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Role of Divalent Metal Ions in Atypical Nonlinear Plasma Protein Binding Behavior of Tigecycline
Journal of Pharmaceutical Sciences xxx (2016) 1-6
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Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Role of Divalent Metal Ions in Atypical Nonlinear Plasma Protein Binding Behavior of Tigecycline Ravi Shankar Prasad Singh, Jatinder Kaur Mukker, Amelia N. Deitchman, Stephanie K. Drescher, Hartmut Derendorf* Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2016 Revised 17 July 2016 Accepted 18 July 2016
In typical nonlinear plasma protein binding (PPB) behavior, the free fraction increases with increasing total concentrations. In contrast, when a drug exhibits atypical nonlinear PPB behavior, the free fraction decreases with increasing total concentrations. Tigecycline, a novel glycylcycline, exhibits atypical nonlinear PPB behavior, but the mechanism of such behavior is currently unknown. Because tigecycline can form complexes with metal ions, an interaction between metal ion, tigecycline, and plasma proteins was hypothesized but not further investigated. The current work explores the role of metal ions in the atypical nonlinear PPB behavior of tigecycline and proposes a plausible mechanism of atypical nonlinear PPB behavior. The addition of ethylenediaminetetraacetic acid resulted in 10- to 30-fold higher unbound fractions, and the atypical behavior was nullified. The saturation of ethylenediaminetetraacetic acid chelation, by addition of excessive divalent metal ions, such as calcium and magnesium, led to the return of the atypical nonlinear PPB behavior. Different possible mechanisms were evaluated by simulation, and a plausible mechanism was proposed. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: tigecycline metal-ion complexation atypical nonlinear plasma protein binding mechanism nonlinear plasma protein binding
Introduction Plasma protein binding (PPB) is a reversible and saturable process that can be described by a Langmuir model.1,2 At lower drug concentrations, protein-bound drug (Cb) increases linearly with increasing unbound drug concentrations (Cu), with unbound fraction of drug (fu) remaining constant. However, the saturation of protein at higher drug concentrations causes Cb to increase less than proportionally to increases in Cu; thereby, fu no longer remains constant and fu increases with the increasing total drug concentration. This type of nonlinear PPB behavior is referred to as “typical” nonlinear PPB. In contrast, “atypical” nonlinear PPB is represented by a decrease in unbound fraction with an increase in total drug concentration (Fig. 1). Because the free fraction of the drug affects its pharmacokinetics and pharmacodynamics,3 PPB determination is vital for dosage regimen design. Fortunately, most drugs follow linear PPB in the therapeutic range, which makes estimation of unbound fraction relatively easy. On the other hand, some drugs such as
This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2016.07.013. * Correspondence to: Hartmut Derendorf (Telephone: þ1-352-2737856; Fax: þ1352-3923249). E-mail address: [email protected]fl.edu (H. Derendorf).
disopyramide,4,5 valproic acid,1 and indisulam2 exhibit typical nonlinear PPB at therapeutic doses. This nonlinearity can pose a challenge in dosing regimen design of these drugs. Tigecycline, a novel glycylcycline antibiotic, with efficacy against a variety of Gram-positive and Gram-negative infections and with indications in complicated intraabdominal and complicated skin and skin structure infections,6 is known to have atypical nonlinear PPB. The in vitro PPB at 0.1, 1, and 15 mg/mL were 29, 11, and 4 percent, respectively.7 Muralidharan et al.8 also reported a decrease in unbound fraction with the increase in total concentration of tigecycline, as determined by ultrafiltration and ultracentrifugation methods. Furthermore, Mukker et al.9 confirmed that the atypical nonlinear PPB behavior is not an artifact of protein binding determination methods. In addition to in vitro determinations, the atypical PPB behavior was also observed clinically in diabetic patients with chronic wound infections.10 Other than tigecycline, an upcoming fluorocycline antibiotic, eravacycline, is also known to exhibit atypical nonlinear PPB in 6 different species.11 Such atypical behavior is counterintuitive, and the mechanism of this atypical nonlinear binding behavior is currently unknown. Given the structural similarity of tigecycline with tetracycline, a complex interaction with metal ions was proposed earlier but was not investigated further.8 The aim of this current work is to investigate the role of metal ions in the PPB of tigecycline and propose a plausible mechanism for its atypical nonlinear PPB behavior.
