Determination Of Protein Binding Research Articles

Simultaneous determination of plasma protein binding of five C-glycosylflavones from TFDS by rapid equilibrium dialysis

The total flavonoids of Desmodium styracifolium (TFDS) are flavonoid-rich extracts obtained from Desmodii Styracifolii Herba, which is approved for the treatment of urolithiasis in China. C-glycosylflavones including schaftoside, vicenin-1, vicenin-2, vicenin-3, and isovitexin are the main active constituents. In this study, the plasma protein binding of these compounds was determined for the first time in rat and human plasma by rapid equilibrium dialysis combined with HPLC-MS/MS method. The developed method was validated in terms of specificity, linearity, accuracy, precision, extraction effect, matrix effect, and stability. Schaftoside, vicenin-1, vicenin-2, and vicenin-3 exhibited moderate plasma protein binding, ranging from 56.6% to 61.5% in rat plasma and 55.0%–62.9% in human plasma. In comparison, isovitexin demonstrated a higher plasma protein binding in the range of 92.3–93.1% and 95.1–96.2% in rat and human plasma, respectively. Furthermore, the potential interactions mediated via plasma protein binding between isovitexin and nonsteroidal anti-inflammatory drugs (NSAIDs) were investigated by rapid equilibrium dialysis. No significant changes were observed, indicating a lower likelihood of interaction between TFDS and NSAIDs due to plasma protein binding in the treatment of urinary system disorders.

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.

Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs’ instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

Pharmacokinetics of N,N-dimethyltryptamine in Humans.

N,N-dimethyltryptamine (DMT) is a psychedelic compound under development for the treatment of major depressive disorder (MDD). This study evaluated the preclinical and clinical pharmacokinetics and metabolism of DMT in healthy subjects. The physiochemical properties of DMT were determined using a series of in vitro experiments and its metabolic profile was assessed using monoamine oxidase (MAO) and cytochrome P450 (CYP) inhibitors in hepatocyte and mitochondrial fractions. Clinical pharmacokinetics results are from the phase I component of a phase I/IIa randomised, double-blind, placebo-controlled, parallel-group, dose-escalation trial (NCT04673383). Healthy adults received single escalating doses of DMT fumarate (SPL026) via a two-phase intravenous (IV) infusion. Dosing regimens were calculated based on pharmacokinetic modelling and predictions with progression to each subsequent dose level contingent upon safety and tolerability. In vitro clearance of DMT was reduced through the inhibition of MAO-A, CYP2D6 and to a lesser extent CYP2C19. Determination of lipophilicity and plasma protein binding was low, indicating that a high proportion of DMT is available for distribution and metabolism, consistent with the very rapid clinical pharmacokinetics. Twenty-four healthy subjects received escalating doses of DMT administered as a 10-min infusion over the dose range of 9-21.5 mg (DMT freebase). DMT was rapidly cleared for all doses: mean elimination half-life was 9-12 min. All doses were safe and well tolerated and there was no relationship between peak DMT plasma concentrations and body mass index (BMI) or weight. This is the first study to determine, in detail, the full pharmacokinetics profile of DMT following a slow IV infusion in humans, confirming rapid attainment of peak plasma concentrations followed by rapid clearance. These findings provide evidence which supports the development of novel DMT infusion regimens for the treatment of MDD. Registered on ClinicalTrials.gov (NCT04673383).

