Atenolol, a hydrophilic beta blocker, has been used as a model drug for studying passive permeability of biological membranes such as the blood-brain barrier (BBB) and the intestinal epithelium. ...However, the extent of S-atenolol (the active enantiomer) distribution in brain has never been evaluated, at equilibrium, to confirm that no transporters are involved in its transport at the BBB.
To assess whether S-atenolol, in fact, depicts the characteristics of a low passive permeable drug at the BBB, a microdialysis study was performed in rats to monitor the unbound concentrations of S-atenolol in brain extracellular fluid (ECF) and plasma during and after intravenous infusion. A pharmacokinetic model was developed, based on the microdialysis data, to estimate the permeability clearance of S-atenolol into and out of brain. In addition, the nonspecific binding of S-atenolol in brain homogenate was evaluated using equilibrium dialysis.
The steady-state ratio of unbound S-atenolol concentrations in brain ECF to that in plasma (i.e., K
) was 3.5% ± 0.4%, a value much less than unity. The unbound volume of distribution in brain (V
) of S-atenolol was also calculated as 0.69 ± 0.10 mL/g brain, indicating that S-atenolol is evenly distributed within brain parenchyma. Lastly, equilibrium dialysis showed limited nonspecific binding of S-atenolol in brain homogenate with an unbound fraction (f
) of 0.88 ± 0.07.
It is concluded, based on K
being much smaller than unity, that S-atenolol is actively effluxed at the BBB, indicating the need to re-consider S-atenolol as a model drug for passive permeability studies of BBB transport or intestinal absorption.
A mathematical equation model was developed by building the relationship between the fu,b/fu,p ratio and the computed physicochemical properties of candidate compounds, thereby predicting Kp,uu,brain ...based on a single experimentally measured Kp,brain value. A total of 256 compounds and 36 marketed published drugs including acidic, basic, neutral, zwitterionic, CNS-penetrant, and non-CNS penetrant compounds with diverse structures and physicochemical properties were involved in this study. A strong correlation was demonstrated between the fu,b/fu,p ratio and physicochemical parameters (CLogP and ionized fraction). The model showed good performance in both internal and external validations. The percentages of compounds with Kp,uu,brain predictions within 2-fold variability were 80.0 %–83.3 %, and more than 90 % were within a 3-fold variability. Meanwhile, “black box” QSAR models constructed by machine learning approaches for predicting fu,b/fu,p ratio based on the chemical descriptors are also presented, and the ANN model displayed the highest accuracy with an RMSE value of 0.27 and 86.7 % of the test set drugs fell within a 2-fold window of linear regression. These models demonstrated strong predictive power and could be helpful tools for evaluating the Kp,uu,brain by a single measurement parameter of Kp,brain during lead optimization for CNS penetration evaluation and ranking CNS drug candidate molecules in the early stages of CNS drug discovery.
A rapid,sensitive,and robust reversed-phase liquid chromatography with tandem mass spectrometry method was developed and validated for the determination of total and unbound ceritinib,a ...secondgeneration ALK inhibitor,in patient plasma and brain tumor tissue samples.Sample preparation involved simple protein precipitation with acetonitrile.Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18column using a4-min gradient elution consisting of mobile phase A(0.1%formic acid inwater)andmobile phase B(0.1%formic acid in acetonitrile),at a flow rate of0.4mL/min.Ceritinib and the internal standard(13C6ceritinib)were monitored using multiple reaction monitoring mode under positive electrospray ionization.The lower limit of quantitation(LLOQ)was1nM of ceritinib in plasma.The calibration curve was linear over ceritinib concentration range of1-2000nM in plasma.The intra-and interday precision and accuracy were within the generally accepted criteria for bioanalytical method(<15%).The method was successfully applied to assess ceritinib brain tumor penetration,as assessed by the unbound drug brain concentration to unbound drug plasma concentration ratio,in patients with brain tumors.
