Science & Technology
Publications
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A Cell-Free Approach Based on Phospholipid Characterization for Determination of the Cell Specific Unbound Drug Fraction (fu,cell)
Treyer A, Walday S, Boriss H, Matsson P, Artursson P. (2019)
Pharm Res. 2019 Nov 7;36(12):178.Purpose
The intracellular fraction of unbound compound (fu,cell) is an important parameter for accurate prediction of drug binding to intracellular targets. fu,cell is the result of a passive distribution process of drug molecules partitioning into cellular structures. Initial observations in our laboratory showed an up to 10-fold difference in the fu,cell of a given drug for different cell types. We hypothesized that these differences could be explained by the phospholipid (PL) composition of the cells, since the PL cell membrane is the major sink of unspecific drug binding. Therefore, we determined the fu,cell of 19 drugs in cell types of different origin.
Method
The cells were characterized for their total PL content and we used mass spectrometric PL profiling to delineate the impact of each of the four major cellular PL subspecies: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The cell-based experiments were compared to cell-free experiments that used beads covered by PL bilayers consisting of the most abundant PL subspecies.
Results
PC was found to give the largest contribution to the drug binding. Improved correlations between the cell-based and cell-free assays were obtained when affinities to all four major PL subspecies were considered. Together, our data indicate that fu,cell is influenced by PL composition of cells.
Conclusion
We conclude that cellular PL composition varies between cell types and that cell-specific mixtures of PLs can replace cellular assays for determination of fu,cell as a rapid, small-scale assay covering a broad dynamic range.
Implication
The TRANSIL Intestinal Absorption Assay kit, or customized variants of this kit that have been used in this study, can used to easily assess the intracellular free fraction of drugs.
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Brain tissue binding of drugs: evaluation and validation of solid supported porcine brain membrane vesicles (TRANSIL) as a novel high-throughput method
Longhi, R.; Corbioli, S.; Fontana S.; Braggio, S.; Helmdach, L.; Schiller, J.; Boriss, H. (2011)
Drug Metab Dispos. 2011 Feb;39(2):312-21.Abstract
Estimating the unbound fraction of drugs in brain has become essential for the evaluation and interpretation of the pharmacokinetics and pharmacodynamics of new central nervous system drug candidates. Dialysis-based methods are considered to be accurate for estimating the fraction unbound in brain; however, these techniques are hampered by a low throughput. In this study, we present a novel, matrix-free, high-throughput method for estimating the unbound fraction, based on a sample pooling approach combining the TRANSIL brain absorption assay with liquid chromatography-mass spectrometry. The base measurement of the TRANSIL approach is the affinity to brain membranes, and this method is used directly to predict the free fraction in brain. The method was evaluated by comparing the free fraction of drugs in brain [f(u,brain) (%)] obtained using the TRANSIL brain absorption assay and equilibrium dialysis methods for a test set of 65 drugs (27 marketed and 38 proprietary drugs). A good correlation (r(2) > 0.93) of f(u,brain) (%) between the TRANSIL brain absorption assay and equilibrium dialysis was observed. Moreover, we compared the lipid composition of rat and porcine brain and analyzed the influence of the brain albumin content on brain tissue binding measurement. The comparison of the lipid composition indicated only minor differences between rat and porcine brain, and albumin appears to have a low impact on brain tissue binding measurements. The TRANSIL brain absorption assay with sample pooling methodology not only significantly reduces the biological matrix required but also increases the throughput, compared with the conventional dialysis methods
Implication
The TRANSIL Brain Absorption Assay kit yields comparable data to dialysis experiments and requires only a fraction of the time and resources.
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Assessing brain free fraction in early drug discovery
Read, K.D.; Braggio, S. (2010)
Expert Opin Drug Metab Toxicol. 2010 Mar;6(3):337-44Abstract
Importance in the field
The incorporation of brain tissue binding routinely in CNS drug discovery screening strategies has markedly changed the way CNS drug discovery is performed and is proving to be a valuable tool in identifying new therapies for CNS diseases. For many years emphasis has been placed on the magnitude of the brain to blood ratio, the bigger the better, even though, in many cases, brain total concentration (C(brain)) has no or, at best, poor correlation with receptor occupancy/pharmacodynamic readout. Today, C(brain) values measured during in vivo experiments are corrected for the fraction unbound measured through in vitro experiments using brain tissue homogenate or brain tissue slice to obtain an estimate of the brain unbound concentration (C(u,brain)), and this has been demonstrated across a range of CNS targets to give a much better correlation with receptor occupancy/pharmacodynamic readout. This apparently simple change in CNS lead optimisation strategy has de facto revolutionised the vision of the brain penetration concepts.
