A sensitive LC-MS/MS assay to quantitate free payload Aur0101 from ADC PYX-201 in rat and monkey plasma.

Aim: Aur0101 is a cytotoxic and small-molecule microtubule depolymerizing agent, and is the payload conjugated to antibody-drug conjugate PYX-201. Developing and validating a sensitive bioanalytical method to quantitate Aur0101 was novel and crucial in preclinical PYX-201 studies. Materials & methods: Reference standard Aur0101 and its stable isotope labelled internal standard Aur0101-d8 were used in this LC-MS/MS method. Results: This sensitive assay was validated at a lower limit of quantitation of 15 pg/ml and successfully applied to support preclinical rat and monkey toxicology studies. Preclinical plasma toxicokinetic parameters were presented. Conclusion: A sensitive and robust LC-MS/MS assay was validated for Aur0101 in rat and monkey plasma.

Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies.

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 - 10,000 ng/mL and 250 - 6000 ng/mL in rat and monkey K2EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix.

Quantification of antibody-drug conjugate PYX-201 in rat and monkey plasma via ELISA and its application in preclinical studies.

Aim: PYX-201 is a novel antibody-drug conjugate targeting the extra domain B splice variant of fibronectin in the tumor microenvironment. Accurate quantification of PYX-201 is critical for PYX-201 pharmacokinetics profiling in preclinical studies. Materials & methods: ELISA was performed using reference standard PYX-201, mouse monoclonal anti-monomethyl auristatin E antibody, mouse IgG1, mouse monoclonal anti-human IgG horseradish peroxidase and donkey anti-human IgG horseradish peroxidase. Results: This assay was validated at 50.0-10,000 ng/ml in rat dipotassium EDTA plasma and 250-10,000 ng/ml in monkey dipotassium EDTA plasma. Conclusion: This is the first time for a PYX-201 bioanalytical assay in any matrix to be reported.

Discovery of mobocertinib, a potent, oral inhibitor of EGFR exon 20 insertion mutations in non-small cell lung cancer.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Clinicopathological study and immunohistochemical analysis of expression of annexin A5 and apelin in human placentae of gestational diabetes mellitus.

BACKGROUND AND AIMS: Gestational diabetes mellitus (GDM) is one of the commonest medical complications of pregnancy. Annexin A5 (ANXA5) is a protein, found in apical surfaces of syncytiotrophoblasts, which prevents fetal and placental vascular thrombosis in GDM. Apelin is a bioactive peptide which has been linked to GDM. The aim of the present study was to correlate macroscopic as well as microscopic changes and immunohistochemical expression of ANXA5 and apelin in placentae of GDM with maternal and neonatal clinical features and also to compare the results with those in matched controls. METHODS: This prospective observational study was undertaken for a period of one year from April 2020 to March 2021. It comprised of 42 patients of GDM. Gross features, microscopic features and intensity and grade of expression of ANXA5 and Apelin were analyzed in placentae of GDM. RESULTS: Morphological changes detected in GDM placentae included increased immature villi (16 cases, 38%), increased syncytial knots (36, 86%), perivillous fibrin deposition (20, 48%), fibrosis of villous stroma (20, 48%), presence of nucleated red blood cells (12, 28.5%) and hypervascularity (34, 81%). The extent of histopathological changes noted in GDM placentae was significantly higher than that in matched controls. GDM placentae showed significantly reduced expression of ANXA5 and Apelin in terms of grade and intensity when compared with matched controls. Reduced expression (mild intensity) of ANXA5 was noted in 22 GDM cases (52.3%) whereas apelin expression was of weak intensity in 26 (61.9%) cases. Among GDM patients, statistically significant association was noted between ANXA5 intensity and neonatal resuscitation, apelin grade and preterm birth as well as low birth weight and apelin intensity and requirement of treatment in sick neonatal care unit. CONCLUSION: The placental expression of the proteins, ANXA5 and Apelin, is altered in GDM though their exact pathogenetic mechanisms are yet to be understood. They can be targets for development of prophylactic and therapeutic agents in future.

