Serum Protein Binding Research Articles

Overcoming Therapeutic Challenges of Antibiotic Delivery with Cubosome Lipid Nanocarriers.

Low discovery rates for new antibiotics, commercial disincentives to invest, and inappropriate use of existing drugs have created a perfect storm of antimicrobial resistance (AMR). This "silent pandemic" of AMR looms as an immense, global threat to human health. In tandem, many potential novel drug candidates are not progressed due to elevated hydrophobicity, which may result in poor intracellular internalization and undesirable serum protein binding. With a reducing arsenal of effective antibiotics, enabling technology platforms that improve the outcome of treatments, such as repurposing existing bioactive agents, is a prospective option. Nanocarrier (NC) mediated drug delivery is one avenue for amplifying the therapeutic outcome. Here, the performance of several antibiotic classes encapsulated within the lipid-based cubosomes is examined. The findings demonstrate that encapsulation affords significant improvements in drug concentration:inhibition outcomes and assists in other therapeutic challenges associated with internalization, enzyme degradation, and protein binding. We emphasize that a currently sidelined compound, novobiocin, became active and revealed a significant increase in inhibition against the pathogenic Gram-negative strain, Pseudomonas aeruginosa. Encapsulation affords co-delivery of multiple bioactives as a strategy for mitigating failure of monotherapies and tackling resistance. The rationale in optimized drug selection and nanocarrier choice is examined by transport modeling which agrees with experimental inhibition results. The results demonstrate that lipid nanocarrier encapsulation may alleviate a range of challenges faced by antibiotic therapies and increase the range of antibiotics available to treat bacterial infections.

Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-β-lactamase, metallo-β-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

Abstract 2055: Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications

Abstract Nucleic acid therapies, including oligonucleotide therapeutics (ONTs) and vaccines, have increased in development, respectively, since their inception as an emerging drug modality and after the first mRNA COVID-19 vaccine was granted EUA approval in 2020. RNA-based therapies, such as antisense oligonucleotides (ASO), RNAi, aptamers, and mRNA, are designed to modulate protein synthesis in patients and have applications in oncology, gene therapy, and infectious disease prevention. Strategies for ONT drug delivery have advanced over recent years with the goal of overcoming limitations when targeting extrahepatic tissues. Improving the “drug-likeness” of ONTs is facilitated by chemical modifications to the sugar-phosphate backbone and/or nitrogenous bases. Modifications are designed to increase ONT drug stability, uptake, and efficacy, but may increase the likelihood of toxicity. First generation chemical modifications include phosphorothioate (PS) modification of the phosphate portion of the backbone, whereas second generation modifications include adding substituent groups to the 2’ position of the ribose portion of the backbone, specifically 2’-O-methyl (OME), 2’-O-methoxy-ethyl (MOE), and 2’-fluoro modifications of RNA. Third generation modifications include peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino phosphoroamidate (PMO) modifications, and others. We have developed, optimized, and validated a library of monoclonal antibody reagents that detect these chemical modifications independent of nucleic acid composition, structure, strandedness, configuration, or platform. These “universal detection reagents” can be used in (1) immunohistochemistry (IHC) to determine biodistribution, (2) immunofluorescence (IF) to determine intracellular localization, (3) ELISA to determine anti-drug antibody binding, immunogenicity, and drug ranking, (4) immunoprecipitation to determine protein binding in serum and/or tissue lysates, (5) in vivo cell culture studies to determine potency, and (6) other immunoassays. While in situ hybridization (ISH) is an established method for the detection and localization of ONTs in cells and tissues, this method has limitations that include lack of utility (as unique probes must be designed individually for each ONT), high costs in time and materials to produce probes, the inability of ISH to bind and detect short ONT drug sequences, and difficulties encountered when repeated sequences are present within the ONT sequence. Here we report differential sensitivity and specificity of panels of antibodies specific for PS and MOE modifications. We demonstrate the performance of these antibodies by various in vitro assays that show the utility of these specialty reagents to facilitate the collection of analytical data to support ADMET studies useful to ensure ONT approval by regulatory agencies. Citation Format: Todd Giardiello, Amy Anderson, Renee Salvo, Carl Ascoli. Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2055.

Quantitative cross-species comparison of serum albumin binding of per- and polyfluoroalkyl substances from five structural classes.