http://dx.doi.org/10.1016/j.xphs.2016.07.013 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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Figure 1. Left panel shows an example of the relationship between bound and unbound concentrations of drugs described by Langmuir model, which can explain linear and typical PPB. Right panel shows the change in fraction unbound with the change in total concentration in case of typical (solid line) and atypical (dashed line) PPB.
Materials and Methods Chemicals, Reagents, and Apparatus The heparinized unfiltered human plasma pooled from both genders (from more than 6 individuals) was obtained from Bioreclamation LLC (Westbury, NY). Ethylenediaminetetraacetic acid (EDTA), methanol, acetonitrile, sodium chloride, and formic acid were obtained from Fisher Scientific (Pittsburgh, PA). The ultrafiltration cartridges were purchased from EMD Millipore (Billerica, MA). Tigecycline was purchased from TSZ CHEM (Framingham, MA). Calcium chloride was obtained from Allied Chemical Corporation (Morristown, NJ), and magnesium chloride was purchased from Sigma-Aldrich Corporation (St. Louis, MO). In Vitro PPB of Tigecycline in the Presence and Absence of EDTA The plasma protein binding of tigecycline in pooled heparinized human plasma was determined in vitro at concentrations of 0.1, 1, and 10 mg/mL in the presence and absence of EDTA. The determinations were done in triplicate. An aliquot of tigecycline stock solutions in water:methanol (50:50) was added to either saline or EDTAcontaining human plasma. To prepare tigecycline-containing plasma samples with EDTA, 50 mL of EDTA (15 mg/mL) was added to 450 mL of plasma-containing tigecycline to achieve final tigecycline concentrations of 0.1, 1, and 10 mg/mL. Similarly, an aliquot of 50 mL of saline was added instead of EDTA solution to prepare tigecycline-containing plasma samples without EDTA. These tigecycline-containing plasma samples (with and without EDTA) were equilibrated at 37 C for 15 min and added to the Millipore Centrifree® cartridges (30 kDa). The cartridges were centrifuged at 1000 g for 15 min, and the filtrates were stored at 80 C until analysis. Effect of Metal Ions on PPB of Tigecycline Divalent metal ions, such as calcium or magnesium, were added to tigecycline solution in plasma (with and without EDTA) in triplicate. The tigecycline-containing pooled human plasma, with and without EDTA, was prepared as described in section In Vitro PPB of Tigecycline in the Presence and Absence of EDTA. The tigecycline-containing plasma was equilibrated for 15 min at 37 C before addition of calcium chloride (equivalent of 10.6-mM Ca2þ) and magnesium chloride (equivalent of 4.2-mM Mg2þ) solutions. After addition of these divalent metal ions, the samples were equilibrated for another
15 min before ultrafiltration using Millipore Centrifree® cartridges. The cartridges with plasma samples were centrifuged at 1000 g at 37 C for 15 min, and the filtrates were stored at 80 C until analysis. Sample Analysis Tigecycline concentrations were quantified by a validated liquid chromatography-mass spectrometry method.9 In brief, an aliquot of collected eluate was mixed with internal standard, and chromatographic separation was achieved using Varian Polaris C18-A (50 3 mm) column with guard cartridge (MetaGuard 2.0-mm Polaris 3 C18-A guard). A gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile:methanol (1:1) over 10 min at the flow rate of 0.8 mL/min was used as mobile phase and the column eluate was split into 1:1 before electrospray ionization. Tigecycline and internal standard, eravacycline, were monitored at 586.3/569.2 and 280.2/84.0, respectively. A linear regression with a 1/x2 weighting factor was used, and the response was linear between 10-1000 ng/mL. Statistical Analysis The percent