Penumbra quantification from MR SWI-DWI mismatch and its comparison with MR ASL PWI-DWI mismatch in patients with acute ischemic stroke.

In acute-ischemic-stroke patients, penumbra assessment plays a significant role in treatment outcome. MR perfusion-weighted imaging (PWI) and diffusion-weighted imaging (DWI) mismatch ratio can provide penumbra assessment. Recently reported studies have shown the potential of susceptibility-weighted imaging (SWI) in the qualitative assessment of penumbra. We hypothesize that quantitative penumbra assessment using SWI-DWI can provide an alternative to the PWI-DWI approach and this can also reduce the overall scan-time. The purpose of the current study was to develop a framework for accurate quantitative assessment of penumbra using SWI-DWI and its validation with PWI-DWI-based quantification. In the current study, the arterial-spin-labelling (ASL) technique has been used for PWI. This retrospective study included 25 acute-ischemic-stroke patients presenting within 24 hours of the last noted baseline condition of stroke onset. Eleven patients also had follow-up MRI within 48 hours. MRI acquisition comprised DWI, SWI, pseudo-continuous-ASL (pCASL), FLAIR and non-contrast-angiography sequences. A framework was developed for the enhancement of prominent hypo-intense vein signs followed by automatic segmentation of the SWI penumbra ROI. Apparent-diffusion-coefficient (ADC) maps and cerebral-blood-flow (CBF) maps were computed. The infarct core ROI from the ADC map and the ASL penumbra ROI from CBF maps were segmented semiautomatically. The infarct core volume, SWI penumbra volume (SPV) and pCASL penumbra volume were computed and used to calculate mismatch ratios MRSWIADC and MRCBFADC . The Dice coefficient between the SWI penumbra ROI and ASL penumbra ROI was 0.96 ± 0.07. MRSWIADC correlated well (r = 0.90, p < 0.05) with MRCBFADC , which validates the hypothesis of accurate penumbra assessment using the SWI-DWI mismatch ratio. Moreover, a significant association between high SPV and the presence of vessel occlusion in the MR angiogram was observed. Follow-up data showed salvation of penumbra tissue (location and volumes predicted by proposed framework) by treatments. Additionally, functional-outcome analysis revealed 93.3% of patients with MRSWIADC > 1 benefitted from revascularization therapy. Overall, the proposed automated quantitative assessment of penumbra using the SWI-DWI mismatch ratio performs equivalently to the ASL PWI-DWI mismatch ratio. This approach provides an alternative to the perfusion sequence required for penumbra assessment, which can reduce scan time by 17% for the protocol without a perfusion sequence.

Mobocertinib (TAK-788): A Targeted Inhibitor of EGFR Exon 20 Insertion Mutants in Non-Small Cell Lung Cancer.

Most EGFR exon 20 insertion (EGFRex20ins) driver mutations in non-small cell lung cancer (NSCLC) are insensitive to approved EGFR tyrosine kinase inhibitors (TKI). To address the limitations of existing therapies targeting EGFR-mutated NSCLC, mobocertinib (TAK-788), a novel irreversible EGFR TKI, was specifically designed to potently inhibit oncogenic variants containing activating EGFRex20ins mutations with selectivity over wild-type EGFR. The in vitro and in vivo activity of mobocertinib was evaluated in engineered and patient-derived models harboring diverse EGFRex20ins mutations. Mobocertinib inhibited viability of various EGFRex20ins-driven cell lines more potently than approved EGFR TKIs and demonstrated in vivo antitumor efficacy in patient-derived xenografts and murine orthotopic models. These findings support the ongoing clinical development of mobocertinib for the treatment of EGFRex20ins-mutated NSCLC. SIGNIFICANCE: No oral EGFR-targeted therapies are approved for EGFR exon 20 insertion (EGFRex20ins) mutation-driven NSCLC. Mobocertinib is a novel small-molecule EGFR inhibitor specifically designed to target EGFRex20ins mutants. Preclinical data reported here support the clinical development of mobocertinib in patients with NSCLC with EGFR exon 20 insertion mutations.See related commentary by Pacheco, p. 1617.This article is highlighted in the In This Issue feature, p. 1601.

