Plasma Binding Research Articles

Antisense Oligonucleotide In Vitro Protein Binding Determination in Plasma, Brain, and Cerebral Spinal Fluid Using Hybridization LC-MS/MS.

The development of quantitative models for prediction of drug pharmacokinetics based on in vitro data has transformed early drug discovery. Drug unbound fraction (ƒu) characterization is a key consideration in pharmacokinetic and pharmacodynamic (PK/PD) modeling, assuming only unbound drug can interact with the target, and therefore has direct implications in the efficacy and potential toxicity of the drug. The current study describes the implementation of a hybridization liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for the direct quantitation of antisense oligonucleotide (ASO) ƒu The method provides substantial improvements, including minimal matrix effects and high specificity when compared with previously used oligonucleotide ƒu detection methods such as ligand binding assays or liquid scintillation. The hybridization LC-MS/MS platform was integrated with ultracentrifugation, ultrafiltration, and equilibrium dialysis, and method performance for each technique was evaluated. Although ASO protein binding has been previously characterized in plasma, there were no studies that quantitated ASO ƒu in brain or cerebral spinal fluid (CSF). As ASOs continue to undergo clinical trials for neurologic and neuromuscular indications, ƒu characterization in brain and CSF can provide invaluable information about ASO distribution and target engagement in the central nervous system, therefore providing support for in vivo PK/PD data characterization. SIGNIFICANCE STATEMENT: A novel hybridization LC-MS/MS-based approach was successfully developed for the determination of ASO in vitro protein binding in plasma, and for the first time brain and CSF. Ultrafiltration, equilibrium dialysis, and ultracentrifugation were assessed for the separation of unbound ASO from biological matrices. The hybridization LC-MS/MS platform provided unique advantages, including minimal matrix effects and high specificity, compared with traditional ligand binding assays or liquid scintillation approaches, which enabled efficient and reliable in vitro protein binding assay.

Clinical Pharmacokinetics and Pharmacodynamics of Roxadustat.

The pharmacokinetics of roxadustat are well characterized, with an apparent volume of distribution after oral administration of 22–57 L, apparent clearance of 1.2–2.65 L/h, and renal clearance of 0.030–0.026 L/h in healthy volunteers; the elimination half-life is 9.6–16 h. Plasma binding is 99% and the fraction eliminated by hemodialysis is 2.34%. As an interpretation of the pharmacodynamics of roxadustat, we proposed a concept with a hypothetical cascade of two subsequent effects, first on erythropoetin (EPO) and second on hemoglobin (delta Hb). The primary effect on EPO is observed within a few hours after roxadustat administration and can be modeled using the sigmoidal Hill equation. The concentration at half-maximum effect can be inferred at 10–36 µg/mL, the Hill coefficient at 3.3, and the effect bisection time at 10–17 h, corresponding to EPO half-life. The subsequent effect on hemoglobin (delta Hb) is observed after several weeks and can be interpreted as an irreversible, dose proportional, unsaturable effect, continuing in agreement with the lifespan of red blood cells of 63–112 days.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01095-x.

1111. Therapeutic Drug Monitoring of Colistin in Cerebrospinal Fluid in the Treatment of Neurosurgical Meningitis caused by Pseudomonas aeruginosa and KPC-producing Enterobacterales