fractions unbound at different concentrations of tigecycline were compared using a one-way ANOVA with Tukey's post hoc test (using R 3.0.1) for groupwise comparison. A p value of less than 0.05 was considered statistically significant. Model Simulation Different possible mechanisms involving divalent metal ions were proposed (Fig. 2), and simulations were performed using R 3.0.1 to select the model that can explain the experimentally observed characteristics of atypical PPB of tigecycline. The models whose simulations failed to meet all the observed characteristics of atypical nonlinear PPB behavior were rejected. Putative Mechanism-1 This first proposed mechanism assumes the independent binding of tigecycline to plasma protein and metal ions and that the observed unbound concentration is the mixture of true unbound concentration (Cu) and metal ionetigecycline complexes (Cb,mi), as drug bound to metal ion is freely filterable in protein binding experiments. A sequence of unbound concentrations between 0.001 and 100 mg/mL were simulated. The protein-bound
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Figure 2. Proposed mechanisms causing atypical nonlinear PPB behavior of tigecycline. In mechanism 4, the dotted line shows the metal ioneassisted positive cooperativity of tigecycline binding. Cb,pp, the concentration tigecycline bound to plasma protein; Cb,mi, the concentration of tigecycline bound to metal ion; Cb,comp, the concentration of tigecyclineemetal ioneprotein complex; Cu, the unbound concentration of tigecycline; MI, metal ion; PP, plasma protein.
concentration (Cb,pp) and concentration of metal ionetigecycline complexes were calculated using Equations 1 and 2, respectively. Amax,pp, Kd,pp, and g1 in Equation 1 refer to the capacity, affinity, and cooperativity factors, respectively, for the tigecyclineeplasma protein interaction; Amax,mi and Kd,mi are capacity and affinity factors for the tigecyclineemetal ion interaction. Cb,pp, Cb,mi, and Cu sum to the total concentration of tigecycline (CT). The apparent 0 unbound concentration (Cu) is the sum of Cb,mi and Cu, and the 0 unbound fraction (fu) was calculated by dividing Cu by CT. A set of values for the different parameters that showed the atypical behavior were used as default parameter values for local sensitivity analysis. The local sensitivity analysis was performed by perturbing one parameter while keeping other parameters fixed at their default values.
Cb;pp ¼
Cb;mi ¼
Amax;pp Cug1 g1 Kd;pp þ Cug1
Amax;mi Cu Kd;mi þ Cu
(1)
(2)
The effect of EDTA was simulated by setting Amax,mi equal to zero as EDTA will chelate metal ions, making them unavailable for binding to tigecycline. We assume that EDTA has higher binding affinity to divalent metal ions than tigecycline. Putative Mechanism-2 The second proposed mechanism assumes the independent binding of tigecycline to plasma protein, metal ions, and metal ioneplasma protein complex (Fig. 2). The simulations were performed as described in section Putative Mechanism-1 with an additional entity drug bound to metal ioneprotein complex (Cb,comp) calculated using Equation 3, where Amax,comp, Kd,comp, and g2 refer to the capacity, affinity, and cooperativity factors of tigecycline binding to metal ioneplasma protein complex. Cb,comp, Cb,pp, Cb,mi, and Cu sum to the total concentration of tigecycline (CT).
Putative Mechanism-3 The third mechanism also builds on Putative Mechanism-1 but also proposes the binding of tigecycline to metal ion forming a metal ionetigecycline complex. Tigecycline and metal ionetigecycline complex independently bind to plasma protein (Fig. 2). The concentration of metal ionetigecyclineeprotein complex (Cb,comp) was calculated using Equation 4, whereas all other steps in simulations were the same as described in Putative Mechanism-1.