Per- and polyfluoroalkyl substances (PFAS) are a class of over 8000 chemicals, many of which are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and the perfluoroalkyl ether acid congener bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model, and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive bioaccumulation and toxicity assessments for PFAS.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2472.

Further evidence for blood-to-brain influx of unconjugated bile acids by passive diffusion: Determination of their brain-to-serum concentration ratios in rats by LC/MS/MS

Bile acids (BAs) reside in the brain and are probably involved in some neurological disorders. The view that most of unconjugated BAs in the brain are derived across the blood–brain barrier from the periphery by passive diffusion depending on their hydrophobicity is currently dominant, but some studies have made conflicting claims. In this study, the correlation analysis between the rat brain and serum levels of unconjugated BAs with a wider range of hydrophobicity was conducted to obtain further evidence about the blood-to-brain influx of unconjugated BAs by passive diffusion. We first developed the precise, accurate and matrix effect-free LC/ESI-MS/MS methods for quantifying eight major unconjugated BAs in the rat brain and serum. Derivatization was employed for increasing the assay sensitivity and specificity. The analysis using these methods reproduced the strong positive correlations between the brain and serum levels, and significant higher concentrations in the serum than in the brain for all the unconjugated BAs. The BA with the higher logPow (hydrophobicity) had the higher brain-to-serum concentration ratio (mono- > di- > trihydroxy BAs). Furthermore, the hydrophobicity was considered as the stronger factor for the blood-to-brain influx of the BAs than the serum protein binding ratio. Thus, this study provided further evidence supporting that passive diffusion is the major mechanism for the blood-to-brain influx of the unconjugated BAs.

Antiretroviral drugs from multiple classes induce loss of excitatory synapses between hippocampal neurons in culture.

Introduction: Antiretroviral (ARV) drugs have improved prognoses for people living with HIV. However, HIV-associated neurocognitive disorders (HAND) persist despite undetectable viral loads. Some ARVs have been linked to neuropsychiatric effects that may contribute to HAND. Synapse loss correlates with cognitive decline in HAND and synaptic deficits may contribute to the neuropsychiatric effects of ARV drugs. Methods: Using an automated high content assay, rat hippocampal neurons in culture expressing PSD95-eGFP to label glutamatergic synapses and mCherry to fill neuronal structures were imaged before and after treatment with 25 clinically used ARVs. Results and Discussion: At a concentration of 10μM the protease inhibitors nelfinavir and saquinavir, the non-nucleoside reverse transcriptase inhibitors etravirine and the 8-OH metabolite of efavirenz, the integrase inhibitor bictegravir, and the capsid inhibitor lenacapavir produced synaptic toxicity. Only lenacapavir produced synapse loss at the nanomolar concentrations estimated free in the plasma, although all 4 ARV drugs induced synapse loss at Cmax. Evaluation of combination therapies did not reveal synergistic synaptic toxicity. Synapse loss developed fully by 24h and persisted for at least 3days. Bictegravir-induced synapse loss required activation of voltage-gated Ca2+ channels and bictegravir, etravirine, and lenacapavir produced synapse loss by an excitotoxic mechanism. These results indicate that select ARV drugs might contribute to neuropsychiatric effects in combination with drugs that bind serum proteins or in disease states in which synaptic function is altered. The high content imaging assay used here provides an efficient means to evaluate new drugs and drug combinations for potential CNS toxicity.

Assessment of bioactive peptides derived from laminin-111 as prospective breast cancer-targeting agents

Breast cancer remains a pressing public health issue primarily affecting women. Recent research has spotlighted bioactive peptides derived from laminin-111, implicated in breast tumor development. Remarkably, the sequences IKVAV, YIGSR, and KAFDITYVRLKF from the α1, β1, and γ1 chains, respectively, have garnered significant attention. This study aims to assess the potential of these radiolabeled peptides as targeting agents for breast cancer. The three peptides were synthesized using the Fmoc strategy, purified via reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized through mass spectrometry. Iodine-131 (131I) radiolabeling was performed using the chloramine T method, exhibiting high radiochemical yield and stability for [131I]I-YIKVAV and [131I]I-YIGSR. Conversely, [131I]I-KAFDITYVRLKF demonstrated low radiochemical yield and stability and was excluded from the biological studies. The lipophilicity of the compounds ranged from − 2.12 to − 1.10. Serum protein binding assay for [131I]I-YIKVAV and [131I]I-YIGSR reached ≅ 48% and ≅ 25%, respectively. Affinity for breast cancer cells was evaluated using MDA-MB-231 and MCF-7 tumor cell lines, indicating the affinity of the radiopeptides with these tumor cells. Ex vivo biodistribution profiles of the radiopeptides were assessed in the MDA-MB-231 breast tumor animal model, revealing tumor tissue accumulation, supported by a high tumor-to-contralateral muscle ratio and autoradiography. These results signify the effective penetration of YIKVAV and YIGSR into tumor tissue. Therefore, the synthesized α1 and β1 peptide fragments exhibit favorable characteristics as potential breast cancer-targeting agents, promising future exploration as radiopharmaceuticals for breast cancer.

