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.

Characteristics associated with response to subcutaneously administered anti-CGRP monoclonal antibody medications in a real-world community cohort of persons living with migraine: A retrospective clinical and genetic study.

OBJECTIVE: To evaluate response to anti-calcitonin gene-related peptide (CGRP) migraine preventives in a real-world community cohort of persons living with migraine and to identify clinical and genetic characteristics associated with efficacious response. BACKGROUND: Erenumab-aooeb, fremanezumab-vrfm, and galcanezumab-gnlm target CGRP or its receptor; however, many patients are non-responsive. METHODS: In this retrospective clinical and genetic study, we identified 1077 adult patients who satisfied the International Classification of Headache Disorders, 3rd edition, criteria for migraine without aura, migraine with aura, or chronic migraine and who were prescribed an anti-CGRP migraine preventive between May 2018 and May 2021. Screening of 558 patients identified 289 with data at baseline and first follow-up visits; data were available for 161 patients at a second follow-up visit. The primary outcome was migraine days per month (MDM). In 198 genotyped patients, we evaluated associations between responders (i.e., patients with ≥50% reduction in MDM at follow-up) and genes involved in CGRP signaling or pharmacological response, and genetic and polygenic risk scores. RESULTS: The median time to first follow-up was 4.4 (0.9-22) months after preventive start. At the second follow-up, 5.7 (0.9-13) months later, 145 patients had continued on the same preventive. Preventives had strong, persistent effects in reducing MDM in responders (follow-up 1: η2 = 0.26, follow-up 2: η2 = 0.22). At the first but not second follow-up: galcanezumab had a larger effect than erenumab, while no difference was seen at either follow-up between galcanezumab and fremanezumab or fremanezumab and erenumab. The decrease in MDM at follow-up was generally proportional to baseline MDM, larger in females, and increased with months on medication. At the first follow-up only, patients with prior hospitalization for migraine or who had not responded to more preventive regimens had a smaller decrease in MDM. Reasons for stopping or switching a preventive varied between medications and were often related to cost and insurance coverage. At both follow-ups, patient tolerance (1: 92.2% [262/284]; 2: 95.2% [141/145]) and continued use (1: 77.5% [224/289]; 2: 80.6% [116/145]) were high and similar across preventives. Response consistency (always non-responders: 31.7% [46/145]; always responders: 56.5% [82/145], and one-time only responders: 11.7% [17/145]) was also similar across preventives. Non-responder status had nominally significant associations with rs12615320-G in RAMP1 (odds ratio [95% confidence interval]: 4.7 [1.5, 14.7]), and rs4680-A in COMT (0.6[0.4, 0.9]). Non-responders had a lower mean genetic risk score than responders (1.0 vs. 1.1; t(df) = -1.75(174.84), p = 0.041), and the fraction of responders increased with genetic and polygenic risk score percentile. CONCLUSIONS: In this real-world setting, anti-CGRP preventives reduced MDM persistently and had similar and large effect sizes on MDM reduction; however, clinical and genetic factors influenced response.

Exploring the Underlying Constructs of Rape-Related Cognition Scales and Their Relationships With Sexual Aggression.

Rape-related cognitions (typically defined as encompassing any number of cognitive constructs) are thought to play a role in sexual aggression. However, rape-related cognition scales often assess these cognitive constructs as one. The purpose of this study is to explore the factor structure of these measures using a sample of 191 community men. We found that items from the Rape Myth Acceptance, RAPE, and Illinois Rape Myth Acceptance (IRMA) scales formed one factor, which was significantly related to sexual aggression. We further found that four and six IRMA subscales were significantly related to past and likelihood of sexual aggression, respectively. Additionally, one IRMA subscale was independently related to past and likelihood of sexual aggression. The results are discussed in terms of implications and direction for future research.

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.

Carpenter Bees (Xylocopa) Harbor a Distinctive Gut Microbiome Related to That of Honey Bees and Bumble Bees.

Eusocial corbiculate bees, including bumble bees and honey bees, maintain a socially transmitted core gut microbiome that contributes to digestion and pathogen defense. In contrast, solitary bees, which have fewer opportunities for direct interhost transmission, typically have less consistent microbiomes dominated by bacteria associated with pollen and food reserves. Carpenter bees (genus Xylocopa) are long-lived bees that are not eusocial but that often live in shared nesting sites. We characterized gut microbiomes for Xylocopa micans, X. mexicanorum, X. tabaniformis parkinsoniae, and X. virginica and for five solitary bee species from other genera (Andrena, Habropoda, Megachile, and Svastra), sampled in the same localities in central Texas. Unexpectedly, all four Xylocopa species had microbiomes dominated by bacterial lineages previously known only from social bees or other insect groups. Microbiomes were similar across three Xylocopa species and included lineages in the families Bifidobacteriaceae, Orbaceae, Lactobacillaceae, Pseudomonadaceae, and Enterobacteriaceae. In contrast, X. virginica had a distinct microbiome dominated by the genus Bombilactobacillus, a group abundant in guts of eusocial bees. Phylogenetic analyses support a past transfer of bacterial lineages into Xylocopa from bumble bees or honey bees. Gut microbiome compositions of Xylocopa species were distinct from those of other co-occurring solitary bees that had variable gut microbiomes dominated by bacteria from environmental sources. IMPORTANCE Gut microbiomes from social bees, such as honey bees and bumble bees, are conserved and consist of host-restricted bacteria that are transmitted among sterile female workers within a colony and that are important to the health of these key insect pollinators. In contrast, solitary bee species typically have more erratic, environmentally acquired microbiomes. Carpenter bees (genus Xylocopa) can be solitary as they lack a worker caste, and each female can excavate nests and raise offspring alone, although females are often social share nests at least in some species. This study showed that the gut microbiomes of four Xylocopa species have distinctive and consistent compositions and are dominated by bacterial lineages previously known from honey bees and bumble bees. Thus, eusociality is not required for bees to maintain a specialized, host-restricted gut microbiome. These findings suggest that gut bacteria are transmitted at shared nesting sites and that they play a role in host ecology.