Brigatinib pharmacokinetics in patients with chronic hepatic impairment

SummaryBrigatinib is an anaplastic lymphoma kinase (ALK) inhibitor approved for the treatment of ALK-positive non-small cell lung cancer. This open-label, parallel-group study investigated the effect of chronic hepatic impairment on the pharmacokinetics (PK) of brigatinib to inform dosing recommendations for these patients. Participants with hepatic impairment classified according to Child-Pugh categories of mild (A), moderate (B), or severe (C) and matched-healthy participants with normal hepatic function received a single oral dose of 90-mg brigatinib. Plasma samples were collected for the determination of brigatinib plasma protein binding and estimation of plasma PK parameters. Twenty-seven participants were enrolled (Child-Pugh A–C, n = 6 each; matched-healthy participants, n = 9). The mean fraction of free plasma brigatinib was comparable for the Child-Pugh A (11.1%), Child-Pugh B (10.8%), and healthy participant groups (8.5%); free brigatinib was higher in the Child-Pugh C group (23.1%). There were no clinically meaningful effects of mild or moderate hepatic impairment on unbound systemic exposures (area under the plasma concentration-time curve [AUC]) of brigatinib (geometric least-squares mean ratios [90% CI] of 89.32% [69.79%–114.31%] and 99.55% [77.78%–127.41%], respectively). In the severe hepatic impairment group, brigatinib unbound AUC was approximately 37% higher (geometric least-squares mean ratio [90% CI] of 137.41% [107.37%–175.86%]) compared with healthy participants with normal hepatic function. Brigatinib was well tolerated in healthy participants and in participants with hepatic impairment. No dose adjustment is required for patients with mild or moderate hepatic impairment. The brigatinib dose should be reduced by approximately 40% for patients with severe hepatic impairment.

Comparison of ultrafiltration and microdialysis for ceftriaxone protein-binding determination.

High protein binding (PB) of antibiotics has an impact on their antimicrobial activity. It has been questioned whether in vitro PB determination can capture the dynamic and concentration-dependent PB of highly bound antibiotics. This clinical study compared in vitro ultrafiltration (UF) and in vivo IV microdialysis (MD) methods to determine ceftriaxone PB. Six healthy male volunteers received a single IV 2 g dose of ceftriaxone. Unbound ceftriaxone plasma concentrations were measured with MD and venous plasma sampling with subsequent UF. Pharmacokinetic parameters were determined using non-compartmental pharmacokinetic analysis. Non-linear mixed-effects modelling was used to quantify the PB. The PTA was estimated. The Cmax of ceftriaxone total plasma concentration (297.42 ± 21.0 mg/L) was approximately 5.5-fold higher than for free concentrations obtained with UF (52.83 ± 5.07 mg/L), and only 3.5-fold higher than for free concentrations obtained with MD (81.37 ± 26.93 mg/L). Non-linear, saturable PB binding was confirmed for both UF and MD. Significantly different dissociation constants (Kd) for the albumin/ceftriaxone complex were quantified: in UF it was 23.7 mg/L (95% CI 21.3-26.2) versus 15.9 mg/L (95% CI 13.6-18.6) in MD. Moreover, the estimated number of binding sites (95% CI) per albumin molecule was 0.916 (0.86-0.97) in UF versus 0.548 in MD (0.51-0.59). The PTA obtained with MD was at most 27% higher than with UF. In vitro UF versus in vivo intravasal MD revealed significantly different PB, especially during the distribution phase. The method of PB determination could have an impact on the breakpoint determination and dose optimisation of antibiotics.

Characterization of binding properties of ephedrine derivatives to human alpha-1-acid glycoprotein

Most drugs, especially those with acidic or neutral moieties, are bound to the plasma protein albumin, whereas basic drugs are preferentially bound to human alpha-1-acid glycoprotein (AGP). The protein binding of the long-established drugs ephedrine and pseudoephedrine, which are used in the treatment of hypotension and colds, has so far only been studied with albumin. Since in a previous study a stereoselective binding of ephedrine and pseudoephedrine to serum but not to albumin was observed, the aim of this study was to check whether the enantioselective binding behavior of ephedrine and pseudoephedrine, in addition to the derivatives methylephedrine and norephedrine, is due to AGP and to investigate the influence of their different substituents and steric arrangement. Discontinuous ultrafiltration was used for the determination of protein binding. Characterization of ligand-protein interactions of the drugs was obtained by saturation transfer difference nuclear magnetic resonance spectroscopy. Docking experiments were performed to analyze possible ligand-protein interactions. The more basic the ephedrine derivative is, the higher is the affinity to AGP. There was no significant difference in the binding properties between the individual enantiomers and the diastereomers of ephedrine and pseudoephedrine.

Accessing Lipophilicity and Biomimetic Chromatography Profile of Biologically Active Ingredients of Botanicals Used in the Treatment of Inflammatory Bowel Disease.

