Therapeutic Entities Research Articles

Does skin permeation kinetics influence efficacy of topical dermal drug delivery system?: Assessment, prediction, utilization, and integration of chitosan biomacromolecule for augmenting topical dermal drug delivery in skin.

Skin permeation is an integral part of penetration of topical therapeutics. Zero order in addition to Higuchi permeation kinetic is usually preferred in topical drug delivery cargo. Penetration of therapeutic entities through epidermal barrier is a major challenge for scientific fraternity. Furthermore, penetration of therapeutic entities determines the transportation and ultimately therapeutic efficacy of topical dermal dosage forms. Apart from experimentation models, mathematical equations, in silico docking, molecular dynamics (MDs), and artificial neural network (Neural) techniques are being used to assess free energies and prediction of electrostatic attractions in order to predict the permeation phenomena of therapeutic entities. Therefore, in the present review, we have summarized the significance of kinetic equations, in silico docking, MDs, and ANN in assessing and predicting the penetration behavior of topical therapeutics through dermal dosage form. In addition, the role of chitosan biomacromolecule in modulating permeation of topical therapeutics in skin has also been illustrated using computational techniques.

Brain Cancer Drug Discovery: Clinical Trials, Drug Classes, Targets, and Combinatorial Therapies

Brain cancer is a formidable challenge for drug development, and drugs derived from many cutting-edge technologies are being tested in clinical trials. We manually characterized 981 clinical trials on brain tumors that were registered in ClinicalTrials.gov from 2010 to 2020. We identified 582 unique therapeutic entities targeting 581 unique drug targets and 557 unique treatment combinations involving drugs. We performed the classification of both the drugs and drug targets based on pharmacological and structural classifications. Our analysis demonstrates a large diversity of agents and targets. Currently, we identified 32 different pharmacological directions for therapies that are based on 42 structural classes of agents. Our analysis shows that kinase inhibitors, chemotherapeutic agents, and cancer vaccines are the three most common classes of agents identified in trials. Agents in clinical trials demonstrated uneven distribution in combination approaches; chemotherapy agents, proteasome inhibitors, and immune modulators frequently appeared in combinations, whereas kinase inhibitors, modified immune effector cells did not as was shown by combination networks and descriptive statistics. This analysis provides an extensive overview of the drug discovery field in brain cancer, shifts that have been happening in recent years, and challenges that are likely to come. SIGNIFICANCE STATEMENT: This review provides comprehensive quantitative analysis and discussion of the brain cancer drug discovery field, including classification of drug, targets, and therapies.

Genetically Engineered Cellular Membrane Vesicles as Tailorable Shells for Therapeutics.

Benefiting from the blooming interaction of nanotechnology and biotechnology, biosynthetic cellular membrane vesicles (Bio‐MVs) have shown superior characteristics for therapeutic transportation because of their hydrophilic cavity and hydrophobic bilayer structure, as well as their inherent biocompatibility and negligible immunogenicity. These excellent cell‐like features with specific functional protein expression on the surface can invoke their remarkable ability for Bio‐MVs based recombinant protein therapy to facilitate the advanced synergy in poly‐therapy. To date, various tactics have been developed for Bio‐MVs surface modification with functional proteins through hydrophobic insertion or multivalent electrostatic interactions. While the Bio‐MVs grow through genetically engineering strategies can maintain binding specificity, sort orders, and lead to strict information about artificial proteins in a facile and sustainable way. In this progress report, the most current technology of Bio‐MVs is discussed, with an emphasis on their multi‐functionalities as “tailorable shells” for delivering bio‐functional moieties and therapeutic entities. The most notable success and challenges via genetically engineered tactics to achieve the new generation of Bio‐MVs are highlighted. Besides, future perspectives of Bio‐MVs in novel bio‐nanotherapy are provided.

Bladder Cancer-related microRNAs With In Vivo Efficacy in Preclinical Models

Progressive and metastatic bladder cancer remain difficult to treat. In this review, we critique seven up-regulated and 25 down-regulated microRNAs in order to identify new therapeutic entities and corresponding targets. These microRNAs were selected with respect to their efficacy in bladder cancer-related preclinical in vivo models. MicroRNAs and related targets interfering with chemoresistance, cell-cycle, signaling, apoptosis, autophagy, transcription factor modulation, epigenetic modification and metabolism are described. In addition, we highlight microRNAs targeting transmembrane receptors and secreted factors. We discuss druggability issues for the identified targets.