Safety margin, a key aspect of any non-clinical toxicity studies, is calculated by dividing the systemic exposure (AUC) at NOAEL (No Adverse Effect Level) in toxicity studies by the clinical ...exposure. The validity of using total plasma concentration (Cp) to calculate AUC is often discussed, as it is the unbound plasma concentration (Cup) that elicits the pharmacological and toxicological effects. Data regarding plasma protein binding across species was collected for 114 MSD small molecule compounds which had been discontinued from development either due to non-clinical toxicity or due to clinical Adverse Effects. A >3-fold difference in unbound fraction in plasma (fup) was selected as a meaningful difference in plasma protein binding between non-clinical species and humans. In rats, dogs and non-human primates, approximately 3–5% of the compounds had a >3-fold difference in plasma protein binding than humans. Following assessment of toxicity profile of these compounds, it was concluded that calculation of safety margins after incorporating fup would have still led to the discontinuation of these compounds. Therefore, although fup can still be used for calculation of safety margin on a case by case basis, the routine use of fup for calculation of safety margins is not warranted.
•The impact of inclusion of plasma protein binding in safety margin calculations for compounds in development was evaluated.•The compounds were discontinued from development due to toxicity in non-clinical studies or in clinical studies.•Including plasma protein binding in safety margin calculations would not lead to continued development for these compounds.
The pharmacological activity of ceftriaxone depends on the unbound concentration. However, direct measurement of unbound concentrations is obstructive, and high individual variability of the unbound ...fraction of ceftriaxone was shown in children. We aim to evaluate and validate a method to predict unbound ceftriaxone concentrations in pediatric patients. Ninety-five pairs of concentrations (total and unbound) from 92 patients were measured by the bioanalysis method that we developed. The predictive performance of the three equations (empirical
equation, disease-adapted equation, and multiple linear regression equation) was assessed by the mean absolute prediction error (MAPE), the mean prediction error (MPE), the proportions of the prediction error within ±30% (
) and ±50% (
), and linear regression of predicted versus actual unbound levels (
). The average total and unbound ceftriaxone concentrations were 126.18 ± 81.46 μg/ml and 18.82 ± 21.75 μg/ml, and the unbound fraction varied greatly from 4.75% to 39.97%. The MPE, MAPE,
,
, and
of the empirical
equation, disease equation, and multiple linear equation were 0.17 versus 0.00 versus 0.06, 0.24 versus 0.15 versus 0.27, 63.2% versus 89.5% versus 74.7%, 96.8% versus 97.9% versus 86.3%, and 0.8730 versus 0.9342 versus 0.9315, respectively. The disease-adapted equation showed the best predictive performance. We have developed and validated a bioanalysis method with one-step extraction pretreatment for the determination of total ceftriaxone concentrations, and a prediction equation of the unbound concentration is recommended. The proposed method can facilitate clinical practice and research on unbound ceftriaxone in children. (This study has been registered at ClinicalTrials.gov under identifier NCT03113344.).
In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (fu) is arguably regarded the effective concentration at the target ...site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK.
We compared three methods to quantify fu: rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log Pow (−0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin.
After RED and UF separation, three polar substances (Log Pow < 2) were largely unbound (fu > 70%), while more lipophilic substances were largely bound (fu < 33%). Compared to RED or UF, UC resulted in a generally higher fu of lipophilic substances. fu obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in fu higher than the reference data. UF, RED and both UF and UC, resulted in lower fu of Flutamide, Ketoconazole and Colchicine, respectively.
For fu quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances.
•Plasma protein binding (PPB) is an important key parameter in toxicokinetics.•Twelve test substances were evaluated using rapid equilibrium dialysis, ultrafiltration, and ultracentrifugation.•Applicability is highly dependent on physicochemical properties of the test substances.
The calcium isotopes are an ideal system to investigate the evolution of shell structure and magic numbers. Although the properties of surface nucleons in calcium have been well studied, probing the ...structure of deeply bound nucleons remains a challenge. Here, we report on the first measurement of unbound states in 53Ca and 55Ca, populated from 54,56Ca(p,pn) reactions at a beam energy of around 216 MeV/nucleon at the RIKEN Radioactive Isotopes Beam Factory. The resonance properties, partial cross sections, and momentum distributions of these unbound states were analyzed. Orbital angular momentum l assignments were extracted from momentum distributions based on calculations using the distorted wave impulse approximation (DWIA) reaction model. The resonances at excitation energies of 5516(41)keV in 53Ca and 6000(250)keV in 55Ca indicate a significant l =3 component, providing the first experimental evidence for the ν0f7/2 single-particle strength of unbound hole states in the neutron-rich Ca isotopes. The observed excitation energies and cross-sections point towards extremely localized and well separated strength distributions, with some fragmentation for the ν0f7/2 orbital in 55Ca. These results are in good agreement with predictions from shell-model calculations using the effective GXPF1Bs interaction and ab initio calculations and diverge markedly from the experimental distributions in the nickel isotones at Z=28.