In the present study, various procedures have been compared for the determination of lipophilicity, hydrophobicity, and plasma protein binding of curcuminoids, boswellic acids, andrographolides, and piperine as biologically active ingredients of botanicals used in IBD treatment. Our results have shown that IAM-HPLC assay is the most suitable one for lipophilicity determination of all analytes regardless of their class and botanical source. HSA-HPAC and AGP-HPAC assays revealed that all investigated compounds have a higher affinity for HSA which is the most abundant protein in human plasma. The high affinity of biologically active compounds to all biological structures (phospholipids and proteins) admonishes that their small portion is available for therapeutic effects in IBD patients. Our experimental research is complemented by various theoretical approaches based on different algorithms for pharmacokinetic properties prediction. The similarities between experimental and calculated values were evaluated using PCA and CA as a statistical tool. The statistical analysis implies that plasma protein binding is a complex process, and theoretical approaches still cannot fully replace experimental ones.

Uncovering the mechanistic basis of MAGEA5/A10 driven resistance to chemotherapeutic drugs

Melanoma Antigen Genes (MAGEs) are defined as cancer‐testis antigens due to their unique expression pattern. There are two families of MAGEs, Type I and Type II. Type I MAGEs are expressed in the testis and in no other normal somatic tissue and then re‐expressed in many cancers. Type II MAGEs on the other hand, are ubiquitous in their expression.. The MAGE‐A family are members of the type 1 family located on the X chromosome. Type I MAGEs, considered to be true cancer‐testis antigens are all located on the X chromosome. Previous studies show that reversible DNA methylation of CpG sites upstream of MAGE‐A promoters regulates their expression. Many members of the MAGE1 subfamily cause increase in proliferative phenotypes. However, MAGEA5 and MAGEA10 expressing cells, demonstrated resistance to high doses of chemotherapeutic agent 5‐flurouracil. We aim to understand the mechanism behind this resistance. We will perform RNA‐Sequencing on cells expressing MAGEA5/A10 and control cells and determine protein binding partners of MAGEA5/A10 using IP‐Mass Spectrometry. In addition, we will test the hypothesis that in the heterogenous tumor cell population, MAGEA5/A10 is expressed in the cancer “stem cells” and that is responsible for driving chemo‐resistance. We will also test resistance of these cells to other chemotherapeutic agents such as taxols.

Determination of plasma protein binding of dalbavancin

Dalbavancin is a lipoglycopeptide with a long half-life, making it a promising treatment for infections requiring prolonged therapy, such as complicated Staphylococcus aureus bacteraemia. Free drug concentration is a critical consideration with prolonged treatment, since free concentration-time profiles may best correlate with therapeutic effect. In support of future clinical trials, we aimed to develop a reliable and reproducible assay for measuring free dalbavancin concentrations. The ultracentrifugation technique was used to determine free dalbavancin concentrations in plasma at two concentrations (50 and 200 mg/L) in duplicate. Centrifuge tubes and pipette tips were treated for 24 h before use with Tween 80 to assess adsorption. Dalbavancin concentrations were analysed from the plasma samples (total) and middle layer samples (free) by LC/MS/MS with isotopically labelled internal standard. Warfarin served as a positive control with known high protein binding. Measurement of free dalbavancin was sensitive to adsorption onto plastic. Treatment of tubes and pipette tips with ≥2% Tween 80 effectively prevented drug loss during protein binding experiments. By the ultracentrifugation method, dalbavancin's protein binding was estimated to be approximately 99%. Dalbavancin has very high protein binding. Given dalbavancin's high protein binding, accurate measurement of free dalbavancin concentrations should be a key consideration in future exposure-response studies, especially clinical trials. Future investigations should confirm if the active fraction is best predicted by the free or total fraction.

Pharmacokinetics of Tropifexor, a Potent Farnesoid X Receptor Agonist, in Participants With Varying Degrees of Hepatic Impairment.