Clinicopathological study of annexin A5 & apelin in pre-eclamptic placentae with emphasis on foetal outcome.

Background & objectives: Pre-eclampsia has remained an elusive disease with serious impacts on both maternal and foetal health. Two novel markers, annexin A5 (ANXA5) and apelin are currently of considerable interest. The present study aimed to determine the placental expression of ANXA5 and apelin in pre-eclamptic placentae and also to elucidate if there is any correlation between the expression of these markers and the clinical features of both, mother and neonate. The comparison between gross and histopathological features of pre-eclamptic placentae and controls was another objective. Methods: A prospective, observational study was undertaken for one year. Placentae of pre-eclamptic patients and matched controls (matched for age, ethnic and socio-economic background) were collected along with the clinical data. Gross and histopathological analyses were done and immunohistochemical study of placental sections with ANXA5 and apelin was also undertaken. Results: 79 pre-eclamptic patients and equal numbers of matched controls were included in the study. The difference in weight and dimensions of placentae between the pre-eclampsia group and matched controls was significant. Histopathological features noted in the pre-eclamptic placentae included decidual vasculopathy, infarction, perivillous fibrin deposition, increased syncytial knots and distal villous hypoplasia. There was a significant reduction in the expression of both ANXA5 and apelin in pre-eclamptic placentae compared to controls. Among pre-eclamptic patients, the intensity of ANXA5 and apelin expression showed a significant association with respect to neonatal resuscitation. Furthermore, the intensity of apelin showed expression a significant correlation with the requirement of sick neonatal care unit treatment. Interpretation & conclusions: The results of the present study suggest that both ANXA5 and apelin levels are reduced in pre-eclamptic placentae. Hence, it is recommended to further explore the impact of these markers on pregnancy outcomes by undertaking randomized controlled trials.

Tenecteplase-induced Nonaneurysmal Subarachnoid Hemorrhage in a Patient with Acute Ischemic Stroke: A Case Report and Literature Review.

Recently, tenecteplase (TNK) has been used for intravenous thrombolysis in acute ischemic stroke (AIS). Although spontaneous subarachnoid hemorrhage (SAH) following thrombolysis with tissue plasminogen activator has been reported, there is a lack of literature regarding TNK-induced nonaneurysmal spontaneous SAH. Our index case received intravenous TNK within an hour of symptom onset of AIS. Following deterioration of sensorium, repeat noncontrast computed tomography was performed, which showed diffuse SAH. Cerebral angiography did not reveal any aneurysm. Nonaneurysmal SAH can be a complication of TNK thrombolysis, which is not reported in literature. Knowledge of this possible adverse reaction is critical for appropriate counseling and management.

Router design for nano-communication using actin quantum cellular automata.

Logic expressions can be designed from actin filaments. It is a protein that makes the cellular structure and plays an important role in intracellular communication. Nano communication technique has been established using actin cellular automata. Among several rules, (1, 30) and (4, 27) rules have been used to design 2 to 1 multiplexer, 4 to 1 multiplexer, 1 to 2 demultiplexer and 1 to 4 demultiplexer. Router or data selector has been made of using multiplexer and demultiplexer. Three novel circuits such as multiplexer, demultiplexer and nano-router have been designed using the projected mechanism. The primary focus of this proposed technique is on different designs of the multiplexer, demultiplexer and minimum cell count with minimum time steps. The different router circuits have been simulated with the help of Simulink by which output has been verified for different circuits. Stuck at fault analysis is also done in this study. Device density and power consumption have also been included in this study. A comparative analysis of the different designs of the router provides a better concept of circuit optimisation. Furthermore, this study analyses convenient forthcoming applications in nano-technology and nano-bio-molecular systems involving the proposed parameters.

GFNB: Gini index-based Fuzzy Naive Bayes and blast cell segmentation for leukemia detection using multi-cell blood smear images.