Areas covered in this review
This review will provide an overview of the use and applications of assessing brain free fraction to determine C(u,brain).
Take home message
Assessing brain free fraction to determine C(u,brain) in CNS lead optimisation strategies is the surrogate of choice for rapidly assessing biophase concentration for the majority of CNS targets.
Implication
The TRANSIL Brain Absorption Assay kit is an ideal tool for optimizing CNS drugs as well as for avoiding potential CNS side-effects in early drug discovery.
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Impact of pH on plasma protein binding in equilibrium dialysis
Kochansky, C.J.; McMasters, D.R.; Lu, P.; Koeplinger, K.A.; Kerr, H.H.; Shou, M.; Korekwa, K.R. (2008)
Mol Pharm. May-Jun 2008;5(3):438-48.Abstract
Many pharmacokinetic analyses require unbound plasma concentrations, including prediction of clearance, volume of distribution, drug-drug interactions, brain uptake analysis, etc. It is most often more convenient to measure the total drug concentration in plasma rather than the unbound drug concentration. To arrive at unbound plasma concentrations, separate in vitro determinations of the plasma protein binding of a drug are usually carried out in serum or in plasma, and the plasma pharmacokinetic results are then mathematically adjusted by this fraction unbound ( f u,p). Plasma protein binding or the drug fraction unbound in plasma ( f u,p) is known to be affected by protein, drug, free fatty acid concentrations, lipoprotein partitioning, temperature, pH, and the presence or absence of other drugs/displacing agents within plasma samples. Errors in f u,p determination caused by lack of adequate pH control in newer assay formats for plasma protein binding (e.g., 96-well equilibrium thin walled polypropylene dialysis plates) will have significant drug-specific impact on these pharmacokinetic calculations. Using a diverse set of 55 drugs and a 96-well equilibrium dialysis plate format, the effect of variable pH during equilibrium dialysis experiments on measured values of f u,p was examined. Equilibrium dialysis of human plasma against Dulbecco's phosphate buffered saline at 37 degrees C under an air or 10% CO 2 atmosphere for 22 h resulted in a final pH of approximately 8.7 and 7.4, respectively. The ratio of f u,p at pH 7.4 (10% CO 2) vs pH 8.7 (air) was >or=2.0 for 40% of the 55 compounds tested. Only one of the 55 compounds tested had a ratio less than 0.9. Select compounds were further examined in rat and dog plasma. In addition, physicochemical properties were calculated for all compounds using ACD/Labs software or Merck in-house software and compared to plasma protein binding results. Changes in plasma protein binding due to pH increases which occurred during the equilibrium dialysis experiment were not species specific but were drug-specific, though nonpolar, cationic compounds had a higher likely hood of displaying pH-dependent binding. These studies underscore the importance of effectively controlling pH in plasma protein binding studies.
Implication
The TRANSIL PPB Binding Assay kit is an ideal tool for early screening of plasma protein binding as it comes in a stable pH environment, doesn't require use of a special CO2 atmosphere and can be run at room temperature.
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High-throughput determination of the free fraction of drugs strongly bound to plasma proteins
Schumacher, J; Kohlsdorfer, C.; Bühner, K.; Brandenburger, T.; Kruk, R. (2004)
J Pharm Sci. 2004 Apr;93(4):816-30.Abstract
Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development. In this publication, we describe the development of a new high-throughput assay for the determination of the free drug fraction in plasma (fu). The new technique is an enhancement of the previously published erythrocytes partition method. It is based on the distribution of drugs between plasma water, plasma proteins, and solid-supported lipid membranes (Transil). The execution of protein binding studies by partitioning is dramatically simplified by substituting erythrocytes with commercially available Transil beads, and makes the method particularly suitable for high-throughput studies. Eight Bayer compounds from different compound classes covering a wide range of lipophilicities (log P = 1.9-5.6) and fu values (0.018-35%) were selected for validation of the assay. The results obtained by the new method and by either the erythrocytes partitioning technique or more conventional methods (ultrafiltration and equilibrium dialysis) are identical, confirming that the new method produces valid results even for drugs that are strongly bound to plasma proteins. Precision and accuracy of the data in the cases of very low and high fu values are comparable, indicating that the method is especially suited for highly lipophilic drugs that tend to adsorb to surfaces compared with other methods, like ultrafiltration or equilibrium dialysis, that may produce biased data. The method is also useful for the determination of binding parameters and the pH dependence of fu. In summary, this assay is well suited for high-throughput determination of protein binding during drug discovery and for extended protein binding studies during drug development.