Tumor binding is an important parameter to derive unbound tumor concentration to explore pharmacokinetics (PK) and pharmacodynamics (PD) relationships for oncology disease targets. Tumor binding was ...evaluated using eleven matrices, including various commonly used ex vivo human and mouse xenograft and syngeneic tumors, tumor cell lines and liver as a surrogate tissue. The results showed that tumor binding is highly correlated among the different tumors and tumor cell lines except for the mouse melanoma (B16F10) tumor type. Liver fraction unbound (f
) has a good correlation with B16F10 tumor binding. Liver also demonstrates a two-fold equivalency, on average, with binding of other tumor types when a scaling factor is applied. Predictive models were developed for tumor binding, with correlations established with LogD (acids), predicted muscle f
(neutrals) and measured plasma protein binding (bases) to estimate tumor f
when experimental data are not available. Many approaches can be applied to obtain and estimate tumor binding values. One strategy proposed is to use a surrogate tumor tissue, such as mouse xenograft ovarian cancer (OVCAR3) tumor, as a surrogate for tumor binding (except for B16F10) to provide an early assessment of unbound tumor concentrations for development of PK/PD relationships.
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The BBB is a protective entity that prevents external substances from reaching the CNS but it also hinders the delivery of drugs into the brain when they are needed. The main ...objective of this work was to improve a previously proposed in vitro cell-based model by using a more physiological cell line (hCMEC/D3) to predict the main pharmacokinetic parameters that describe the access and distribution of drugs in the CNS: Kpuu,brain, fu,plasma, fu,brain and Vu,brain. The hCMEC/D3 permeability of seven drugs was studied in transwell systems under different conditions (standard, modified with albumin and modified with brain homogenate). From the permeability coefficients of those experiments, the parameters mentioned above were calculated and four linear IVIVCs were established. The best ones were those that relate the in vitro and in vivo Vu,brain and fu,brain (r2 = 0.961 and r2 = 0.940) which represent the binding rate of a substance to the brain tissue, evidencing the importance of using brain homogenate to mimic brain tissue when an in vitro brain permeability assay is done. This methodology could be a high-throughput screening tool in drug development to select the CNS promising drugs in three different in vitro BBB models (hCMEC/D3, MDCK and MDCK-MDR1).
Recent developments (since the last review in J. of Physics G by I. Tanihata in 1996 1) at RIB facilities opened possibilities of detailed studies of halo nuclei. New facilities have been constructed ...to provide higher intensity beams of radioactive nuclei in a wide range of energies. At the time of the last review, only secondary beams by projectile fragmentation were the production source of halo nuclei for use in reaction studies. Since then, re-acceleration facilities have been developed and thus high-quality low-energy beams become available for the reaction studies. The wide variety of new data are thus available on halo nuclei and nuclei on and outside of proton and neutron drip lines.
Low energy beams provided a means to determine the masses and charge radii of halo nuclei (6,8He, 11Li). Also transfer reactions have been measured in many nuclei far from the stability line. In fragmentation facilities, new experimental methods such as gamma ray detection in coincidence with breakup fragments of halo nuclei have been developed. Also the reaction cross sections have been measured in a wide range of beam energies. In addition, proton elastic scattering of halo nuclei has been measured at high energies. All together, studies of density distribution, identification of shell orbitals and spectroscopic factors of halo wave function became possible. Such studies reveal many new important information such as the change of magic numbers in nuclei far from the stability line.
In this article, we would like to review the experimental developments on halo nuclei and other related drip line nuclei. Also the new view of the nuclear structure learned from such studies will be discussed. Development of selected theories on related nuclear structure problems will be mentioned briefly.
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