Tropifexor, a non-bile acid farnesoid X receptor (FXR) agonist, has dose-proportional pharmacokinetics and no obvious major enterohepatic circulation. This open-label study investigated the effect of hepatic impairment (HI), as determined by Child-Pugh grade, on tropifexor's pharmacokinetics, safety, and tolerability following a 200-μg dose in the fasted state. Blood samples were collected through 168 hours after dosing for quantification and plasma protein-binding determination. Total tropifexor exposure was comparable across participants with HI vs those with normal hepatic function. Tropifexor was highly protein bound (>99%) in human plasma across participants of all groups. The average unbound fractions (percentage free) were 0.14% in participants with normal hepatic function and mild HI, which increased to 0.17% and 0.24% in participants with moderate and severe HI, respectively. Similar unbound drug exposure was noted in participants with mild HI and normal hepatic function. Participants with moderate HI (N = 8) had a 1.6-fold increase in unbound exposure (area under the plasma concentration-time curve from time 0 to infinity [AUCinf,u ]) and a 1.3-fold increase in maximal exposure (Cmax,u ) vs those with normal hepatic function (geometric mean ratio: AUCinf,u , 1.64 [90%CI, 1.25-2.16]; Cmax,u , 1.30 [90%CI, 0.96-1.76]). Participants with severe HI (N = 8) had a 1.6-fold increase in AUCinf,u (1.61 [90%CI, 1.04-2.49]) and comparable Cmax,u (1.02 [90%CI, 0.60-1.72]) compared to participants with normal hepatic function. Tropifexor was well tolerated. The relative insensitivity of tropifexor to HI offers the potential to treat patients with severe liver disease without dose adjustment.

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.

Enantioselective determination of plasma protein binding of common amphetamine-type stimulants

Amphetamine-type stimulants (ATS) like amphetamine (‘speed’), methamphetamine (‘crystal meth’) and 3,4-methylenedioxy-N-methylamphetamine (MDMA, ‘ecstasy’) represent some of the most frequently abused drugs worldwide. Another less frequently abused ATS is 4-fluoroamphetamine (4-FA). The enantiomers of these four compounds exhibit different pharmacokinetic and pharmacodynamic properties. According to the free drug theory, the pharmacological properties of a substance are dependent on its plasma protein binding (PPB). However, data on PPB of stimulant enantiomers in humans are rare or non-existent.Human plasma samples were spiked with racemic mixtures of the stimulants and subjected to ultrafiltration to extract the unbound fraction. Enantioselective liquid chromatography − tandem mass spectrometry (LC-MS/MS) methods were applied using a chiral Phenomenex® Lux3 μm AMP column. Method validation showed satisfactory selectivity, linearity (0.5 250 ng/mL), accuracy and precision. Enantiomers were quantified before and after ultracentrifugation to determine PPB.For all analytes, low to medium plasma protein binding was found. For (R)-amphetamine a slightly but significantly higher PPB was found compared to the (S)-enantiomer (31.7 % vs 29.0 %). (R)-MDMA also showed only slightly but significantly significantly higher PPB than (S)-MDMA, although the mean difference was negligible (21.6 % vs 21.3 %). For the enantiomers of methamphetamine and 4-FA, no significant differences in PPB were found.In summary, there were no or only minor differences in PBB for the enantiomers of all investigated compounds. The different pharmacological properties of the stimulant enantiomers can therefore not be explained by differences in PPB.

Optimizing a High-Throughput Solid-Phase Microextraction System to Determine the Plasma Protein Binding of Drugs in Human Plasma.

Plasma protein binding refers to the binding of a drug to plasma proteins after entering the body. The measurement of plasma protein binding is essential during drug development and in clinical practice, as it provides a more detailed understanding of the available free concentration of a drug in the blood, which is in turn critical for pharmacokinetics and pharmacodynamics studies. In addition, the accurate determination of the free concentration of a drug in the blood is also highly important for therapeutic drug monitoring and in personalized medicine. The present study uses C18-coated solid-phase microextraction 96-pin devices to determine the free concentrations of a set of drugs in plasma, as well as the plasma protein binding of drugs with a wide range of physicochemical properties. It should be noted that the extracted amounts used to calculate the binding constants and plasma protein bindings should be measured at respective equilibrium for plasma and phosphate buffer. Therefore, special attention is placed on properly determining the equilibration times required to correctly estimate the free concentrations of drugs in the investigated systems. The plasma protein binding values obtained with the 96-pin devices are consistent with those reported in the literature. The 96-pin device used in this research can be easily coupled with a Concept96 or other automated robotic systems to create an automated plasma protein binding determination protocol that is both more time and labor efficient compared to conventional equilibrium dialysis and ultrafiltration methods.

68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice.

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvβ3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [68Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [68Ga]Ga-DFO-c(RGDyK) can be used for αvβ3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.