HPLC method for the determination of thymoquinone in growth cell medium.

Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 μM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

Radiosynthesis Standardization and Preclinical Assessment of the [68Ga]Ga-DOTA-Ubiquicidin29-41: A Translational Study Targeting Differential Diagnosis of Infectious Processes.

Human bacterial infections significantly contribute to the increase in healthcare-related burdens. This scenario drives the study of novel techniques for the early and precise diagnosis of infectious processes. Some alternatives include Nuclear Medicine- and Molecular Imaging-based strategies. However, radiopharmaceuticals that are available for routine assessments are not specific to differentiating infectious from aseptic inflammatory processes. In this context, [68Ga]Ga-DOTA-Ubiquicidin29-41 was synthesized using an automated module and radiochemical; in vivo and in vitro studies were performed. The radiopharmaceutical remained stable in saline (up to 180 min) and in rodent serum (up to 120 min) with radiochemical purities > 99 and 95%, respectively. Partition coefficient and serum protein binding at 60 min were determined (-3.63 ± 0.17 and 44.06 ± 1.88%, respectively). Ex vivo biodistribution, as well as in vivo microPET/CT images in mice, showed rapid blood clearance with renal excretion and reduced uptake in other organs in Staphylococcus aureus-infected animals. Higher uptake was observed in the target as compared to the non-target tissue (p < 0.0001) at 60 min post administration. The presented in-human clinical case demonstrates uptake of the radiopharmaceutical by Staphyloccocus aureus bacteria. These results indicate the potential of [68Ga]Ga-DOTA-Ubiquicidin29-41 as a radiopharmaceutical that can be obtained in a hospital radiopharmacy for the diagnosis of infectious processes using PET/CT.

In vitro screening of per- and polyfluorinated substances (PFAS) for interference with seven thyroid hormone system targets across nine assays

The US Environmental Protection Agency is evaluating the ecological and toxicological effects of per- and polyfluorinated chemicals. A number of perfluorinated chemicals have been shown to impact the thyroid axis in vivo suggesting that the thyroid hormone system is a target of these chemicals. The objective of this study was to evaluate the activity of 136 perfluorinated chemicals at seven key molecular initiating events (MIE) within the thyroid axis using nine in vitro assays. The potential MIE targets investigated are Human Iodothyronine Deiodinase 1 (hDIO1), Human Iodothyronine Deiodinase 2 (hDIO2), Human Iodothyronine Deiodinase 3 (hDIO3), Xenopus Iodothyronine Deiodinase (xDIO3); Human Iodotyrosine Deiodinase (hIYD), Xenopus Iodotyrosine Deiodinase (xIYD), Human Thyroid Peroxidase (hTPO); and the serum binding proteins Human Transthyretin (hTTR) and Human Thyroxine Binding Globulin (hTBG). Of the 136 PFAS chemicals tested, 85 had sufficient activity to produce a half-maximal effect concentration (EC50) in at least one of the nine assays. In general, most of these PFAS chemicals did not have strong potency towards the seven MIEs examined, apart from transthyretin binding, for which several PFAS had potency similar to the respective model inhibitor. These data sets identify potentially active PFAS chemicals to prioritize for further testing in orthogonal in vitro assays and at higher levels of biological organization to evaluate their capacity for altering the thyroid hormone system and causing potential adverse health and ecological effects.