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.

Structured Clinical Documentation to Improve Quality and Support Practice-Based Research in Headache.

OBJECTIVE: To use the electronic medical record (EMR) to optimize patient care, facilitate documentation, and support quality improvement and practice-based research, in a headache specialty clinic. BACKGROUND: Many physicians enter data into the EMR as unstructured free text and not as discrete data. This makes it challenging to use data for quality improvement or research initiatives. METHODS: We describe the process of building a customized structured clinical documentation support toolkit, specific for patients seen in a headache specialty clinic. The content was developed through frequent physician meetings to reach consensus on elements that define clinical Best Practices. Tasks were assigned to the care team and data mapped to the progress note. RESULTS: The toolkit collects hundreds of fields of discrete, standardized data. Auto scored and interpreted score tests include the Generalized Anxiety Disorder 7-item, Center for Epidemiology Studies Depression Scale, Migraine Disability Assessment questionnaire, Insomnia Sleep Index, and Migraine-Specific Quality of Life. We have developed Best Practice Advisories (BPA) and other clinical documentation support tools that alert physicians, when appropriate. As of April 1, 2018, we have used the toolkits at 4346 initial patient visits. We provide screenshots of our toolkits, details of data fields collected, and diagnoses of patients at the initial visit. CONCLUSIONS: The EMR can be used to effectively structure and standardize headache clinic visits for quality improvement and practice-based research. We are sharing our proprietary toolkit with other clinics as part of the Neurology Practice-Based Research Network. These tools are also facilitating clinical research enrollment and a pragmatic trial of comparative effectiveness at the point-of-care among migraine patients.

Structured clinical documentation in the electronic medical record to improve quality and to support practice-based research in epilepsy.

OBJECTIVE: Using the electronic medical record (EMR) to capture structured clinical data at the point of care would be a practical way to support quality improvement and practice-based research in epilepsy. METHODS: We describe our stepwise process for building structured clinical documentation support tools in the EMR that define best practices in epilepsy, and we describe how we incorporated these toolkits into our clinical workflow. RESULTS: These tools write notes and capture hundreds of fields of data including several score tests: Generalized Anxiety Disorder-7 items, Neurological Disorders Depression Inventory for Epilepsy, Epworth Sleepiness Scale, Quality of Life in Epilepsy-10 items, Montreal Cognitive Assessment/Short Test of Mental Status, and Medical Research Council Prognostic Index. The tools summarize brain imaging, blood laboratory, and electroencephalography results, and document neuromodulation treatments. The tools provide Best Practices Advisories and other clinical decision support when appropriate. The tools prompt enrollment in a DNA biobanking study. We have thus far enrolled 231 patients for initial visits and are starting our first annual follow-up visits and provide a brief description of our cohort. SIGNIFICANCE: We are sharing these EMR tools and captured data with other epilepsy clinics as part of a Neurology Practice Based Research Network, and are using the tools to conduct pragmatic trials using subgroup-based adaptive designs.

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.

In Vivo Formation of Cubic Phase in Situ after Oral Administration of Cubic Phase Precursor Formulation Provides Long Duration Gastric Retention and Absorption for Poorly Water-Soluble Drugs.

Lipid-based liquid crystalline systems based on the combination of digestible and nondigestible lipids have been proposed as potential sustained release delivery systems for oral delivery of poorly water-soluble drugs. The potential for cubic phase liquid crystal formation to induce dramatically extended gastric retention in vivo has been shown previously to strongly influence the resulting pharmacokinetics of incorporated drug. In vitro studies showing the in situ formation of cubic phase from a disordered precursor comprising a mixture of digestible and nondigestible lipids under enzymatic digestion have also recently been reported. Combining both concepts, here we show the potential for such systems to form in vivo, increasing gastric retention, and providing a sustained release effect for a model poorly water-soluble drug cinnarizine. A mixture of phytantriol and tributyrin at an 85:15 mass ratio, shown previously to form cubic phase under the influence of digestion, induced a similar pharmacokinetic profile to that in the absence of tributyrin, but completely different from tributyrin alone. The gastric retention of the formulation, assessed using micro-X-ray CT imaging, was also consistent with the pharmacokinetic behavior, where phytantriol alone and with 15% tributyrin was greater than that of tributyrin in the absence of phytantriol. Thus, the concept of precursor lipid systems that form cubic phase in situ during digestion in vivo has been demonstrated and opens new opportunities for sustained release of poorly water-soluble drugs.