Implication
The TRANSIL High Sensitivity Binding kit implements the concept developed by this publication and provides a more rigourous mathematical data analysis and lots of internal quality controls. The assay requires an incubation time of only 30 minutes at room temperature and thereby minizes any plasma instabilities.
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Brain Availability Is the Key Parameter for Optimising the Permeability of Central Nervous System Drugs
Boriss, H. (2010)
Drug Discovery 7:57-60.Abstract
Drug disposition across the blood–brain barrier is frequently determined by measuring the distribution coefficient of drug between brain and plasma in vivo or by predicting the distribution coefficient from in vitro experiments. To render the distribution coefficient between brain and plasma (B/P ratio) a useful parameter for lead optimisation, it needs to be complemented with a measure of brain tissue binding, because it has been shown that only the free concentration of a drug in the brain relates to its pharmacodynamics. Methods for assessing drug disposition and brain tissue binding were reviewed and a new concept of brain availability has been introduced. Drug availability in the brain is defined as the product of the B/P ratio and the unbound fraction of drug in the brain. An easily accessible brain efficacy scale is introduced based on the proposed brain availability. Brain efficacy is defined as the ratio of brain availability over the drug’s inhibition constant. This relates the drug’s ability to penetrate the brain to its potency. Hence, this efficacy scale provides a new means of ranking drug candidates according to their dose-independent effect in the brain.
Implication
The TRANSIL Brain Absorption Assay kit measure brain tissue binding (i.e. the affinity to brain membranes) and predicts both the brain to plasma distribution as well as the availablity of drugs in brain.
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Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids
Treyer, A.; Mateus, A.; Wisniewski, J.R.; Boriss, H.; Matsson, P.; Artusson, P. (2018)
Mol Pharm. 2018 Jun 4;15(6):2224-2233Abstract
Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( Fic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower Fic. The induction of NL did not further increase drug binding but led to altered Fic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.
Implication
The TRANSIL Intestinal Absorption Assay kit, or customized variants of this kit that have been used in this study, is a useful tool to easily assess the intracellular free fraction of drugs.
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Updates on contemporary protein binding techniques
Chuang, V.T.G.; Maruyama, T.; Otagiri, M. (2009)
Drug Metab Pharmacokinet. 2009;24(4):358-64.Abstract
Automated high-throughput techniques have become key to improving existing as well as new techniques associated with protein binding analysis. A wide variety of methods and experimental conditions are used for estimating protein binding as well as binding affinity, such as ultrafiltration and affinity chromatography. These methods rely either on the separation of a bound and free drug for subsequent conventional analysis or change in intrinsic parameters such as conformational properties of the protein. More recently developed techniques include surface plasmon resonance and solid-phase microextraction. Photoaffinity labeling, site-directed mutagenesis and x-ray crystallography are valuable techniques to identify the locations of binding sites on a protein. A new high-throughput assay based on the distribution of a drug among plasma water, plasma proteins, and solid-supported lipid membranes (Transil) has been reported to produce valid results, even for drugs that are strongly bound to plasma proteins. This new method may be suited for examining highly lipophilic drugs that adsorb onto surfaces due to their low solubility in aqueous media. Such a method may promote drug discovery and development for high-throughput determination of protein binding.
Implication
The TRANSIL High Sensitivity Binding kit is the method recommended by the authors for determining plasma protein binding of highly lipophilic drugs.
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Solid-supported lipid membranes as a tool for determination of membrane affinity: high-throughput screening of a physicochemical parameter
Loidl-Stahlhofen, A.; Eckert, A.; Hartmann, T.; Schöttner, M. (2001)
J Pharm Sci. 2001 May;90(5):599-606.Abstract
Quantification of membrane affinity is an important early screening step in modern drug design. However, current approaches using different lipid membrane models usually are time-consuming or show severe experimental drawbacks. In this paper we describe the use of solid-supported lipid membranes (TRANSIL) as a new tool for the determination of membrane affinity. Eighteen pharmaceuticals (neutrals, acids, and bases) have been analyzed for their lipophilicity at physiological pH in an automated setup; phase separation of lipid and aqueous phase can be achieved simply by a short low-speed centrifugation or filtration. The membrane affinity is then calculated by quantification of the total drug concentration and the amount of drug remaining in the aqueous phase after incubation with solid-supported lipid membranes. Lipophilicity parameters relying on solid-supported lipid membranes correlate well with octanol-water partition coefficients K(ow) for neutral organic compounds (range of log K(ow) = 1.5-5, n = 7, r = 0.93). Data acquisition with this lipid membrane model system is highly re-producible. Even in the case of ionizable drugs, where K(ow) tends to underestimate membrane affinity, the latter can be correctly quantified using solid-supported lipid membranes: data comparison shows good agreement of the presented approach with established but time-consuming standardized lipid/buffer systems. Solid-supported lipid membranes allow a fast and reliable quantification of membrane affinity, enabling high-throughput screening of this physicochemical parameter.