BackgroundCentral nervous system (CNS) infections caused by carbapenem-resistant Enterobacterales (CRE) and Difficult-to-treat resistant (DTR)-P. aeruginosa (PA) present a therapeutic dilemma. Therapies are limited due to antibiotic resistance and inadequate CNS diffusion. Intraventricular polymyxins are utilized in this setting despite a lack in pharmacokinetic data after CNS injection. We describe the utilization of intravenous and intrathecal polymyxin E [colistimethate (CMS)] therapeutic drug monitoring (TDM) in 3 cases of post-neurosurgical meningitis. MethodsBacterial identification and susceptibility testing were performed using MicroScan. TDM was employed by dosing CMS at 125,000 IU (i.e., 4.1 mg CBA or 10 mg) administered via external ventricular drain twice daily and 4.5 MIU (133.2 CBA or 360 mg) CMS administered over 30 minutes IV twice daily. Four pairs of CSF and blood samples were collected for each patient (Table 1). Samples were placed on ice to minimize in-vitro conversion of CMS to Colistin. Colistin binding in plasma and CSF was measured using ultracentrifugation. Concentrations of CMS and Colistin in CSF and human plasma were determined by liquid chromatography/mass spectrometry. Patients A, B and C received 20, 15, and 12 doses of CMS, respectively, prior to TDM.ResultsBacterial cultures revealed DTR PA, blaKPCE. cloacae and blaOXA-48K. pneumoniae for patients A, B and C, respectively. Colistin minimum inhibitory concentrations (MIC) were 0.5 µg/ml, 0.125 µg/ml, and 0.125 µg/ml, respectively. The measured CSF and plasma concentrations of CMS, Colistin, and binding are shown in Table 1. Clinical resolution and microbiological cure were attained in all patients. Therapeutic Drug Monitoring of Unchanged CMS and Formed Colistin in CSF samples for patient A, B, and C Therapeutic Drug Monitoring of Unchanged CMS and Formed Colistin in Plasma Samples for patient A, B, and C ConclusionFavorable concentrations of formed Colistin and CMS in CSF were achieved in 3 patients with complicated CNS infection. To the best of our knowledge, this is the first study to report the binding of Colistin in CSF in humans. A TDM method was effectively applied to demonstrate that Colistin achieves and maintains the PK/PD target (fAUC/MIC) [ratio of area under the plasma concentration curve of unbound drug to MIC] that best correlates with killing activity. Overall, our results support intraventricular polymyxins for treating DTR Gram-negative CNS infections.Disclosures Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

Protease Domain Exosites Regulate Plasma Recovery and Extravascular Distribution of Factor IX(a) Variants in Hemophilic Mice