Strategies for targeting undruggable targets

ABSTRACT Introduction Undruggable targets refer to clinically meaningful therapeutic targets that are ‘difficult to drug’ or ‘yet to be drugged’ via traditional approaches. Featuring characteristics of lacking defined ligand-binding pockets, non-catalytic protein–protein interaction functional modes and less-investigated 3D structures, these undruggable targets have been targeted with novel therapeutic entities developed with the progress of unconventional drug discovery approaches, such as targeted degradation molecules and display technologies. Area covered This review first presents the concept of ‘undruggable’ exemplified by RAS and other targets. Next, detailed strategies are illustrated in two aspects: innovation of therapeutic entities and development of unconventional drug discovery technologies. Finally, case studies covering typical undruggable targets (Bcl-2, p53, and RAS) are depicted to further demonstrate the feasibility of the strategies and entities above. Expert opinion Targeting the undruggable expands the scope of therapeutically reachable targets. Consequently, it represents the drug discovery frontier. Biomedical studies are capable of dissecting disease mechanisms, thus broadening the list of undruggable targets. Encouraged by the recent approval of the KRAS inhibitor Sotorasib, we believe that merging multiple discovery approaches and exploiting various novel therapeutic entities would pave the way for dealing with more ‘undruggable’ targets in the future.

In silico study of some selective phytochemicals against a hypothetical SARS-CoV-2 spike RBD using molecular docking tools

BackgroundThis world is currently witnessing a pandemic outbreak of ‘COVID-19′ caused by a positive-strand RNA virus ‘SARS-CoV-2’. Millions have succumbed globally to the disease, and the numbers are increasing day by day. The viral genome enters into the human host through interaction between the spike protein (S) and host angiotensin-converting enzyme-2 (ACE2) proteins. S is the common target for most recently rolled-out vaccines across regions. A recent surge in single/multiple mutations in S region is of great concern as it may escape vaccine induced immunity. So far, the treatment regime with repurposed drugs has not been too successful. HypothesisNatural compounds are capable of targeting mutated spike protein by binding to its active site and destabilizing the spike-host ACE2 interaction. Materials and methodsA hypothetical mutated spike protein was constructed by incorporating twelve different mutations from twelve geographical locations simultaneously into the receptor-binding domain (RBD) and docked with ACE2 and seven phytochemicals namely allicin, capsaicin, cinnamaldehyde, curcumin, gingerol, piperine and zingeberene. Molecular Dynamic (MD) simulation and Principal Component Analysis (PCA) were finally used for validation of the docking results. ResultThe docking results showed that curcumin and piperine were most potent to bind ACE2, mutated spike, and mutated spike-ACE2 complex, thereby restricting viral entry. ADME analysis also proved their drug candidature. The docking complexes were found to be stable by MD simulation. ConclusionThis result provides a significant insight about the phytochemicals' role, namely curcumin and piperine, as the potential therapeutic entities against mutated spike protein of SARS-CoV-2.

O-EP004. Targeting epilepsy with novel therapeutic entities to investigate the biochemical and molecular mechanisms

Introduction . Analysis of anticonvulsant action of novel therapeutic entity (NTE) in kindling (Pentylenetetrazole (PTZ)) and acute (Kainic Acid (KA)) seizure mice (male swiss albino) models. Methods . The NTE (NP-VM1) was screened from a class of 5 hydrazone derivatives of 2,4-disubstituted thiazole through subcutaneous PTZ and neurotoxicity screening followed by log-probit analysis for determining ED50, TD50 and protective-index (PI). Neuroprotective potential of the NTE was analyzed in both models and was validated through behavioral screening tests and oxidative stress estimation (MDA, GPx and SOD levels). Results were analyzed by ANOVA single factor and Tukey’s post-hoc test. Results . NP-VM1 showed significant biological activity in screening. It significantly reduced the seizure activity in both models. Significant increase in the spatial learning ability in Morris Water Maze test was further confirmed by increased recognition index in Object Recognition Test. Reduced transfer latency and time spent in open arm in Elevated Plus Maze test along with increased rearings and movement through the centre in Open Field Test signified restoration of behavior normalcy and locomotion ability. Rotarod test showed that NTE significantly negated the neurotoxic and ataxic effects which was further proved by enhanced GPx and SOD levels and attenuated MDA levels in the hippocampus. The results of sucrose consumption test were however not statistically significant. Conclusion . NP-VM1 significantly augmented the ROS detoxification thereby fortifying the neurons from chronic damage, which was evident by the restoration of behavioral normalcy and learning, motor coordination and locomotion abilities.