Probing the biomolecular interactions of oxidized Ti3C2Tx nanosheets: How surface chemistry influences serum protein binding

Ti3C2Tx has promising applications in the biomedical field due to its biocompatibility, hydrophilicity, and antibacterial activity. However, its effects on health are still being explored. This study analyzes the interaction of bovine serum albumin (BSA) with Ti3C2Tx at different levels of oxidation. Zeta potential measurements showed that the colloidal stability of Ti3C2Tx varied with the degree of oxidation, with unoxidized Ti3C2Tx (U-Ti3C2Tx) being the most stable, followed by fully oxidized Ti3C2Tx (F-Ti3C2Tx) and partially oxidized Ti3C2Tx (P-Ti3C2Tx). UV–Vis absorption and fluorescence spectroscopy confirmed that the formation of Ti3C2Tx-BSA complexes resulted in fluorescence quenching. In addition, synchronous fluorescence spectroscopy, Fourier transform infrared spectroscopy and circular dichroism showed that the interaction between F-Ti3C2Tx and BSA altered the secondary conformation of BSA, and that U-Ti3C2Tx had a weaker effect on the conformation of BSA. Molecular docking results emphasized that hydrogen bonding is the main driving force between Ti3C2Tx and BSA.

Radiochemical and biological assessments of a PSMA-I&S cold kit for fast and inexpensive 99mTc-labeling for SPECT imaging and radioguided surgery in prostate cancer.

The expression of prostate-specific membrane antigen (PSMA) is upregulated in prostate cancer (PCa) cells and PSMA-ligands have been radiolabeled and used as radiopharmaceuticals for targeted radionuclide therapy (TRT), single photon emission computed tomography (SPECT) or positron emission tomography (PET) molecular imaging, and radioguided surgery in PCa patients. Herein, we aimed at radiolabeling the PSMA-I&S cold kit with 99mTc, resulting in a radiopharmaceutical with high radiochemical yield (RCY) and stability for SPECT imaging and radioguided surgery in PCa malignancies. Various pre-clinical assays were conducted to evaluate the [99mTc]Tc-PSMA-I&S obtained by the cold kit. These assays included assessments of RCY, radiochemical stability in saline, lipophilicity, serum protein binding (SPB), affinity for LNCaP-PCa cells (binding and internalization studies), and ex vivo biodistribution profile in naive and LNCaP-PCa-bearing mice. The radiopharmaceutical was obtained with good RCY (92.05% ± 2.20%) and remained stable for 6h. The lipophilicity was determined to be -2.41 ± 0.06, while the SPB was ∼97%. The binding percentages to LNCaP cells were 9.41% ± 0.57% (1h) and 10.45% ± 0.45% (4h), with 63.12 ± 0.93 (1h) and 65.72% ± 1.28% (4h) of the bound material being internalized. Blocking assays, employing an excess of unlabeled PSMA-I&S, resulted in a reduction in the binding percentage by 2.6 times. The ex vivo biodistribution profile confirmed high accumulation of [99mTc]Tc-PSMA-I&S in the tumor and the tumor-to-contralateral muscle ratio was ∼6.5. In conclusion, [99mTc]Tc-PSMA-I&S was successfully obtained by radiolabeling the cold kit using freshly eluted [99mTc]NaTcO4, exhibiting good RCY and radiochemical stability. The preclinical assays demonstrated that the radiopharmaceutical shows favorable characteristics for SPECT imaging and radioguided surgery in PCa patients.

THU610 Distribution Of Cortisol In Human Serum In Vitro: Role Of Competitive Ligand-protein Interactions In Women On Oral Contraceptives