Introduction:Among the coagulation protease, factor IX (FIX) is unique in that a substantial pool of extravascular factor exists that contributes to in vivo hemostasis. Extravascular distribution of FIX contributes to poor plasma recovery of infused FIX in hemophilia B (HB). The effect of mutations in the heparin (K126A/K132A) and antithrombin (R150A) binding exosites of FIX and FIXa on the distribution of these variants between the plasma and extravascular binding sites was examined in HB and hemophilia A (HA) mice, respectively.Methods:Recombinant human FIX wild type (18 U/kg) or equimolar FIX K126A/K132A was administered to HB mice (n=8) via tail vein injection with sacrifice 5 min post-injection following retro-orbital blood collection. Equimolar doses of human FIXa wild type (WT), K126A/K132A and R150A were similarly injected into HA mice (n=4-8), with blood collection and sacrifice 5 min post-injection. Plasma was isolated by centrifugation, organs (liver, spleen, kidneys, heart, lung, and brain) harvested, rinsed with PBS, weighed, with a portion made into tissue lysates and remainder frozen. FIX(a) content of tissue lysates was determined by a species-specific human FIX(a) ELISA with variant-specific standard curves. Tissue concentration was determined by total FIX(a) present in lysate (ng) divided by respective tissue weight (mg). Organ distribution was determined by extrapolating FIX(a) tissue concentration to total organ weight. The FIX(a) present in a specific organ was divided by the administered FIX(a) dose to determine the % organ distribution.Results:PK studies in HB mice demonstrated that FIX WT and R150A demonstrated a similar pattern and time course of elimination. In contrast, FIX K126A/K132A demonstrated ~2.4 fold higher plasma recovery relative to FIX WT. Based on plasma concentrations at 5 min post-injection (plasma volume 40 ml/kg), ~12.9% of FIX WT and 30.5% of FIX K126A/K132A was localized to the plasma compartment. Tissue lysates from the liver, spleen, kidney and brain demonstrated that liver had the highest FIX content by far, with ~41% of the FIX WT dose at 5 min post-injection. Other tissues demonstrated markedly less FIX WT content, including kidney (1%), spleen (0.1%) and brain (undetectable). FIX tissue concentration was significantly higher in the liver (0.69 ng/mg tissue) compared to other tissues, followed by kidney (0.07 ng/mg), spleen (0.02 ng/mg) and brain (undetectable). Comparison of FIX K126A/K132A tissue content to WT demonstrated reduced liver localization (25% dose) with concomitant reduction in tissue concentration (0.45 ng/mg). In contrast, FIX K126A/K132A increased localization to the kidney (1.7%) and spleen (0.37%) relative to WT.In protease PK studies, FIXa K126A/K132A and R150A both enhanced plasma recovery (2.2-2.5 fold) in HA mice compared to FIXa WT. Based on plasma concentrations at 5 min post-injection, ~16.1% of FIXa WT and 41.1% of FIXa K126A/K132A dose localized in the plasma compartment. Similar to zymogen, tissue lysates demonstrated that liver had the highest FIXa WT content (28%). Other tissues contained markedly less FIXa, including kidney (2.4%), spleen (0.2%), heart (0.5%) and brain (undetectable). Similarly, FIXa tissue concentration was 4-fold higher in the liver (0.47 ng/mg) compared to other tissues, followed by kidney (0.12 ng/mg), spleen and heart (0.06 ng/mg) and brain (undetectable). Localization of FIXa K126A/K132A to the liver was reduced by nearly half compared to FIXa WT (16%) with reduction in liver tissue concentration (0.29 ng/mg). FIXa K126A/K132A had increased localization to the kidney (3.3%) relative to WT. In contrast, FIXa R150A (not shown) had similar liver distribution (31.9% dose) and tissue concentration (0.53 ng/mg) to FIXa WT, despite increased localization to the plasma (36.2%).Conclusions:Liver is the predominant organ for extravascular FIX binding and the heparin binding exosite contributes to this localization, similar to the collagen IV binding site in the Gla domain. Heparan sulfate and collagen IV co-localize in the basement membrane suggesting synergistic roles. FIXa also localizes to extravascular sites via the heparin-binding exosite, although that binding may be modestly limited by endogenous FIX in the HA mouse. Disruption of the antithrombin binding exosite re-distributes FIXa to the plasma compartment by an independent mechanism, as liver content is unchanged. DisclosuresSheehan: Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; BioMarin: Consultancy, Research Funding.

TRIM31 facilitates K27-linked polyubiquitination of SYK to regulate antifungal immunity

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

Fulvestrant-3-Boronic Acid (ZB716) Demonstrates Oral Bioavailability and Favorable Pharmacokinetic Profile in Preclinical ADME Studies.

Fulvestrant-3-boronic acid (ZB716), an oral selective estrogen receptor degrader (SERD) under clinical development, has been investigated in ADME studies to characterize its absorption, metabolism, and pharmacokinetics. ZB716 was found to have high plasma protein binding in human and animal plasma, and low intestinal mucosal permeability. ZB716 had high clearance in hepatocytes of all species tested. ZB716 was metabolized primarily by CYP2D6 and CYP3A. In human liver microsomes, ZB716 demonstrated relatively low inhibition of CYP1A2, 2C8, 2C9, 2C19, 2D6, and 3A4 (when testosterone was used as the substrate), and no inhibition of CYP2B6 and 3A4 (when midazolam was used as the substrate). In assays for enzyme activity, ZB716 induced CYP1A2, 2B6, and 3A4 in a concentration-dependent manner. Single-dose and repeated-dose pharmacokinetic studies in rats and dogs showed oral bioavailability, dose-proportional drug exposure, and drug accumulation as measured by maximum concentration and area under the concentration–time curve (AUC).

A New Guidance for the Prediction of Hepatic Clearance in the Early Drug Discovery and Development from the in Vitro-to-in Vivo Extrapolation Method and an Approach for Exploring Whether an Albumin-Mediated Hepatic Uptake Phenomenon Could be Present Under in Vivo Conditions.