Insights on the polypyrrole based nanoformulations for photodynamic therapy

This review is written to endow updated information on polypyrrole based photosensitizers for the treatment of deadly diseases such as cancer and microbial infection. Tetrapyrrolic macromolecules such as porphyrins and phthalocyanines hold unique photophysical properties which make them very useful compounds for various biomedical applications. Besides their properties, they also have some limitations such as low water solubility, bioavailability, biocompatibility and lack of specificity, etc. Researchers are trying to overcome these limitations by incorporating photosensitizers into the different types of nanoparticles and improve the quality of photodynamic therapy. We have contributed to this field by synthesizing and developing polypyrrolic photosensitizer based nanoparticles for potential applications in antimicrobial and anticancer photodynamic activity. Throughout this review, newly synthesized and existing PSs conjugated/encapsulated/doped/incorporated with nanoparticles are emphasized, which are essential for current and future research themes. Also in this review, we briefly summarized the research work carried over the past few years by considering the porphyrin based photosensitizers as alternative therapeutic entities for the treatment of microbial infections, cancers, and many other diseases.

Molecular Perspective of Nanoparticle Mediated Therapeutic Targeting in Breast Cancer: An Odyssey of Endoplasmic Reticulum Unfolded Protein Response (UPRER) and Beyond.

Breast cancer (BC) is the second most frequent cause of death among women. Representing a complex and heterogeneous type of cancer, its occurrence is attributed by both genetic (gene mutations, e.g., BRCA1, BRCA2) and non-genetic (race, ethnicity, etc.) risk factors. The effectiveness of available treatment regimens (small molecules, cytotoxic agents, and inhibitors) decreased due to their poor penetration across biological barriers, limited targeting, and rapid body clearance along with their effect on normal resident cells of bone marrow, gastrointestinal tract, and hair follicles. This significantly reduced their clinical outcomes, which led to an unprecedented increase in the number of cases worldwide. Nanomedicine, a nano-formulation of therapeutics, emerged as a versatile delivering module for employment in achieving the effective and target specific delivery of pharmaceutical payloads. Adoption of nanotechnological approaches in delivering therapeutic molecules to target cells ensures not only reduced immune response and toxicity, but increases the stability of therapeutic entities in the systemic circulation that averts their degradation and as such increased extravasations and accumulation via enhanced permeation and the retention (EPR) effect in target tissues. Additionally, nanoparticle (NP)-induced ER stress, which enhances apoptosis and autophagy, has been utilized as a combative strategy in the treatment of cancerous cells. As nanoparticles-based avenues have been capitalized to achieve better efficacy of the new genera of therapeutics with enhanced specificity and safety, the present study is aimed at providing the fundamentals of BC, nanotechnological modules (organic, inorganic, and hybrid) employed in delivering different therapeutic molecules, and mechanistic insights of nano-ER stress induced apoptosis and autophagy with a perspective of exploring this avenue for use in the nano-toxicological studies. Furthermore, the current scenario of USA FDA approved nano-formulations and the future perspective of nanotechnological based interventions to overcome the existing challenges are also discussed.