Solid-phase microextraction for assessment of plasma protein binding, a complement to rapid equilibrium dialysis.

Aim: Determination of plasma protein binding (PPB) is considered vital for better understanding of pharmacokinetic and pharmacodynamic activities of drugs due to the role of free concentration in pharmacological response. Methodology &results: Solid-phase microextraction (SPME) was investigated for measurement of PPB from biological matrices and compared with a gold standard approach (rapid equilibrium dialysis [RED]). Discussion & conclusion: SPME-derived values of PPB correlated well with literature values, and those determined by RED. Respectively, average protein binding across three concentrations by RED and SPME was 33.1 and 31.7% for metoprolol, 89.0 and 86.6% for propranolol and 99.2 and 99.0% for diclofenac. This study generates some evidence for SPME as an alternative platform for the determination of PPB.

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.

Effect of Hepatic Impairment on the Pharmacokinetics of Itacitinib.

Itacitinib is a potent, selective JAK-1 inhibitor currently in development for the treatment of chronic graft-vs-host-disease in combination with corticosteroids. Itacitinib is primarily eliminated via cytochrome P450 3A metabolism with minimal renal elimination. The purpose of this open-label study was to investigate the effect of hepatic impairment, as determined by Child-Pugh grade, on itacitinib pharmacokinetics. All participants received a single 300-mg dose of itacitinib orally in the fasted state. Blood samples were collected serially through 96 hours after dosing; 4 hours after dosing, an additional sample was collected for protein binding determination. Participants with moderate hepatic impairment (N = 8) had an approximate 2.5-fold increase in total exposure (area under the plasma concentration-time curve from time 0 to infinity [AUC0-∞ ]) and an approximate 2-fold increase in maximal exposure (Cmax ) compared to those with normal hepatic function (N = 8) (geometric mean ratio, 2.51 [90% confidence interval (CI), 1.54-4.08] for AUC0-∞ and 1.95 [90%CI, 1.14-3.35] for Cmax ). Participants with severe hepatic impairment (N = 6) had an approximate 4-fold increase in total exposure (AUC0-∞ ) and an approximate 3.5-fold increase in maximal exposure compared to participants with normal hepatic function (geometric mean ratio, 4.08 [90%CI, 2.41-6.89] for AUC0-∞ and 3.48 [90%CI, 1.94-6.23] for Cmax ). Protein binding was similar between participants with moderate or severe hepatic impairment and participants with normal hepatic function, with average unbound fractions (percent free) of 25.7%, 31.5%, and 25.6%, respectively. There were no serious or fatal treatment-related adverse events. The results of this study combined with exposure, efficacy, and safety data from the pivotal study in the relevant patient population will inform final dosing recommendations.

Enabling direct and definitive free fraction determination for highly-bound compounds in protein binding assay

Protein binding determination for highly-bound compounds using equilibrium dialysis remains a challenge in drug discovery. The reasons are mainly three-fold; 1. due to their slow diffusion rate, highly-bound compounds require a much longer incubation time to reach dialysis equilibrium than typically needed; 2. highly-bound compounds are often hydrophobic and prone to non-specific binding in dialysis; 3. free drug concentration in the post incubation dialysate is too low for reliable analytical quantification. Modified equilibrium dialysis approaches include using diluted plasma for dialysis, or pre-saturating the non-specific binding sites in the dialysis device with compounds of interest prior to dialysis.In this study, we developed a customized equilibrium dialysis assay with an extended incubation time of 24 h, followed by microflow (μF) LC-MS/MS for bioanalysis, for direct and definitive free fraction determination of highly protein-bound compounds. The extended incubation time ensured the dialysis to reach equilibrium and saturating the non-specific binding sites, while μFLC-MS/MS provided far better sensitivity than the conventional LC-MS/MS typically used for post incubation bioanalysis. For a group of commercially available, highly protein-bound compounds, the free fraction data generated by the developed assay correlated very well with the literature values generated with diluted plasma method or pre-saturation method. This novel assay approach has been successfully used to generate protein binding results for highly-bound compounds to support ongoing drug discovery research.

68Ga-labelled desferrioxamine-B for bacterial infection imaging

PurposeWith the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections.MethodsIn vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens.ResultsDFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters.ConclusionDFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.