The in vitro-to-in vivo extrapolation (IVIVE) methods for predicting the hepatic clearance (CL) of drugs based on microsomal or hepatocyte data have certainly advanced; however, there is still place for improving the extrapolations from in vitro assays containing no plasma proteins. Accordingly, there is a discussion on whether the free drug hypothesis or an albumin (ALB)-mediated hepatic uptake phenomenon is the best scaling method. Therefore, the objectives of this study were to guide the prediction of CL and to diagnose which scaling method between the free drug hypothesis and ALB-mediated uptake could be more accurate; this, irrespective of the mechanism(s) governing CL if the drugs can get to the hepatocyte membrane. The analysis of several datasets demonstrated that almost all values of CL in vivo fall within the two calculated values of CL use as boundaries from: 1) the free drug hypothesis, and 2) ALB-mediated uptake. The average value from these two CL boundaries predicted the CL in vivo with an incredible accuracy. Validating these boundaries in preclinical species prior going to human as well as considering the fractional binding in plasma increased the accuracy. Overall, this study is another step towards guiding the CL prediction in drug discovery and development.

Effect of severe renal impairment on the pharmacokinetics of brigatinib

SummaryBackground Brigatinib, a next-generation anaplastic lymphoma kinase (ALK) inhibitor, targets activated, mutant forms of ALK and overcomes mechanisms of resistance to the ALK inhibitors crizotinib, ceritinib, and alectinib. Brigatinib is approved in multiple countries for treatment of patients with ALK-positive non–small cell lung cancer. Based on population pharmacokinetic (PK) analyses, no dosage adjustment is required for patients with mild or moderate renal impairment. Methods An open-label, single-dose study was conducted to evaluate the PK of brigatinib (90 mg) in patients with severe renal impairment (estimated glomerular filtration rate < 30 mL/min/1.73 m2; n = 8) and matched healthy volunteers with normal renal function (estimated glomerular filtration rate ≥ 90 mL/min/1.73 m2; n = 8). Plasma and urine were collected for the determination of plasma protein binding and estimation of plasma and urine PK parameters. Results Plasma protein binding of brigatinib was similar between patients with severe renal impairment (92 % bound) and matched healthy volunteers with normal renal function (91 % bound). Unbound brigatinib exposure (area under the plasma concentration-time curve from time zero to infinity) was approximately 92 % higher in patients with severe renal impairment compared with healthy volunteers with normal renal function. The renal clearance of brigatinib in patients with severe renal impairment was approximately 20 % of that observed in volunteers with normal renal function. Conclusions These findings support a brigatinib dosage reduction of approximately 50 % in patients with severe renal impairment.Trial registry: Not applicable.

Nonspecific Binding Considerations in the Rational Design and Development of Small Molecule COVID-19 Therapeutics.

In their recent manuscript, Boffito et al. caution readers not to forget the fundamentals of pharmacology in the rush to identify antiviral therapeutics to combat the ongoing COVID-19 pandemic[1]. Emphasis on the appropriate use of nonspecific binding data is warranted since the underlying, oft-misunderstood concepts are critically important to effective research and development.

Successful treatment by doravirine with cobicistat-boosted darunavir for end-stage renal failure under chronic haemodialysis

Sir, Doravirine is an NNRTI that was newly approved for use in Japan on 14 January 2020. The pharmacokinetics of doravirine are not greatly influenced by sex, age, race or hepatic impairment.1,2 Doravirine is primarily metabolized by cytochrome P450 3A4 and its clearance is reduced by strong inhibitors of this enzyme. However, no dose adjustment is recommended when administered in combination with PIs such as ritonavir or cobicistat.3 In addition, no dose adjustment is required for renal impairment;4 however, pharmacokinetic data during haemodialysis (HD) remain quite limited. Furthermore, doravirine has a relatively low molecular weight (425.75 g/mol, which is <500 g/mol) and exhibits low protein binding in plasma (0.76, which is <0.90),5 which suggests that doravirine may be removed by dialysis.6 Here, we present one case of end-stage renal failure treated with a doravirine-containing regimen where drug concentrations were measured during HD. This...

