed us to determine binding of SAR441566 to TNF on the plasma membrane of living cells .in vivo model of rheumatoid arthritis used in drug discovery to identify anti-arthritic drugs. The model is induced by sensitizing mice to chicken type II collagen, as described previously  in our model. For SAR441566, intracellular binding is assumed to solely depend on drug concentration and the binding kinetics that are equivalent to extracellular TNF binding. To understand the rate of intracellular binding of TNF by SAR441566, the intracellular pre-binding assay was used to measure the appearance of newly formed TNF-compound complex on the cell surface (a and kd) inside and outside of the cell but taking into account accumulation factors that are specific for each compound. An accumulation factor of 50 explained most of the data for SAR441566 while other reference compounds had higher binding kinetics but lower estimated accumulation factors and, overall, slower TNF-compound complex appearance rates on the cell surface. An accumulation factor of 50 is between the tissue to plasma ratios  or BID dosing in human.Preclinical PK parameters were analyzed in rat, dog and cynomolgus monkey (cyno) after IV and PO dosing  and werein vitro and in vivo . The 3D images clearly show a visual difference between SAR441566, and vehicle treated groups  to assess the potential efficacious concentration of SAR441566. QSP predicted that a reduction in free soluble TNF in synovial tissue to less than 10% would be achieved with SAR441566 at a free Cav of 8 nM and this was comparable to data for infliximab and etanercept. This suggests that SAR441566 is likely to have antibody like potency in the clinic at this exposure.For human dose predictions, we routinely apply other methods to complement in vitro, in vivo and QSP systems. Given the complexities and variabilities of these individual methods it is extremely encouraging that the target efficacy is comparable across all systems, giving a relatively narrow predicted target range of 8–24 nM free SAR441566 Cav.In summary, to determine suitable doses for human trials we generated efficacy data on SAR441566 using a multi-system approach across SAR441566 possesses the drug like properties to make it suitable for therapeutic use. It has good permeability, low metabolic clearance, high tissue distribution, high fraction unbound, moderate to high bioavailability and accumulates intracellularly where it can pre-bind TNF  prior toThe search for a small molecule that could do the same as a biologic in diseases like RA has been a goal for many years. Small molecules not only have an advantage in being administered orally but are expected to have better tissue penetration and can be chemically engineered to enter compartments that protein-based drugs traditionally cannot access. Having described small molecules that block TNF by the unusual mode of stabilizing it in an inactive conformation, we now show the outcome of those initial studies. The resulting molecule, SAR441566, has the potential to be more accessible to patients in the pre-biologic treatment space or after first line drug failure when a swift change in disease management is required. We expect it to deliver a more stable treatment option with no immunogenicity, a reliable exposure and efficacy profile, a reduced propensity to require a swap or switch of medication, as well as the potential elimination of ADA related adverse events whilst still maintaining anti-TNF biologic like disease modifying activity. With these characteristics, SAR441566 has the potential to become the anti-TNF of choice in the pre-biologic phase of treatment and is currently advancing in clinical trials.