Cb;comp ¼
g2 Amax;comp Cb;mi g2 g2 Kd;comp þ Cb;mi
(4)
The effect of EDTA was simulated by setting Amax,mi and Amax,comp equal to zero with the assumption that EDTA has a higher affinity for metal ions than tigecycline; and thereby, preventing the formation of tigecyclineemetal ion complex and subsequent binding of this complex to plasma protein. Putative Mechanism-4 The fourth mechanism proposes metal ioneassisted positive cooperativity of tigecycline binding to plasma protein (Fig. 2). The unbound concentrations of tigecycline were simulated in the range of 0.001 to 100 mg/mL, and the bound concentration was calculated using Equation 1. To simulate metal ioneassisted positive cooperativity, g1 was considered as 2 for simulations. Based on local sensitivity analyses, g1 must be 1.5 to exhibit atypical nonlinear behavior (Supplementary Fig. 4). The total concentration (CT) was obtained by summing the unbound concentration (Cu) and plasma protein-bound tigecycline concentration (Cb,pp). The fraction unbound was then calculated by dividing Cu by CT. The effect of metal-ion chelation was simulated by assuming loss of metal ioneassisted positive cooperativity factor (by assuming g1 ¼ 1 and a lowered PPB affinity [increase in Kd,pp]). Results In Vitro Plasma Protein Binding of Tigecycline
Cb;comp ¼
Amax;comp Cug2 g2 Kd;comp þ Cug2
(3)
The effect of EDTA was simulated using Amax,mi and Amax,comp equal to zero, with the assumption that EDTA has higher affinity to metal ion and metal ioneplasma protein complexes than tigecycline.
In vitro plasma protein binding was determined in the presence and absence of EDTA to evaluate the effect of metal ions on the protein binding of tigecycline. The percent unbound of tigecycline at total concentrations of 0.1, 1, and 10 mg/mL, when no EDTA was added, were 13.8 ± 2.2, 7.4 ± 6.7, and 2.2 ± 2.2 percent, respectively, whereas in the presence of EDTA, the percent unbound for each
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Figure 3. The percent-free fractions of tigecycline in heparinized human plasma pooled from both genders (n >6) at different concentrations of tigecycline in presence (1.5 mg/mL) of EDTA alone or in combination with calcium (10.6 mM) or magnesium (4.2 mM) ions. Equal volume of saline or water was added instead of EDTA in controls. The p values for comparisons are summarized in Table 1.
total tigecycline concentration were 39.9 ± 9.2, 35.4 ± 8.9, and 33.1 ± 8.4 percent, respectively. Without the addition of EDTA, free fractions of tigecycline at 0.1 and 10 mg/mL were significantly different (p < 0.001). However, in the presence of EDTA, there were no significant differences (p ¼ 0.415) between free fractions at different concentrations. In addition, the free fractions of tigecycline increased significantly (p < 0.001) in the presence of EDTA.
atypical nonlinear PPB behavior for a limited set of parameter values, but in no case did the chelation of metal ions increase the unbound fraction and nullify the atypical behavior. Simulations of mechanism 4, which assumes metal ion-assisted cooperativity, also showed a U-shaped fraction unbound curve of atypical nonlinear PPB. In addition, the chelation of metal ions not only nullified this atypical nonlinear PPB behavior but also increased the unbound fractions of tigecycline (Fig. 4).
Effect of Metal Ions An excess of metal ions, such as calcium and magnesium, were added to saturate EDTA to reverse its effect on PPB (described in section In Vitro Plasma Protein Binding of Tigecycline). Similar to the effect seen in section In Vitro Plasma Protein Binding of Tigecycline, the addition of EDTA resulted in >3-fold higher free fraction and no significant differences (p < 0.001) in free fractions at different concentrations. The addition of calcium and magnesium in addition to EDTA significantly decreased the free fraction and resulted in reversion to the atypical nonlinear binding behavior. Significant differences in unbound fractions were observed for all concentrations when calcium was added, whereas the addition of magnesium resulted in significant decreases at 1 and 10 mg/mL only. Addition of saline did not change the unbound concentrations significantly (Fig. 3). Tukey's comparisons of different groups are presented in Table 1.