IKK regulates the deubiquitinase CYLD at the postsynaptic density.

K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca(2+)-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca(2+) in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca(2+), but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.

Infant formula as a solid lipid dose form for enhancement of the oral bioavailability of cannabidiol for paediatric patients.

Cannabinoids can save paediatric patients from harmful psychological conditions caused by epilepsy. However, the limited aqueous solubility of the drug presents a limitation to oral absorption and bioavailability. Previous studies have shown the enhancement of oral bioavailability for poorly water-soluble drugs using milk or milk-based products like infant formula as a novel lipid-based formulation, due to digestion of the lipids to enhance drug solubility that is particularly well suited to infants and in low economy settings. Therefore, this study has investigated the in vitro solubilisation enhancement of cannabidiol (CBD) in milk-based products during digestion using synchrotron small angle X-ray scattering, followed by pharmacokinetic studies to determine the relative oral bioavailability. The in vitro results, coupled with in vivo data, demonstrate a two-fold increase in the oral bioavailability of CBD in bovine milk as well as infant formula. The results of this study indicate the potential for infant formula to be considered as a novel formulation approach for CBD. Further study is encouraged for more drugs with infant formula to strengthen the correlation between the solubilisation of drug and their oral bioavailability.

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.

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.

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.

ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

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.

Controlling drug release by introducing lipase inhibitor within a lipid formulation.

Drug overdose connected to marketed pharmaceutical products, particularly opioids, occurs at an alarming rate. Novel strategies through innovative formulation approaches that reduce the likelihood of overdose while allowing safe therapeutic outcomes are urgently required. The current study provides a proof-of-concept for a new formulation approach by co-formulating drug with a lipase inhibitor within a solid lipid formulation in order to prevent or reduce the harmful effects of taking multiple doses of an oral solid dose form. Lipase inhibitor controlled-release (LICR) formulations were created using a simple hot melt method to co-formulate the inhibitor (orlistat) and ibuprofen, as the model drug, within the lipid matrix. The digestion and drug release kinetics were determined using an in vitro lipolysis model. Above a threshold level of orlistat there was decreased digestibility of multiple doses of the LICR formulations, leading to reduced drug release. Upon administration of the formulations in capsules to rats, the LICR formulation displayed the lowest exposure of ibuprofen during the pharmacokinetic studies. This novel formulation approach shows promise in preventing accidental drug overdose after oral administration of multiple doses of formulation.

Towards mesoporous silica as a pharmaceutical treatment for obesity - impact on lipid digestion and absorption.

Mesoporous silica particles (MSPs) are emerging as an interesting option to reduce calorific uptake as a treatment for obesity and other metabolic conditions. However, their further development under the pharmaceutical regulatory framework is hindered by poor understanding of the mechanisms by which they exert their effects. In the current study the interaction of MSPs with the lipid digestion process is investigated, specifically interactions with lipase enzymes and lipid digestion products as a key contributing factor to lipid absorption and calorific intake. The impact of exposing lipase to MSPs on the enzyme activity was assessed directly using the tributyrin digestion test. The extent of interaction of digestion products with MSPs was studied using selectively radiolabeled bile components and lipids, while the impact on in vivo absorption of lipids was studied by incorporation of radiolabelled lipid (triolein) into milk and administration with and without particles. The studies showed that particles that inhibited lipase activity also tended to interact more extensively with lipid digestion products. In vitro X-ray scattering studies revealed the interaction of some MSPs with lipid digestion products through changes in lipid self-assembly during digestion. The MSPs led to reduced lipid absorption in vivo compared to the control particles and MSP-free milk. While the specific properties of the MSPs that drive the differences between the behavior of MSPs during lipid digestion remain elusive, the studies highlight that interactions with the lipid digestion and absorption pathways are a likely mechanism for reducing calorific uptake.

Formation of Self-Assembled Mesophases During Lipid Digestion.

Lipids play an important role in regulating bodily functions and providing a source of energy. Lipids enter the body primarily in the form of triglycerides in our diet. The gastrointestinal digestion of certain types of lipids has been shown to promote the self-assembly of lipid digestion products into highly ordered colloidal structures. The formation of these ordered colloidal structures, which often possess well-recognized liquid crystalline morphologies (or "mesophases"), is currently understood to impact the way nutrients are transported in the gut and absorbed. The formation of these liquid crystalline structures has also been of interest within the field of drug delivery, as it enables the encapsulation or solubilization of poorly water-soluble drugs in the aqueous environment of the gut enabling a means of absorption. This review summarizes the evidence for structure formation during the digestion of different lipid systems associated with foods, the techniques used to characterize them and provides areas of focus for advancing our understanding of this emerging field.