Effect of Topical Oxygen Therapy on Chronic Wounds

Over the past three decades, there has been a growing interest in the use of oxygen therapy to promote wound healing. Although the most commonly recognized oxygen therapy for the treatment of chronic wounds is hyperbaric oxygen therapy, topical oxygen therapy has a greater level of evidence supporting its use in chronic wound care. Still, it is imperative that these two treatment modalities be recognized not merely as competitors, but as distinct therapeutic entities. Through personal experience and a thorough literature review, we investigated the use of topical oxygen therapy in the management of chronic wounds. The benefits of using topical oxygen therapy have been demonstrated in patients with diabetic foot ulcers, ischemic ulcers, post-revascularization ulcers, and pressure ulcers. There are several topical oxygen devices currently on the market that are versatile, relatively low-risk, and generally well-tolerated by patients. While these devices have been used in the treatment of chronic wounds at different locations and of different etiologies, other uses of these devices are still being investigated. Topical oxygen therapy is yet another tool in our arsenal to be used in treating difficult to heal chronic wounds and could potentially be used more readily.

Treating Bladder Cancer: Engineering of Current and Next Generation Antibody-, Fusion Protein-, mRNA-, Cell- and Viral-Based Therapeutics.

Bladder cancer is a frequent malignancy and has a clinical need for new therapeutic approaches. Antibody and protein technologies came a long way in recent years and new engineering approaches were applied to generate innovative therapeutic entities with novel mechanisms of action. Furthermore, mRNA-based pharmaceuticals recently reached the market and CAR-T cells and viral-based gene therapy remain a major focus of biomedical research. This review focuses on the engineering of biologics, particularly therapeutic antibodies and their application in preclinical development and clinical trials, as well as approved monoclonal antibodies for the treatment of bladder cancer. Besides, newly emerging entities in the realm of bladder cancer like mRNA, gene therapy or cell-based therapeutics are discussed and evaluated. As many discussed molecules exhibit unique mechanisms of action based on innovative protein engineering, they reflect the next generation of cancer drugs. This review will shed light on the engineering strategies applied to develop these next generation treatments and provides deeper insights into their preclinical profiles, clinical stages, and ongoing trials. Furthermore, the distribution and expression of the targeted antigens and the intended mechanisms of action are elucidated.

Opening a debate on open-source modeling tools: Pouring fuel on fire versus extinguishing the flare of a healthy debate.

As model‐informed drug development becomes an integral part of modern approaches to the discovery of new therapeutic entities and showing their safety and effectiveness, modalities of incorporating the paradigm into widespread practice require a revisit. Traditionally, modeling and simulation (M&S) have been performed by specialized teams who create bespoke models for each case and have reservations about letting modeling be done by the greater mass of scientists engaged in various stages of drug development. An analogy can be drawn between M&S and automobiles: typical drivers of ordinary cars use them for daily tasks, such as going from point A to B whereas specialized Formula 1 drivers using bespoke individually made cars to test the latest technologies. The reliability and robustness of ordinary cars for the first group requires elements related to quality and endurance that are very different from those applicable to any Formula 1 car supported by a large team of engineers. In this commentary, we frame and analyze the problems concerning the structure and setup of various M&S tools, and their pros and cons. We demonstrate that many misconceptions have precluded having an open discussion on what each modality of M&S tools strives to achieve, and we provide data and evidence that support the move of M&S to main stream use by many, as opposed to specialized usage by few. Parallels are drawn in many other areas involving laboratory instrumentation, statistical analyses, and so on.

2019-2020 Drug Updates in Solid Tumors.

During JADPRO Live Virtual 2020, Kirollos S. Hanna, PharmD, BCPS, BCOP, updated the audience on the pharmacology, new indications, and the clinical trial data of novel medications as well as expanded indications in solid tumors. Dr. Hanna also discussed signs and symptoms of potential serious and life-threatening adverse events, and how new therapeutic entities will impact the advanced practice setting.

A Systematic Review of the Preventive and Therapeutic Effects of Naringin Against Human Malignancies.