Discussion The unbound fractions of drugs are expected to increase nonlinearly at higher drug concentrations as drug-binding sites on plasma proteins are saturated. Contrary to this expected behavior, tigecycline shows atypical nonlinear protein binding behavior in which the fraction unbound decreases with the increase in total concentration over a certain concentration range. Evaluation of plasma protein binding by several methods, including ultrafiltration, ultracentrifugation, and microdialysis,9 showed that the atypical behavior of tigecycline is not an artifact. In addition, the atypical behavior has also been observed in vivo10 in diabetic patients, but the mechanism of such counterintuitive behavior is currently unknown. Given the structural similarity of tigecycline with tetracycline, earlier investigations8 indicated that a complex interaction
Model Simulation Simulations were performed to investigate the underlying mechanism of atypical nonlinear PPB behavior; if correct, the simulation should be able to satisfy the experimentally observed properties of the atypical nonlinear PPB of tigecycline. The 3 experimentally observed properties of the atypical nonlinear PPB of tigecycline were (a) the unbound fraction decreases with increases in the total concentration, and the unbound fraction increases at very high total concentrations (“U” shape), (b) the chelation of metal ions nullifies this atypical PPB effect, and (c) the chelation of metal ions increases the unbound fraction. Four different possible mechanisms involving metal ions were proposed, and local sensitivity analyses of the models were performed. All 4 mechanisms exhibited a decrease in unbound concentration with increasing total concentrations of tigecycline for a set of parameters. Simulations of putative mechanisms based on different sets of parameters showed varying profiles (Supplementary Figs. 1-4). Local sensitivity analyses of mechanisms 1, 2, and 3 exhibited
Table 1 Pairwise Comparison of Unbound Fractions Between Different Groups Using Tukey's Test to Show the Effect of Metal-Ion Chelation on Tigecycline's Plasma Protein Binding Groups Comparisonsa
p Values at Total Concentration (mg/mL) 0.1 mg/mL
1 mg/mL
10 mg/mL
EDTA þ Ca versus EDTA EDTA þ Mg versus EDTA Saline versus EDTA Water versus EDTA Saline versus EDTA þ Ca Water versus EDTA þ Ca Saline versus EDTA þ Mg Water versus EDTA þ Mg Water versus saline
<0.001 0.533 <0.001 <0.001 0.132 0.061 <0.001 <0.001 0.985
<0.001 0.007 <0.001 <0.001 0.683 0.732 <0.001 <0.001 1.000
<0.001 <0.001 <0.001 <0.001 1.000 1.000 <0.001 <0.001 1.000
a EDTA ¼ 1.5 mg/mL of EDTA; EDTA þ Ca ¼ 1.5 mg/mL of EDTA þ 10.6-mM calcium ion; EDTA þ Mg ¼ 1.5 mg/mL of EDTA þ 4.2-mM magnesium ion; saline and water ¼ volume equivalent to EDTA solution was replaced with saline and water, respectively.
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Figure 4. Simulation of model representing mechanism 4 in absence (left panel, parameter values: Amax,pp ¼ 1000, Kd,pp ¼ 10, g ¼ 2) and presence (right panel, parameter values: Amax,pp ¼ 1000, Kd,pp ¼ 500, g ¼ 1) of metal-ion (MI) chelating agent.