Abstract Disclosure: R.I. Dorin: None. I. Perogamvros: None. C.R. Qualls: None. Background: The relationship between serum concentrations of total (XTotF) and free (XF) cortisol (test tube equilibrium at 37°C) is influenced by many factors. These include concentrations and affinities of serum binding proteins (BP), such as CBG and albumin (A). Among women taking oral contraceptives (OC), XF was found to be higher than predicted using ligand-BP association parameters developed in healthy volunteers (HV). We hypothesized that ligand(s) (XP) competing for XF binding to CBG may contribute to altered relationships between XTotF and XF observed in OC relative to HV. Methods: Feldman’s system of iterative, non-linear equilibrium equations1 were developed in (nxm) matrix format. Data included measured XTotF and XF at each time point (0-480 min) after 20 mg hydrocortisone2. Groups were stratified by (i) mode of administration (iv vs. po) and (ii) condition (HV, n=13 vs. OC, n=12). The matrix was interrupted to obtain iterative equilibrium solutions for parameters of interest using (1x2) minimal model (MM) and (2x2) ligand competition model (LCM). Solutions were optimized by minimizing least squares differences between measured and model predicted XF. Albumin-bound cortisol was parameterized as a constant ratio of XF (N) to simplify the matrix problem. Comparisons and interactions within the 2x2x2 design (delivery, group, and model) were analyzed by ANOVA. Results: In OC, LCM provided significantly better fit to measured XF relative to MM (13 vs. 19% error, P=0.003). MM solutions for CBG-cortisol affinities were significantly higher in HV (KD=26.6) vs. OC (KD=71.9 nmol/L), but LCM yielded similar affinities in both groups (KD = 23.9 in HV and 16.1 nmol/L in OC, interaction P&amp;lt;0.001). LCM solutions for concentrations and CBG-binding affinities of XP were significantly increased in OCP vs. HV (both P&amp;lt;0.001). LCM solutions for HV yielded higher N for po (3.1±1.2) vs. iv (1.4±0.4) (P=0.007). Limitations: Model solutions depend on the precision and accuracy of measurements made in clinical samples. Although the competitor (XP) is treated as a single compound in LCM, it may represent the combined effects (e.g., harmonic means) of multiple ligands. Conclusions: (i) matrix notation was useful to development of non-linear, iterative equations for competitive ligand-BP interactions, which were applicable to both forward and inverse solutions of the matrix problems (ii) LCM provided a better fit to experimental data for OC compared to MM, providing (indirect) evidence that competing ligand(s) (XP) influence the distribution of cortisol in serum samples obtained from women on OC, (iii) inter- and intra-subject variability for N was observed for both models, suggesting that assignment of a single population value for cortisol-albumin binding constant may be unrealistic.

Prediction of Human Pharmacokinetics of Phosphorodiamidate Morpholino Oligonucleotides in Duchenne Muscular Dystrophy Patients Using Viltolarsen.

Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike other nucleic acids, are not negatively charged. Thus, PMOs differ from other ASOs in their pharmacokinetic (PK) properties. Drugs with a PMO backbone have been administered to Duchenne muscular dystrophy pediatric patients; however, appropriate methodologies are not currently available to predict their human PK from nonclinical data. In this study, we used viltolarsen as a representative PMO to investigate the applicability of the allometric scaling approach to human PK prediction. We first summarized the nonclinical and clinical PK data for viltolarsen as showing high total clearance, low serum protein binding, metabolic resistance, and urinary excretion as the unchanged drug in both animals and humans. We then investigate the PK of viltolarsen in mice, rats, cynomolgus monkeys, and dogs and used the results, with body weight, to extrapolate to humans by several methods. The estimate of human total clearance obtained from cynomolgus monkeys was the best, and body weight may be the key factor in accurately predicting human total clearance. In contrast, all of the well-known prediction methods for the volume of distribution at steady state gave underestimates. However, the human PK profiles predicted from the PK parameters in cynomolgus monkeys fit the observed human plasma concentrations well. These results are expected to contribute to the further development of PMOs. SIGNIFICANCE STATEMENT: We investigated how to predict the human PK of phosphorodiamidate morpholino oligomers from nonclinical data. The estimates of human PK parameters and profiles determined from cynomolgus monkeys by an allometric scaling approach were the most suitable, and the cynomolgus monkey body weight may be the key factor in accurately predicting human total clearance.

Abstract 5544: Association of circulating markers of gut barrier damage and inflammation with colorectal neoplasia stage

