Impact of bariatric surgery on cytochrome P 450 enzyme activity.

Bariatric surgeries are becoming more prevalent as obesity rates continue to rise. Being that it is an effective weight-loss procedure, it can induce significant anatomical, physiological, and metabolic alterations, which affect the pharmacokinetics of various medications. Cytochrome (CYP) P450 is a group of enzymes that are primarily responsible for metabolizing most medications. Bariatric surgery may affect CYP activity and consequently alter metabolism of various medications, and the resulting weight loss may influence the metabolism of various drugs. This study investigates the impact of bariatric surgery on which CYP enzymes are affected and their effects medications. Authors of this study did an extensive literature review and research in databases including PubMed and EMBASE. The evidence was gathered for medication efficacy influenced by enzyme fluctuations to advocate for further studies for patients that undergo bariatric surgery. The search was limited to English-language results and is deemed up to date as of September 2023. There are numerous studies that indicated alterations of the CYP enzyme activity, which affects the pharmacokinetics of medications used to treat acute and chronic conditions after bariatric surgery. There are various mechanisms involved in CYP enzyme activity leading to fluctuations and the clearance of medications and subsequently compromising the efficacy and safety of these agents. It is imperative to conduct more prospective randomized control studies with longer duration to guide clinicians on how to manage medications with various CYP activity for patients' post-bariatric surgery.

Smarca2 genetic ablation is phenotypically benign in a safety assessment of tamoxifen-inducible conditional knockout rats.

SMARCA2 and SMARCA4 are the ATPases of the SWI/SNF chromatin remodeling complex, which play a significant role in regulating transcriptional activity and DNA repair in cells. SMARCA2 has become an appealing synthetic-lethal, therapeutic target in oncology, as mutational loss of SMARCA4 in many cancers leads to a functional dependency on residual SMARCA2 activity. Thus, for therapeutic development, an important step is understanding any potential safety target-associated liabilities of SMARCA2 inhibition. To best mimic a SMARCA2 therapeutic, a tamoxifen-inducible (TAMi) conditional knockout (cKO) rat was developed using CRISPR technology to understand the safety profile of Smarca2 genetic ablation in a model system that avoids potential juvenile and developmental phenotypes. As the rat is the prototypical rodent species utilized in toxicology studies, a comprehensive toxicological and pathological assessment was conducted in both heterozygote and homozygous knockout rats at timepoints up to 28 days, alongside relevant corresponding controls. To our knowledge, this represents the first TAMi cKO rat model utilized for safety assessment evaluations. No significant target-associated phenotypes were observed when Smarca2 was ablated in mature (11- to 15-week-old) rats; however subsequent induction of SMARCA4 was evident that could indicate potential compensatory activity. Similar to mouse models, rat CreERT2-transgene and TAMi toxicities were characterized to avoid confounding study interpretation. In summary, a lack of significant safety findings in Smarca2 cKO rats highlights the potential for therapeutics targeting selective SMARCA2 ATPase activity; such therapies are predicted to be tolerated in patients without eliciting significant on-target toxicities.

Effects of bariatric surgery on drug pharmacokinetics-Preclinical studies.

With the rising worldwide obesity rates, bariatric surgeries are increasing. Although the surgery offers an effective treatment option for weight loss, the procedure causes dramatic physiological and metabolic changes. Animal models in rodents provide a valuable tool for studying the systemic effects of the surgery. Since the surgery may significantly influence the pharmacokinetic properties of medications, animal studies should provide essential insight into mechanisms underlying changes in how the body handles the drug. This review summarizes research work in rodents regarding the impact of standard bariatric procedures on pharmacokinetics. A qualitative literature search was conducted via PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), and EMBASE. Studies that examined bariatric surgery's effects on drug pharmacokinetics in rodent models were included. Clinical studies and studies not involving drug interventions were excluded. A total of 15 studies were identified and assessed in this review. These studies demonstrate the possible impact of bariatric surgery on drug absorption, distribution, metabolism, excretion, and potential mechanisms. Pharmacokinetic changes exhibited in the limited pre-clinical studies highlight a need for further investigation to fully understand the impact and mechanism of bariatric surgery on drug responses.

Advanced Technologies and Automation in mES Cell Workflow.

Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. To streamline and increase the efficiency in our ES cell pipeline and decrease the timeline for mouse models produced via ES cells, automation is introduced in the pipeline. Below, we describe a novel and effective approach utilizing ddPCR, dPCR, automated DNA purification, MultiMACS, and adenovirus recombinase combined screening workflow that reduces the time between therapeutic target identification and experimental validation.

Accelerated embryonic stem cell screening with a highly efficient genotyping pipeline.

INTRODUCTION: Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. METHODS AND RESULTS: The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. Here, we optimized and functionally linked magnetic activated cell sorting (MACS) technology as well as multiplex droplet digital PCR (ddPCR) to our ES cell screening process to achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. We describe this improved workflow and illustrate the reduction in time between therapeutic target identification and experimental validation. CONCLUSION: In sum, we describe a novel and effective implementation of ddPCR, multiMACS, and adenovirus recombinase into a streamlined screening workflow that significantly reduces timelines for gene targeting in mouse ES cells.

Prolonged Plasma Exposure of the Kv1.3-Inhibitory Peptide HsTX1[R14A] by Subcutaneous Administration of a Poly(Lactic-co-Glycolic Acid) (PLGA) Microsphere Formulation.