between drug, metal ion, and plasma protein could possibly be responsible for the observed atypical PPB effect, but no investigations were performed. Earlier reports showed a decreasing unbound fraction of tigecycline with increasing total concentrations, but these PPB determinations were only determined up to a maximum concentration of 15 mg/mL. However, the unbound fraction increased at very high concentrations (100 mg/mL) of tigecycline, thus giving a “U-shape” curve overall.9 This U-shaped unbound fraction curve was also observed for doxycycline and minocycline in the plasma of rats, mice, and monkeys.12 Other than these 2 characteristics of atypical nonlinear PPB, nothing about this behavior was known. The current work aimed to study the interactions between tigecycline, metal ions, and plasma proteins. In the first set of experiments, the metal ions present in the plasma were chelated using EDTA. The fraction unbound increased several fold, and there were no significant differences between plasma protein binding at different concentrations, indicating that the divalent metal ions present in the plasma play an important role in the atypical PPB behavior of tigecycline. This increase was in concurrence with the results obtained by Chen et al.,13 who observed higher unbound fractions of tigecycline when EDTA was used as anticoagulant in comparison to heparin. Because EDTA is capable of chelating several metal ions present in the plasma, the increased unbound fraction shows that metal ions play a vital role in atypical nonlinear PPB behavior. By saturating EDTA's chelating capacity, one can determine if the EDTA effect, the nullification of atypical nonlinear PPB behavior, is reversible. Excess divalent metal ions (Ca2þ and Mg2þ) were added to saturate the EDTA added to plasma samples, and the atypical behavior was restored. This showed that the effect of EDTA is reversible and further supported the involvement of divalent metal ions in the atypical PPB behavior of tigecycline. To understand the interplay of metal ions, tigecycline, and plasma proteins, different possible interactions were proposed and simulated to determine if the observed characteristics of tigecycline's atypical nonlinear PPB were present. Based on our experimental results, the following characteristics of atypical nonlinear PPB behavior of tigecycline can be delineated: (a) the unbound fraction shows a “U” shaped behavior with increasing total concentrations of tigecycline; (b) the presence of metal ions is required for this atypical nonlinear PPB behavior and chelation of metal ions nullifies this atypical nonlinear behavior; and (c) the chelation of metal ions increases the unbound fraction. Different possible
mechanisms were proposed (Fig. 2), simulated, and local sensitivity analyses were performed to evaluate these models based on the known characteristics of atypical PPB. Local sensitivity analyses showed that out of 4 different proposed mechanisms, mechanisms 1-3 were able to show the “U” shaped curve with a limited range of parameters, but the unbound fraction did not increase when metal ions were removed from the system. Mechanism 4, which assumed metal ioneassisted cooperativity in tigecycline binding, was able to satisfy all the observed characteristics of atypical nonlinear binding and cooperativity (g 1.5) was required for the atypical nonlinear behavior. Cooperativity of metal ions may influence the protein binding characteristics of ligands (drugs). Brannvall et al. observed that the cooperativity between different divalent metal ions controlled the ribozyme cleavage rate and cleavage site recognition.14 Stojanovic et al. observed higher binding constants for tigecycline with human serum albumin in the presence of Ca2þ, Cu2þ, and Fe3þ using fluorescence, UV-Vis spectroscopic, and molecular docking methods.15 Thus, we propose metal ion cooperativity with tigecycline as a possible reason for atypical nonlinear PPB behavior. However, direct experimental evidence of cooperativity between metal ions and tigecycline will be required to confirm this hypothesis. The current work has a few limitations: the proposed mechanisms assume interactions between a single plasma protein with metal ions and tigecycline. As plasma protein is a mixture of several proteins, more complex interactions between different proteins may also be possible. In addition, the change in unbound fraction was used as indirect evidence of the interactions between tigecycline, divalent metal ions, and plasma proteins; a study of interactions at the molecular level will be confirmatory evidence in the elucidation of the mechanism of atypical nonlinear PPB.