Abstract A disturbance in the microbiome facilitated by gut barrier dysfunction and inflammation can influence the progression of colorectal neoplasia. One of the most studied indicators of gut barrier status is lipopolysaccharide (LPS). Increased permeability of the gut barrier leads to extensive translocation of microbes into the lamina propria, which is associated with higher circulating levels of LPS. Additionally, increased serum concentrations of inflammatory cytokines including IFNy, IL6, IL10, and TNFα have been associated with impaired gut integrity and its progressive dysfunction. We investigated the role of gut barrier permeability and inflammation in colorectal cancer (CRC) development by assessing markers of intestinal barrier dysfunction and inflammation in two different patient cohorts; Irish (discovery) and Czech (validation). ELISA assays were used to assess circulating concentrations of lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP) in serum from patients with CRC (n=30), colorectal adenoma (n=40), and negative after colonoscopy/blood donor controls (n=35). Circulating levels of inflammatory cytokines and calprotectin were assessed using an MSD MultiSPOT platform and an MSD R-plex assay (Mesocale Discovery, Rockville, Md, United States), respectively. Differences between pathology groups were assessed using the Kruskal Wallis or Mann-Whitney U test. Receiver operating curve (ROC) analysis was implemented to identify single and combinations of biomarkers capable of discriminating between CRC patients and controls with maximum sensitivity (SE) and specificity (SP) (RStudio, PBC, Boston, MA URL http://www.rstudio.com/). In the discovery cohort, levels of LBP, TNFα, IL6, calprotectin and CRP were significantly higher in CRC patients than in controls after false discovery rate correction (p=0.02, p=0.03, p=0.006, p=0.01, p=0.006, respectively). The area under the ROC curve (AUC) was 0.76 (95% CI: 0.64-0.87, p = 0.003) for LBP, 0.69 (95% CI: 0.55-0.82, p = 0.007) for calprotectin and 0.87(95% CI: 0.74-1.00, p = 0.003) for CRP. The optimal biomarker combination for discriminating between CRC and controls was the combination of the three markers (SP= 0.75, SE = 0.75). In the validation cohort, levels of calprotectin and TNFα, were significantly higher in cancer compared to control (p=0.05, p=0.02, respectively). Furthermore, IFNγ and IL10 were also significantly elevated in cancer compared to control (p= 0.02, p=0.003, respectively). These results support the hypothesis that a dysfunctional gut barrier and attendant inflammation may influence CRC development and progression. Additionally, LBP, CRP and calprotectin may have potential as diagnostic biomarkers for CRC or for risk stratification. Gut bacterial and barrier wall modulation and stabilization has potential for preventing colorectal adenomas and hence colorectal cancer. Citation Format: Flavia Genua, Petr Holý, Pavel Souček, Václav Liška, David J. Hughes. Association of circulating markers of gut barrier damage and inflammation with colorectal neoplasia stage. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5544.

Abstract 2799: Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology

Abstract Transforming growth factor-beta (TGF-ß) is a master regulatory cytokine with pleotropic effects important in tumor development. TGF-ß1 binds TGF-ß receptor 2, which recruits and phosphorylates TGF-ß receptor 1 (TGFBR1). TGFBR1 in turn phosphorylates SMAD2 and SMAD3. Phosphorylated SMAD2/3 (pSMAD2/3) complexes with SMAD4 to translocate to the nucleus and enact transcriptional programs. In late-stage tumors, TGF-ß signaling is oncogenic, promoting growth, invasion, immune suppression, tissue remodeling, and epithelial to mesenchymal transition (EMT). In an effort to counter the oncogenic effects of TGF-ß signaling, Sumitomo Pharma Oncology, Inc. is developing TP-6379, an investigational, orally available, small molecule, selective TGFBR1 inhibitor. TP-6379 has a biochemical IC50 of 1.07 nM and has been shown to reduce pSMAD2 in Rhabdomyosarcoma cells with an IC50 of 108 nM. A dose dependent increase of TP-6379 was observed in plasma and tumors of A549 tumor bearing athymic nude mice (10, 50, 75, and 100 mg/kg PO). In another study using the same mouse model, pSMAD2 has shown an inverse correlation with pharmacokinetics showing &amp;gt; 90% inhibition for up to 4 hours (75 mg/kg PO). Pharmacodynamic (PD) biomarkers are critical for determining biological activity, but procuring serial tumor biopsies for PD analysis is challenging for many tumor types. We hypothesize that pSMAD2 can be measured in non-tumor tissues as a surrogate for activity within the tumor. Surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin punches, and even circulating tumor cells (CTCs) are much less invasive and can be sampled over multiple timepoints. We observed a measurable decrease of PD biomarkers pSMAD2/3 in PBMCs and skin after treatment with TP-6379 using a bead-based immunoassay (Luminex). Healthy human donor PBMCs treated ex vivo for 2 hours with up to 1 µM TP-6379 showed a 96% reduction in pSMAD2/3 signal. Although TP-6379 was still active in reducing pSMAD2 in PBMCs when treated in whole blood for 2 hours, it required around 30-fold more drug to achieve a similar effect, possibly due to serum protein binding. Skin samples from in vivo dosed tumor bearing mice also show significant inhibition of pSMAD2/3, which correlated with significant pSMAD2/3 inhibition in tumors from both EMT6 and 4T1 syngeneic triple-negative breast cancer mouse tumor models. We confirmed that the Luminex assay is sensitive enough to reliably measure pSMAD2/3 in healthy human doner skin punches down to 3 mm in diameter. Lastly, CTCs from breast cancer patients’ whole blood treated ex vivo for 24 hours with as low as 1 µM TP-6379 showed a decrease in pSMAD2 and EMT marker SNAI1 by as much as 96% and 86%, respectively, when measured by immunofluorescence. In summary, our research has shown that pSMAD2/3 and EMT marker SNAI1 are valid PD biomarkers for TP-6379 therapy. We propose that these biomarkers would be best measured clinically using PBMCs, CTCs and/or skin punches. Citation Format: Richard E. Heinz, Curtis A. Allred, David A. Kircher, Yuta Matsumura, Bettina Franz, Zhizhou Ye, Jinny Lee, Clifford J. Whatcott, Jason M. Foulks, Adam Siddiqui, Steven L. Warner. Pharmacodynamic biomarkers for TGFBR1 inhibition in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2799.