Population model analysis of chiral inversion and degradation of bupropion enantiomers, and application to enantiomer specific fraction unbound determination in rat plasma and brain

Pharmacologic effects elicited by drugs most directly relate to their unbound concentrations. Measurement of binding in blood, plasma and target tissues are used to estimate these concentrations by determining the fraction of total concentration in a biological matrix that is not bound. In the case of attempting to estimate R- and S-bupropion concentrations in plasma and brain following racemic bupropion administration, reversible chiral inversion and irreversible degradation of the enantiomers were hypothesized to confound attempts at unbound fraction estimation. To address this possibility, a kinetic modeling approach was used to quantify inversion and degradation specific processes for each enantiomer from separate incubations of each enantiomer in the two matrices, and in pH 7.4 buffer, which is also used in binding experiments based on equilibrium dialysis. Modeling analyses indicated that chiral inversion kinetics were two to four-fold faster in plasma and brain than degradation, with only inversion observed in buffer. Inversion rate was faster for S-bupropion in the three media; whereas, degradation rates were similar for the two enantiomers in plasma and brain, with overall degradation in plasma approximately 2-fold higher than in brain homogenate. Incorporation of degradation and chiral inversion kinetic terms into a model to predict enantiomer-specific binding in plasma and brain revealed that, despite existence of these two processes, empirically derived estimates of fraction unbound were similar to model-derived values, leading to a firm conclusion that observed extent of plasma and brain binding are accurate largely because binding kinetics are faster than parallel degradation and chiral inversion processes.

Method for Simultaneous Determination of Free Concentration, Total Concentration, and Plasma Binding Capacity in Clinical Samples.

Most quantitative research methods are based on measuring either the total or the free concentration of an analyte in a sample. However, this is often insufficient for the study of complex biological systems. The main objective of this research was to develop new methods for providing more information from samples: the free concentration (Cf), the total concentration (Ct), and the plasma binding capacity (PBC). Samples were processed using microextraction and ultrafiltration. For each of these techniques, two quantification procedures were used: addition of isotopically labeled standard and repeated analysis of the same sample. The new methods were validated by analyzing clinical samples and samples with known concentrations. Methods based on addition of labeled compound were found to be the fastest, and most reproducible. For analysis of clinical samples, methods based on microextraction were more sensitive and more accurate than those based on ultrafiltration. For analysis of pooled plasma samples, the overall accuracy of all approaches to determine PBC, testosterone Cf, and testosterone Ct was between 94 and 109%, 87-113%, and 94-122% respectively. The new approach goes beyond a simple concentration measurement, giving more information from clinical samples, with great potential for personalizing drug dosage and therapy to the needs of individual patients.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Toward Consensus on Correct Interpretation of Protein Binding in Plasma and Other Biological Matrices for COVID-19 Therapeutic Development.

The urgent global public health need presented by severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) has brought scientists from diverse backgrounds together in an unprecedented international effort to rapidly identify interventions. There is a pressing need to apply clinical pharmacology principles and this has already been recognized by several other groups. However, one area that warrants additional specific consideration relates to plasma and tissue protein binding that broadly influences pharmacokinetics and pharmacodynamics. The principles of free drug theory have been forged and applied across drug development but are not currently being routinely applied for SARS‐CoV‐2 antiviral drugs. Consideration of protein binding is of critical importance to candidate selection but requires correct interpretation, in a drug‐specific manner, to avoid either underinterpretation or overinterpretation of its consequences. This paper represents a consensus from international researchers seeking to apply historical knowledge, which has underpinned highly successful antiviral drug development for other viruses, such as HIV and hepatitis C virus for decades.

In Vitro to In Vivo Extrapolation of Metabolic Clearance for UGT Substrates Using Short-Term Suspension and Long-Term Co-cultured Human Hepatocytes.