Implication
This study illustrates the utility of the TRANSIL Intestinal Absorption Assay kit, for tissue distribution studies in pharmaceutical research.
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Lipophilic and polar interaction forces between acidic drugs and membrane phospholipids encoded in IAM-HPLC indexes: their role in membrane partition and relationships with BBB permeation data
Grumetto, L.; Carpentiero, C.; Di Vaio, P.; Frecentese, F.; Barbato, F. (2019)
J Pharm Biomed Anal. 2013 Mar 5;75:165-72.Abstract
The membrane phospholipid affinity data, log k(w)(IAM), for 18 acidic and unionized drugs spanning a wide lipophilicity range were measured by HPLC on two different phospholipid stationary phases, i.e. IAM.PC.MG and IAM.PC.DD2. These data related weakly with both log P(N) values, the n-octanol/water partition coefficients of the neutral forms, and log D(7.4) values, the n-octanol/water partition coefficients of the mixtures of neutral and ionized forms at pH 7.4. The lack of collinearity confirms that, differently from partition in n-octanol/water, partition in phospholipids encodes not only lipophilic/hydrophobic intermolecular recognition forces but also ionic bonds, due to electrostatic interactions between electrically charged species and phospholipids, according to the "pH piston hypothesis". Since, differently from bases, electrostatic interactions between acids and phospholipids take place at the surface of phospholipid layers (choline moieties), and not near their lipophilic core (phosphate moieties), they were parameterized by a new procedure yielding "Δ'log k(w)(IAM)" parameters, i.e. the difference between the IAM retention factors observed for the analytes and those of neutral compounds with the same n-octanol partition values displayed by the analytes at pH 7.4. All acidic analytes, but one, and all unionized analytes, but the unionizable ones, showed positive Δ'log k(w)(IAM) values, indicating that they partition stronger in phospholipids than in n-octanol. Log BB values (capability to pass BBB) weakly related with both lipophilicity and phospholipid affinity values; in contrast they are inversely related with Δ'log k(w)(IAM) values. The relationships between log BB and Δ'log k(w)(IAM) practically overlapped the previously found log BB/Δlog k(w)(IAM) relationships for bases. The excess of polar interaction component between acidic drugs and phospholipids, mainly electrostatic forces, although enhancing partition in phospholipids, hinders membrane passage, analogously to the behavior previously reported for bases. The study suggests that IAM-HPLC is an effective technique to perform simple and fast measurements of the intermolecular recognition forces related to membrane partition and permeation. This can contribute to better understand the mechanisms governing both partition of charged species in cell membranes and passage through them, also allowing the possible optimization of the pharmacokinetic properties of the drugs at the early stages of their development
Implication
The publication provides the background why the TRANSIL Brain Absorption Assay kit provides better estimates of brain pentration than traditional logP or octanol/water partitioning approaches. While the TRANSIL assay is even easier to perform than these mehtods, it yields comparable, if not more reproducible, results for brain tissue binding and the brain to plasma distribution (logBB).
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Capillary electrochromatography as a new tool to assess drug affinity for membrane phospholipids
Barbato, F.; Grumetto, L.; Carpentiero, C.; Rocco, A.; Fanali, S. (2011)
J Pharm Biomed Anal. 2011 Apr 5;54(5):893-9.Abstract
This work proposes a new capillary electrochromatography (CEC) method for determination of drug partition in membrane phospholipids. CEC experiments were carried out in a 100 μm (ID) fused-silica capillary, partially packed with a chromatographic phospholipid stationary phase, so-called Immobilized Artificial Membrane, IAM.PC.DD2. The observed retention values were corrected by both the electro-osmotic and electrophoretic mobility values, measured by capillary electrophoresis (CE) experiments, assuming the values of the logarithms of "chromatographic" affinity factors, log k(CEC) as indexes of affinity for phospholipids. Analogously to biochromatography, all the values were determined with a totally aqueous mobile phase, or extrapolated to 100% aqueous buffer. The analytes were 16 structurally unrelated compounds, of basic, neutral, and acidic nature. To evaluate the effectiveness of CEC data to describe partition in phospholipids, log k(CEC) were related to both log P and log k(w)(IAM) values. log P are the lipophilicity values expressed as the logarithms of n-octanol/water partition coefficients and log k(w)(IAM) are the retention data measured by High Performance Liquid Chromatography (HPLC) on an IAM.PC.DD2 column, assumed as the reference values for phospholipid affinity. Phospholipid affinity scale by CEC related to that achieved by HPLC, but only if two different subclasses were considered separately, i.e. protonated and unprotonated analytes; indeed, all the compounds protonated at the experimental pH value (7.0) were retained stronger in CEC than in HPLC. This discrepancy may be due to the use of different buffers in CEC and HPLC since, to avoid the occurrence of a high current, the eluent in CEC experiments was of different composition and lower ionic strength than in HPLC. CEC analyses were faster and required lower amounts of both solvent and stationary phase than HPLC; moreover, with the exception of only three analytes, all analyses were performed with 100% aqueous eluents avoiding time-consuming and tedious extrapolation procedures.