[Protective role and mechanism of tubastatin A on renal and intestinal injuries after cardiopulmonary resuscitation in swine].

To investigate the protective effect and potential mechanism of tubastatin A (TubA), a specific inhibitor of histone deacetylase 6 (HDAC6), on renal and intestinal injuries after cardiopulmonary resuscitation (CPR) in swine. Twenty-five healthy male white swine were divided into Sham group (n = 6), CPR model group (n = 10) and TubA intervention group (n = 9) using a random number table. The porcine model of CPR was reproduced by 9-minute cardiac arrest induced by electrical stimulation via right ventricle followed by 6-minute CPR. The animals in the Sham group only underwent the regular operation including endotracheal intubation, catheterization, and anesthetic monitoring. At 5 minutes after successful resuscitation, a dose of 4.5 mg/kg of TubA was infused via the femoral vein within 1 hour in the TubA intervention group. The same volume of normal saline was infused in the Sham and CPR model groups. Venous samples were collected before modeling and 1, 2, 4, 24 hours after resuscitation, and the levels of serum creatinine (SCr), blood urea nitrogen (BUN), intestinal fatty acid binding protein (I-FABP) and diamine oxidase (DAO) in serum were determined by enzyme-linked immunoadsordent assay (ELISA). At 24 hours after resuscitation, the upper pole of left kidney and terminal ileum were harvested to detect cell apoptosis by TdT-mediated dUTP-biotin nick end labeling (TUNEL), and the expression levels of receptor-interacting protein 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) were detected by Western blotting. After resuscitation, renal dysfunction and intestinal mucous injury were observed in the CPR model and TubA intervention groups when compared with the Sham group, which was indicated by significantly increased levels of SCr, BUN, I-FABP and DAO in serum. However, the serum levels of SCr and DAO starting 1 hour after resuscitation, the serum levels of BUN starting 2 hours after resuscitation, and the serum levels of I-FABP starting 4 hours after resuscitation were significantly decreased in the TubA intervention group when compared with the CPR model group [1-hour SCr (μmol/L): 87±6 vs. 122±7, 1-hour DAO (kU/L): 8.1±1.2 vs. 10.3±0.8, 2-hour BUN (mmol/L): 12.3±1.2 vs. 14.7±1.3, 4-hour I-FABP (ng/L): 661±39 vs. 751±38, all P < 0.05]. The detection of tissue samples indicated that cell apoptosis and necroptosis in the kidney and intestine at 24 hours after resuscitation were significantly greater in the CPR model and TubA intervention groups when compared with the Sham group, which were indicated by significantly increased apoptotic index and markedly elevated expression levels of RIP3 and MLKL. Nevertheless, compared with the CPR model group, renal and intestinal apoptotic indexes at 24 hours after resuscitation in the TubA intervention group were significantly decreased [renal apoptosis index: (21.4±4.6)% vs. (55.2±9.5)%, intestinal apoptosis index: (21.3±4.5)% vs. (50.9±7.0)%, both P < 0.05], and the expression levels of RIP3 and MLKL were significantly reduced [renal tissue: RIP3 protein (RIP3/GAPDH) was 1.11±0.07 vs. 1.39±0.17, MLKL protein (MLKL/GAPDH) was 1.20±0.14 vs. 1.51±0.26; intestinal tissue: RIP3 protein (RIP3/GAPDH) was 1.24±0.18 vs. 1.69±0.28, MLKL protein (MLKL/GAPDH) was 1.38±0.15 vs. 1.80±0.26, all P < 0.05]. TubA has the protective effect on alleviating post-resuscitation renal dysfunction and intestinal mucous injury, and its mechanism may be related to inhibition of cell apoptosis and necroptosis.