The use of micro-patterned co-cultured hepatocytes for human hepatic clearance predictions has previously been demonstrated using drugs metabolized by cytochrome P450 enzymes. The present study evaluates the in vitro to in vivo extrapolation (IVIVE) performance for UDP-glucuronosyltransferase (UGT) substrates. In vitro intrinsic clearances for 13 drugs mainly cleared by UGTs were determined using HepatoPac and suspended hepatocytes. The in vivo intrinsic clearance was predicted from in vitro intrinsic clearance and compared with weighted mean in vivo intrinsic clearance estimated from several clinical studies. A conventional scaling methodology accounting for protein binding in plasma and incubation medium was used for the IVIVE assuming that only free drug is accessible at the site of metabolism. The in vivo hepatic intrinsic clearance was predicted within threefold error for six and nine out of thirteen drugs using suspended hepatocytes and HepatoPac, respectively. A reduced under-estimation of hepatic intrinsic clearance was observed in the average fold error (AFE) in HepatoPac (AFE, 0.69) compared with the suspended hepatocytes (AFE, 0.37). The current study shows reasonable performance of hepatic clearance prediction of drugs mainly metabolized by UGT enzymes using HepatoPac with a similar under-prediction bias as obtained in the reported IVIVEs for cytochrome P450 substrates. This study provides a validation of the approach for drugs cleared via UGT conjugation mechanisms and discusses potential causes for outlier behavior considering pharmacokinetic or physicochemical properties.

Physiologically Based Pharmacokinetic Modeling Involving Nonlinear Plasma and Tissue Binding: Application to Prednisolone and Prednisone in Rats.

The pharmacokinetics (PK) of prednisolone (PNL) exhibit nonlinearity related to plasma protein binding, tissue binding, metabolic interconversion with prednisone (PN), and renal elimination. Blood and 11 tissues were collected from male Wistar rats after steady-state (SS) infusion and after subcutaneous boluses of 50 mg/kg of PNL. Concentrations of PNL and PN were measured by liquid chromatography-tandem mass spectrometry. Plasma and tissue profiles were described using a complex physiologically based pharmacokinetics (PBPK) model. Concentrations of PN and PNL were in rapid equilibrium in plasma and tissues. The tissue partition coefficients (K p ) of PNL calculated from most subcutaneously dosed tissue and plasma areas were similar to SS infusion and in silico values. The blood-to-plasma ratio of PNL was 0.71 with similar red blood cell and unbound-plasma concentrations. Plasma protein binding (60%-90%) was related to corticosteroid-binding globulin (CBG) saturation. Tissue distribution was nonlinear. The equilibrium dissociation constant (K d ) of PNL shared by all tissues was 3.01 ng/ml, with the highest binding in muscle, followed by liver, heart, intestine, and bone and the lowest binding in skin, spleen, fat, kidney, lung, and brain. Fat and bone distribution assumed access only to interstitial space. Brain PNL concentrations (K p = 0.05) were low owing to presumed P-glycoprotein-mediated efflux. Clearances of CBG-free PNL were 1789 from liver and 191.2 ml/h from kidney. The PN/PNL ratio was nonlinear for plasma, spleen, heart, intestine, bone, fat, and linear for the remaining tissues. Our PBPK model with multiple complexities well described the PK profiles of PNL and PN in blood, plasma, and diverse tissues. SIGNIFICANCE STATEMENT: Because steroids, such as prednisolone and prednisone, have similar and complex pharmacokinetics properties in various species, receptors in most tissues, and multiple therapeutic and adverse actions, this physiologically based pharmacokinetics (PBPK) model may provide greater insights into the pharmacodynamic complexities of corticosteroids. The complex properties of these compounds require innovative PBPK modeling approaches that may be instructive for other therapeutic agents.

Characteristics of Dolutegravir and Bictegravir Plasma Protein Binding: a First Approach for the Study of Pharmacologic Sanctuaries.

This study aimed to characterize in vitro dolutegravir (DTG) and bictegravir (BIC) binding. They had a preferential binding to human serum albumin (HSA) with two classes of albumin sites. Human alpha-1-acid glycoprotein (HAAG) binding of DTG and BIC showed an atypical nonlinear binding. The low-affinity site on HSA, the main plasma binding protein, suggests that the high protein binding rate should not impair passive diffusion.