Background: Natural product-based cancer preventive and therapeutic entities, such as flavonoids and their derivatives, are shown to have a noticeable capability to suppress tumor formation and cancer cell growth. Naringin, a natural flavanone glycoside present in various plant species, has been indicated to modulate different signaling pathways and interact with numerous cell signaling molecules, which allows for an extensive variety of pharmacological actions, such as amelioration of inflammation, oxidative stress, metabolic syndromes, bone disorders, and cancer. The purpose of this systematic review is to present a critical and comprehensive assessment of the antitumor ability of naringin and associated molecular targets in various cancers. Methods: Studies were identified through systematic searches of Science Direct, PubMed, and Scopus as well as eligibility checks according to predefined selection criteria. Results: Eighty-seven studies were included in this systematic review. There was strong evidence for the association between treatment with naringin alone, or combined with other drugs and antitumor activity. Additionally, studies showed that naringin-metal complexes have greater anticancer effects compared to free naringin. It has been demonstrated that naringin employs multitargeted mechanisms to hamper cancer initiation, promotion, and progression through modulation of several dysregulated signaling cascades implicated in cell proliferation, autophagy, apoptosis, inflammation, angiogenesis, metastasis, and invasion. Conclusion: The results of our work show that naringin is a promising candidate for cancer prevention and treatment, and might offer substantial support for the clinical application of this phytocompound in the future. Nevertheless, further preclinical and clinical studies as well as drug delivery approaches are needed for designing novel formulations of naringin to realize the full potential of this flavonoid in cancer prevention and intervention.

Protease Enzymes: Highlights on Potential of Proteases as Therapeutics Agents

In the era of advancement of biotechnology and molecular biology, the structure, catalytic site specification, role in biochemical processes, and many diseases of an enzyme have been discovered. Enzymes are protein in nature and are formulated in the human body from amino acids. Each enzyme carries its specific functions in the body and has particular substrates to work with. The enzymes are present in the optimal amount in the body to carry out essential functions for life and health. Proteases or proteinases or peptidases are the largest family of proteolytic enzymes whose primary function is cleaving proteins into smaller fragments. Due to their structural and functional diversity, these enzymes carry out many functions, including intracellular protein formation or recycling, nutrient digestion, and immune system cascade formation. Proteases have been used for years commercially in leather industries, in beer industries to prevent agglomeration of proteins, and detergent formulations. Still, in recent years their therapeutic functions have been discovered and utilized in the pharmaceutical industry. They have been used in the curing, diagnosis, and replacement therapies, etc. Proteases with the development of microbiology and protein engineering have been hitting the market now quite frequently as drugs/therapeutic agents. The U.S. FDA (Food and Drug Administration) now has approved various therapies utilizing protease enzymes as a therapeutic entities, and many next-generation or completely new proteases are in clinical development. There are multiple proteases in the development stage of clinical and preclinical trials. The utilization of enzymes has not achieved 100 percent of its potential mainly due to two reasons: the first reason is the economical difficulties in obtaining pure and homogeneous enzymes leading to higher cost issues, and second the immunogenicity, fast degradation, and inactivation in the body by inhibitors and secretions and due to Physiological pH and temperature barriers. The present article attempts to introduce various therapeutic roles of these enzymes and methods to utilize them to the maximum of their potential in the medical/pharmaceutical field.

A Phytopharmacological Review on a Medicinal Plant: Cordia africana Lam.

The use of phytoconstituents single or combined with standard medicines has been utilized in cure of different diseases. Many plants of genus Cordia comprise of trees and shrubs are widely distributed in warmer regions and have been utilized in management of various diseases. Cordia africana Lam. (family- Boraginaceae) is a small to medium-sized evergreen tree, 4 to 15 (30) m high, heavily branched with a spreading, umbrella-shaped or rounded crown. Bole typically curved or crooked. Bark grayish-brown to dark brown, smooth in young trees, but soon becoming rough and longitudinally fissured with age; young branch lets with sparse long. Uses of C. africana: firewood, timber (furniture, beehives, boxes, mortars, church, drums), food (fruit), medicine (bark, roots), fodder (leaves), bee forage, mulch, soil conservation, ornamental, shade. Various phytoconstituents like flavonoids, alkaloids, tannins, terpenoids, saponins, steroids, anthraquinones, carbohydrates and protins having different activities were screened and isolated from different parts of Cordia africana. Various important Pharmacological properties including Antioxidant, Cytotoxicity, anti-inflammatory, anthelmintic, antimicrobial, anti-nociceptive, and others have been well documented for this plant. Therefore, we have briefly reviewed the various bioactivities of Cordia africana to improve our knowledge on plant phytochemicals as therapeutic entities. The present review describes the various phytoconstituents and therapeutic potential of Cordia africana that can be followed for future research on this plant for human health benefits.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC).Purpose:We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved.Methods:We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice.Results:We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE.Conclusions:These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Current Challenges for the Effective Management of the COVID-19 Pandemic.