The blood cell counting and classification ensures the evaluation and diagnosis of a number of diseases. The analysis of white blood cells (WBCs) permits us to detect the acute lymphoblastic leukemia (ALL), a type of blood cancer that causes fatality when untreated. At present, the morphological analysis of blood cells is performed manually by skilled operators, which holds numerous drawbacks. The manual techniques for leukemia detection are time-consuming and show less accurate results. Hence, there is a need for an automatic method for detecting leukemia. In order to overcome the demerits associated with the manual methods of counting and classifying, an automatic method of blast cell counting and leukemia classification is progressed. This paper proposes a leukemia detection method, using the Gini index-based Fuzzy Naive Bayes (GFNB) classifier that is the integration of Gini index and Fuzzy Naive Bayes classifier. Initially, the input multi-cell blood smear image is subjected to pre-processing, and the blast cell is segmented using the adaptive thresholding. Then, the blast cells are counted using the proposed classifier so as to decide the presence of leukemia for the effective diagnosis. Experimental analysis using the ALL-IDB1 database confirms that the proposed method operates better than the existing methods in terms of accuracy, specificity, and sensitivity that are found to be 0.9591, 0.9599, and 1, respectively. The experimental results reveal that the proposed method is reliable and accurate. Also, the proposed system can help the physicians to improve and speed up their process.Graphical abstract Leukemia is caused by the excess production of the immature leucocytes in the bone marrow that expose the human body to lose the tendency to fight against the diseases. Leukemia classification is highly needed as in the later stage, failure of the diagnosis steps may lead to the death of the person. Moreover, some countries do not have any study against the diagnosis steps of leukemia and it highly exists among the low-income people. In order to analyze the type of leukemia and to provide an effective diagnosis strategy, the paper presents a fast and highly accurate classification method. The main aim of the paper is to propose a method to perform the leukemia classification through the segmentation and classification of the WBC cells using the multi-cell blood smear images. The major steps involved in the leukemia classification are pre-processing, segmentation, feature extraction, and classification. The input blood smear image is enhanced in the pre-processing step and the pre-processed image is subjected to segmentation using the LUV color transformation and Adaptive Thresholding strategy. The features are extracted from the individual segments and they are presented to the classifier for the classification. The features extracted are shape, texture, and count of the blast cells, for which the grid-based shape extraction, local gradient pattern (LGP)-based texture features, and pixel threshold-based counting of the blast cells are employed. The proposed classifier is developed using the Gini index and Fuzzy Naive Bayes classifier.

Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

An Unusual Cause of Posterior Reversible Encephalopathy Syndrome.

Posterior reversible encephalopathy syndrome (PRES) is a clinical radiographic diagnosis of heterogeneous etiologies. The pathogenesis of PRES remains unclear, but may be related to impaired cerebral autoregulation and endothelial dysfunction. We present a case of intravascular nonionic contrast-induced PRES observed after cerebral angiography. The index patient was a follow-up case of large vertebrobasilar artery-dissecting aneurysm for which endovascular coiling was done 6 months back. She improved completely within a week. Contrast-induced PRES is a reversible benign condition, knowledge of which is crucial for appropriate management.

Dissecting Aneurysm of Vertebral Artery Involving the Origin of Posteroinferior Cerebellar Artery Treated with Retrograde Stent Placement and Coil Embolization in the Era of Flow Diverter.

Ruptured vertebral artery (VA) dissecting aneurysm carries high risk of rerupture and mortality if not treated immediately. Dissecting aneurysm of the VA involving the posteroinferior cerebellar artery (PICA) origin is difficult to treat by surgical and endovascular route. With the availability of flow diversion device for reconstructive procedure, endovascular treatment has now become easy to treat difficult aneurysm while maintaining the patency of the PICA. However, instead of using flow diverter (FD) in our case, we successfully treated dissecting VA aneurysm involving the PICA origin with retrograde stent placement from distal right VA to the left PICA to maintain the patency of PICA and occlusion of dissecting aneurysm of VA with detachable coils instead of performing surgical bypass and FD placement.