Evaluation of HIV-1 neutralizing and binding antibodies in maternal-infant transmission in Thailand

Despite anti-retroviral therapy (ART) interventions for HIV+ pregnant mothers, over 43,000 perinatal infections occur yearly. Understanding risk factors that lead to mother-to-child transmission (MTCT) of HIV are critical. We evaluated maternal and infant plasma binding and neutralizing antibody responses in a drug-naïve, CRF01_AE infected MTCT cohort from Thailand to determine associations with transmission risk. Env V3-specific IgG and neutralizing antibody responses were significantly higher in HIV- infants, as compared to HIV+ infants. In fact, infant plasma neutralizing antibodies significantly associated with non-transmission. Conversely, increased maternal Env V3-specific IgG and neutralizing antibody responses were significantly associated with increased transmission risk, after controlling for maternal viral load. Our results highlight the importance of evaluating both maternal and infant humoral immune responses to better understand mechanisms of protection, as selective placental antibody transport may have a role in MTCT. This study further emphasizes the complex role of Env-specific antibodies in MTCT of CRF01_AE HIV.

Deep Proteome Profiling of Semen of Indian Indigenous Malnad Gidda (Bos indicus) Cattle.

Malnad Gidda is a dwarf indigenous cattle breed of India, which is known for its uniqueness of calving every year under a low input grazing system of rearing. Bulls of Malnad Gidda are known to be highly fertile even in stress conditions. However, the proteomic profiling of semen of this breed has not been investigated so far, which might provide a platform for a better understanding of its semen quality and male fertility. Therefore, we made an effort to characterize and quantify the proteome of seminal plasma and spermatozoa components of Malnad Gidda semen using a high-resolution mass spectrometry platform. We identified 2814 proteins from spermatozoa and 1974 proteins from the seminal plasma of this breed. Furthermore, >90% of proteins from each fraction were quantified using the intensity-based absolute quantification. We observed signal peptides in 33% of seminal plasma proteins, indicating their secretory nature. Gene Ontology analysis revealed their involvement in cytoskeletal assembly associated with sperm head, sperm motility, acrosome reaction, seminal plasma binding, and spermatogenesis-associated protein. An in-depth proteome profiling of semen of a unique indigenous cattle breed of India was carried out. Our findings could provide a reference for further studies on sperm functions, semen quality, and reproductive health of Bos indicus cattle. Mass spectrometry data generated in this study is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014172.

Tetrazine-TCO Ligation: A Potential Simple Approach to Improve Tumor Uptake through Enhanced Blood Circulation.

Targeted imaging via peptides has been extensively investigated for both diagnostic and therapeutic applications. However, peptides can be cleared out from the blood circulation quickly, leading to only moderate to low accumulation in regions of interest. Previously, 18F-sTCO-DiPhTz-RGDyK demonstrated relatively high blood retention with increased tumor uptake at the late time point (5.3 ± 0.4 and 8.9 ± 0.5%ID/g tumor uptake at 1 h p.i. and 4 h p.i., respectively). In this study, we aim to develop a novel platform based on TCO/tetrazine ligation that could be used not only to label the peptides with F-18 for PET but also to lead to increased or persistent tumor uptake potentially due to enhanced blood circulation of the labeled peptides. We first constructed systems containing different combinations of TCOs/tetrazines and found that the tetrazine moiety played a more important role to facilitate the enhanced blood circulation compared with TCO moiety. Four clinically relevant peptides including NT20.3, RGD, BBN, and exendin-4 were then evaluated, and the increased tumor uptake at a late time point was demonstrated by the combination of 18F-sTCO-DiPhTz system to NT20.3, RGD, and exendin-4. The plasma binding components of the PET agents were investigated by electrophoresis and autoradiography and indicated that transferrin and hemopexin could be the major proteins in blood plasma for binding with 18F-sTCO-DiPhTz conjugates. In summary, we have discovered a TCO/tetrazine system that could not only be used for PET probe construction but also potentially improve the tumor uptake and retention of fast-clearing peptides. Although additional binding experiments are still needed for some of the constructs, the promising preliminary result suggested that this strategy may be used to convert fast-clearing bioligands into relatively long circulating agents with enhanced tumor uptake/retention for either imaging or therapy applications.