This study evaluated the impact of poly(lactic-co-glycolic acid) (PLGA) microsphere formulations on in vitro release and in vivo plasma exposure of HsTX1[R14A], a potent inhibitor of the voltage-gated potassium channel Kv1.3, with potential to treat autoimmune conditions. Microspheres containing HsTX1[R14A] were prepared using different PLGA materials, including Resomer® RG502H, RG503H and PURASORB® PDLG 5004 (Purac). After assessing encapsulation efficiency and in vitro release, plasma concentrations of HsTX1[R14A] were quantified by LCMS/MS following subcutaneous administration of HsTX1[R14A]-loaded RG503H microspheres (15 mg/kg) or HsTX1[R14A] solution (4 mg/kg) to Sprague-Dawley rats. Microspheres prepared with Purac exhibited the greatest encapsulation efficiency (45.5 ± 2.4% (mean ± SD)) and RG502H the lowest (22.0 ± 6.4%). Release of HsTX1[R14A] was fastest in vitro for RG502H microspheres (maximum release at 31 days) and slowest for Purac (82 days). With a relatively rapid burst release of 20.0 ± 0.4% and a controlled release profile of up to 41 days, HsTX1[R14A]-loaded RG503H microspheres were selected for subcutaneous administration, resulting in detectable plasma concentrations for 11 days relative to 8 h following subcutaneous administration of HsTX1[R14A] solution. Therefore, subcutaneous administration of RG503H PLGA microspheres is a promising approach to be exploited for delivery of this immune modulator.

Integration of innate immune signalling by caspase-8 cleavage of N4BP1.

Mutations in the death receptor FAS1,2 or its ligand FASL3 cause autoimmune lymphoproliferative syndrome, whereas mutations in caspase-8 or its adaptor FADD-which mediate cell death downstream of FAS and FASL-cause severe immunodeficiency in addition to autoimmune lymphoproliferative syndrome4-6. Mouse models have corroborated a role for FADD-caspase-8 in promoting inflammatory responses7-12, but the mechanisms that underlie immunodeficiency remain undefined. Here we identify NEDD4-binding protein 1 (N4BP1) as a suppressor of cytokine production that is cleaved and inactivated by caspase-8. N4BP1 deletion in mice increased the production of select cytokines upon stimulation of the Toll-like receptor (TLR)1-TLR2 heterodimer (referred to herein as TLR1/2), TLR7 or TLR9, but not upon engagement of TLR3 or TLR4. N4BP1 did not suppress TLR3 or TLR4 responses in wild-type macrophages, owing to TRIF- and caspase-8-dependent cleavage of N4BP1. Notably, the impaired production of cytokines in response to TLR3 and TLR4 stimulation of caspase-8-deficient macrophages13 was largely rescued by co-deletion of N4BP1. Thus, the persistence of intact N4BP1 in caspase-8-deficient macrophages impairs their ability to mount robust cytokine responses. Tumour necrosis factor (TNF), like TLR3 or TLR4 agonists, also induced caspase-8-dependent cleavage of N4BP1, thereby licensing TRIF-independent TLRs to produce higher levels of inflammatory cytokines. Collectively, our results identify N4BP1 as a potent suppressor of cytokine responses; reveal N4BP1 cleavage by caspase-8 as a point of signal integration during inflammation; and offer an explanation for immunodeficiency caused by mutations of FADD and caspase-8.

Comparison across Three Hybrid Lipid-Based Drug Delivery Systems for Improving the Oral Absorption of the Poorly Water-Soluble Weak Base Cinnarizine.

Three state-of-the-art drug delivery vehicles engineered by nanostructuring lipid colloids within solid particle matrices were fabricated for the oral delivery of the poorly water-soluble, weak base, cinnarizine (CIN). The lipid and solid phase of each formulation was varied to systematically analyze the impact of key material characteristics, such as nanostructure and surface chemistry, on the in vitro and in vivo fate of CIN. The three systems formulated were: silica-stabilized lipid cubosomes (SSLC), silica-solid lipid hybrid (SSLH), and polymer-lipid hybrid (PLH) particles. Significant biopharmaceutical advantages were presented for CIN when solubilized in the polymer (poly(lactic-co-glycolic) acid; PLGA) and lipid phase of PLH particles compared to the lipid phases of SSLC and SSLH particles. In vitro dissolution in simulated intestinal conditions highlighted reduced precipitation of CIN when administered within PLH particles, given by a 4-5-fold improvement in the extent of CIN dissolution compared to the other delivery vehicles. Furthermore, CIN solubilization was enhanced 1.5-fold and 6-fold under simulated fasted state lipid digestion conditions when formulated with PLH particles compared to SSLH and SSLC particles, respectively. In vivo pharmacokinetics correlated well with in vitro solubilization data, whereby oral CIN bioavailability in rats, when encapsulated in the corresponding formulations, increased from SSLC < SSLH < PLH. The pharmacokinetic data obtained throughout this study indicated a synergistic effect between PLGA nanoparticles and lipid droplets in preventing CIN precipitation and thus, enhancing oral absorption. This synergy can be harnessed to efficiently deliver challenging poorly water-soluble, weak bases through oral administration.

The kinase TPL2 activates ERK and p38 signaling to promote neutrophilic inflammation.

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.

Matrix disruptions, growth, and degradation of cartilage with impaired sulfation.

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-µm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.