A Study on Pharmacokinetic Functionalities and Safety Margins of an Optimized Simvastatin Nanoformulation.

A pharmaceutical formulation with favorable pharmacokinetic parameters is more likely to be efficacious and safe to overcome the failures of the drug resulting from lack of efficacy, poor bioavailability, and toxicity. In this view, we aimed to evaluate the pharmacokinetic functionalities and safety margin of an optimized CS-SS nanoformulation (F40) by in vitro/in vivo methods. The everted sac technique was used to evaluate the improved absorption of a simvastatin formulation. In vitro protein binding in bovine serum and mice plasma was performed. The formulation's liver and intestinal CYP3A4 activity and metabolic pathways were investigated by the qRT-PCR technique. The excretion of cholesterol and bile acids was measured to demonstrate the formulation's cholesterol depletion effect. Safety margins were determined by histopathology as well as fiber typing studies. In vitro protein binding results revealed the existence of a high percentage of free drugs (22.31 ± 3.1%, 18.20 ± 1.9%, and 16.9 ± 2.2%, respectively) compared to the standard formulation. The controlled metabolism in the liver was demonstrated from CYP3A4 activity. The formulation showed enhanced PK parameters in rabbits such as a lower Cmax, clearance, and a higher Tmax, AUC, Vd, and t1/2. qRT-PCR screening further proved the different metabolic pathways followed by simvastatin (SREBP-2) and chitosan (PPAR-γ pathway) in the formulation. The results from qRT-PCR and histopathology confirmed the toxicity level. Hence, this pharmacokinetic profile of the nanoformulation proved it has a unique synergistic hypolipidemic modality.

Can blood proteome diversity among fish species help explain perfluoroalkyl acid trophodynamics in aquatic food webs?

Per- and polyfluoroalkyl substances (PFAS) are a diverse family of industrially significant synthetic chemicals infamous for extreme environmental persistence and global environmental distribution. Many PFAS are bioaccumulative and biologically active mainly due to their tendency to bind with various proteins. These protein interactions are important in determining the accumulation potential and tissue distribution of individual PFAS. Trophodynamics studies including aquatic food webs present inconsistent evidence for PFAS biomagnification. This study strives to identify whether the observed variability in PFAS bioaccumulation potential among species could correspond with interspecies protein composition differences. Specifically, this work compares the perfluorooctane sulfonate (PFOS) serum protein binding potential and the tissue distribution of ten perfluoroalkyl acids (PFAAs) detected in alewife (Alosa pseudoharengus), deepwater sculpin (Myoxocephalus thompsonii), and lake trout (Salvelinus namaycush) of the Lake Ontario aquatic piscivorous food web. These three fish sera and fetal bovine reference serum all had unique total serum protein concentrations. Serum protein-PFOS binding experiments showed divergent patterns between fetal bovine serum and fish sera, suggesting potentially two different PFOS binding mechanisms. To identify interspecies differences in PFAS-binding serum proteins, fish sera were pre-equilibrated with PFOS, fractionated by serial molecular weight cut-off filter fractionation, followed by liquid chromatography–tandem mass spectrometry analysis of the tryptic protein digests and the PFOS extracts of each fraction. This workflow identified similar serum proteins for all fish species. However, serum albumin was only identified in lake trout, suggesting apolipoproteins are likely the primary PFAA transporters in alewife and deepwater sculpin sera. PFAA tissue distribution analysis provided supporting evidence for interspecies variations in lipid transport and storage, which may also contribute to the varied PFAA accumulation in these species. Proteomics data are available via ProteomeXchange with identifier PXD039145.