Implication
This research illustrates why it is important to consider the actual membrane structure for assessing tissue distribution and drugs' membrane permeability. TRANSIL assay kits for membrane binding and permeability address this issue and provide easy and versatile tools for measureing the interaction with various membrane types.
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The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N-terminal region of the enzyme
Thorolfsson, M.; Doeskeland, A.P.; Muga, A.; Martinez, A. (2002)
FEBS Lett. 2002 May 22;519(1-3):221-6.Abstract
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL). The use of TRANSIL beads has allowed the determination of apparent dissociation constants (Kd) for the binding of the enzyme to negatively charged bilayers (Kd=230-380 microM, at pH 6.0-7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal alpha-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but alpha-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH.
Implication
The paper exemplifies the broad spectrum of applications of the TRANSIL Intestinal Absorption Assay kit, which goes way beyond tissue distribution, intestinal permeability, and the estimation of the volume of distribution.
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Determination of the Plasma Protein Binding of Liraglutide Using the EScalate Equilibrium Shift Assay
Ungewiss, J; Gericke, S.; Boriss, H. (2019)
J Pharm Sci. 2019 Mar;108(3):1309-1314Purpose
The plasma protein binding capability of drug substances represents an important assay parameter in drug discovery and development. For very strong plasma protein binding molecules, however, the free fraction in plasma fu is very small and therefore difficult to determine with standard methods. To solve this problem, the EScalate equilibrium shift in vitro assay was developed.
Method
Escalating concentrations of plasma were found to shift the binding equilibrium in solution between the test item and immobilized human serum albumin. Following liquid chromatography coupled to mass spectrometry analysis of the samples, the test compound's fu in plasma is calculated with a 2-dimensional fitting procedure. Comparability of EScalate assay results was demonstrated for 4 extensively studied small molecule drugs (carbamazepine, desipramine, pyrimethamine, and warfarin) as well as for liraglutide, a fatty acid-conjugated peptide drug with very strong plasma protein binding. The results were in good agreement with published data. A free fraction of 0.51% was determined for liraglutide. Our results confirm the compound's very strong plasma protein binding properties in a novel and robust assay system.
Implication
The paper shows that the TRANSIL technology can be used for optimizing peptide half-life as it enables easy measurement of plasma protein binding.
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Bioanalysis for plasma protein binding studies in drug discovery and drug development: views and recommendations of the European Bioanalysis Forum
Buscher, B.; Laakso, S.; Mascher, H.; Pusecker, K.; Doig, M.; Dillen, L.; Wagner-Redeker, W.; pfeifer, T.; Delrat, P.; Timmerman, P. (2014)
Pharm Res. 2019 Nov 7;36(12):178.Purpose
Plasma protein binding (PPB) is an important parameter for a drug’s efficacy and safety that needs to be investigated during each drug-development program. Even though regulatory guidance exists to study the extent of PPB before initiating clinical studies, there are no detailed instructions on how to perform and validate such studies. To explore how PPB studies involving bioanalysis are currently executed in the industry, the European Bioanalysis Forum (EBF) has conducted three surveys among their member companies
Conclusion
The EBF recommends a tiered approach to the design of PPB studies and the bioanalysis of PPB samples: ‘PPB screening’ experiments in (early) drug discovery versus qualified and validated procedures in drug development. When in vitro or in vivo plasma protein binding of highly lipophilic and sticky drugs is analyzed many companies resort to the Transil High Sensitivity binding kit.
Best practice & recommendations for in vitro PPB in drug development
Even though regulatory guidance exists that requires PPB to be investigated prior to the initiation of clinical studies there are no instructions on how to perform such studies. The technical aspects of the different techniques used to separate the free fraction are not the primary scope of this paper, but it must be stressed that different techniques, all with their own specific limitations, may give different results. The decision of which technique to use must be carefully evaluated based on the physical and chemical characteristics of the compound, and the experience gained in drug discovery.