Within a short period, the coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) spread all over the globe and became the first pandemic of the present century. Early diagnostic tools and effective drugs are urgently needed to effectively manage the COVID-19 pandemic. Based on current literature, we provide recent updates on SARS-CoV-2 biology, available diagnostic methods, and therapeutic options for the management of COVID-19 pandemic. A literature survey was done using Google and PubMed and Web of Science to summarize the current updates on this topic. Current coronavirus diagnostic tests are reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR (qRT-PCR), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) which detects the presence of specific genome sequence of virus. Existing antiviral drugs or new therapeutic options such as neutralizing antibody or plasma therapy are mostly used to restrict the virus growth with a limited success. As there is no specific treatment or vaccine available to limit the infection of SARS-CoV-2, we need to rely on the existing way to limit the disease. The first priority to fight COVID-19 is development of early diagnostic tools so that infected persons can be identified and further viral transmission can be blocked. Evaluation of existing drugs or identification of new therapeutic entities becomes the major challenge to deal with the present pandemic.

Tofacitinib inhibits inflammatory cytokines from ulcerative colitis and healthy mucosal explants and is associated with pSTAT1/3 reduction in T-cells.

Poor translatability of animal disease models has hampered the development of new inflammatory bowel disorder (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from patients with ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor tofacitinib (TOFA) as a model therapeutic. Colorectal biopsies from HC participants and patients with UC were cultured and stimulated with multiple mitogens ± TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3+CD4+ and CD3+CD8+ T-cells was determined by flow cytometry in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMCs) following the activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed-effects modeling, t test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection. Under baseline conditions, 27 of 29 biomarkers from patients with UC were increased versus HC participants. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMCs and MMCs. Immunogen stimulation increases biomarker release in similar patterns for HC participants and patients with UC, while enhancing the dynamic range for pharmacological effects. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.NEW & NOTEWORTHY Using colorectal biopsy material from healthy volunteers and patients with clinically defined IBD supports translational research by informing the evaluation of therapeutic efficacy and target engagement for the development of new therapeutic entities. Combining experimental readouts from intact and dissociated tissue enhances our understanding of the tissue-resident immune system that contribute to disease pathology. Bayesian logistic regression modeling is an effective tool for predicting ex vivo explant biomarker release patterns.

Developability Assessment of an Isolated CH2 Immunoglobulin Domain.

The IgG CH2 domain continues to hold promise for the development of new therapeutic entities because of its bifunctional role as a biomarker and effector protein. The need for further understanding of molecular stability and aggregation in therapeutic proteins has led to the development of a breakthrough quantum cascade laser microscope to allow for real-time comparability assessment of an array of related proteins in solution upon thermal perturbation. Our objective was to perform a comprehensive developability assessment of three similar monoclonal antibody (mAb) fragments: CH2, CH2s, and m01s. The CH2 construct consists of residues Pro238 to Lys340 of the IgG1 heavy chain sequence. CH2s has a 7-residue deletion at the N-terminus and a 16-residue C-terminal extension containing a histidine tag. The m01s construct is identical to CH2s, except for two cysteines introduced at positions 242 and 334. A series of hyperspectral images was acquired during thermal perturbation from 28 to 60 °C for all three proteins in an array. Co-distribution and two-dimensional infrared correlation spectroscopies yielded the mechanism of aggregation and stability for these three proteins. The level of detail is unprecedented, identifying the regions within CH2 and CH2s that are prone to self-association and establishing the differences in stability. Furthermore, CH2 helical segments, β-sheets, β-turns, and random coil regions were less stable than in CH2s and m01s because of the presence of the N-terminal 310-helix and β-turn type III. The engineered disulfide bridge in m01s eliminated the self-association process and rendered this mAb fragment the most stable.