Conclusion The interactions between metal ions and tigecycline with plasma proteins were investigated. Divalent metal ions are necessary for the atypical nonlinear PPB behavior of tigecycline, and the removal of metal ions nullifies the atypical nonlinear behavior and increases the unbound fraction. This work identified a plausible mechanism of atypical nonlinear binding behavior of tigecycline. The metal ioneassisted positive cooperativity in tigecycline's binding with plasma protein is likely an explanation for the atypical
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nonlinear PPB behavior of tigecycline. However, further direct evidence is needed for confirmation of this mechanism. References 1. Kumar S, Wong H, Yeung SA, Riggs KW, Abbott FS, Rurak DW. Disposition of valproic acid in maternal, fetal, and newborn sheep. II: metabolism and renal elimination. Drug Metab Dispos. 2000;28(7):857-864. 2. Zandvliet AS, Copalu W, Schellens JH, Beijnen JH, Huitema AD. Saturable binding of indisulam to plasma proteins and distribution to human erythrocytes. Drug Metab Dispos. 2006;34(6):1041-1046. 3. Schmidt S, Gonzalez D, Derendorf H. Significance of protein binding in pharmacokinetics and pharmacodynamics. J Pharm Sci. 2010;99(3):1107-1122. 4. Lima JJ, Boudoulas H, Blanford M. Concentration-dependence of disopyramide binding to plasma protein and its influence on kinetics and dynamics. J Pharmacol Exp Ther. 1981;219(3):741-747. 5. Meffin PJ, Robert EW, Winkle RA, Harapat S, Peters FA, Harrison DC. Role of concentration-dependent plasma protein binding in disopyramide disposition. J Pharmacokinet Biopharm. 1979;7(1):29-46. 6. U.S. Food and Drug Administration. Tygacil (Tigecycline) label. Available at: http://www.accessdata.fda.gov/drugsatfda_docs/label/2016/021821s042lbl. pdf. Accessed August 10, 2016. 7. U.S. Food and Drug Administration. Tygacil (tigecycline) dclinical pharmacology and biopharmaceutics review(s). Washington, DC: FDA; 2005. Available at: http://www.accessdata.fda.gov/drugsatfda_docs/nda/2005/021821Orig1 s000ClinPharmR.pdf. Accessed August 10, 2016.
8. Muralidharan G, Micalizzi M, Speth J, Raible D, Troy S. Pharmacokinetics of tigecycline after single and multiple doses in healthy subjects. Antimicrob Agents Chemother. 2005;49(1):220-229. 9. Mukker JK, Singh RP, Derendorf H. Determination of atypical nonlinear plasmaprotein-binding behavior of tigecycline using an in vitro microdialysis technique. J Pharm Sci. 2014;103(3):1013-1019. 10. Bulik CC, Wiskirchen DE, Shepard A, Sutherland CA, Kuti JL, Nicolau DP. Tissue penetration and pharmacokinetics of tigecycline in diabetic patients with chronic wound infections described by using in vivo microdialysis. Antimicrob Agents Chemother. 2010;54(12):5209-5213. 11. Singh RS, Falcao N, Sulcliffe JA, Derendorf H. Plasma Protein Binding of Eravacycline in Mouse, Rat, Rabbit, Cynomolgus Monkey, African Green Monkey and Human Using Microdialysis. ICAAC. Denver, Colorado: American Society of Microbiology; 2013. 12. Chaturvedi P, Esposito C, Koroma J, Cannon EP, Tanaka SK. In vitro assessment of plasma protein binding and metabolic stability of BAY 73-6944/PTK 0796. In: Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). 2003. 13. Chen Q, Tung EC, Ciccotto SL, et al. Effect of the anticoagulant ethylenediamine tetra-acetic acid (EDTA) on the estimation of pharmacokinetic parameters: a case study with tigecycline and ciprofloxacin. Xenobiotica. 2008;38(1):76-86. 14. Brannvall M, Kirsebom LA. Metal ion cooperativity in ribozyme cleavage of RNA. Proc Natl Acad Sci U S A. 2001;98(23):12943-12947. Jeli 15. Stojanovi c SD, Jankovi c SM, Matovi c ZD, Jakovljevi c IZ, c RM. Interaction between tigecycline and human serum albumin in aqueous solution. Monatshefte für Chem - Chem Monthly. 2015;146(2):399-409.
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Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Role of Divalent Metal Ions in Atypical Nonlinear Plasma Protein Binding Behavior of Tigecycline Ravi Shankar Prasad Singh, Jatinder Kaur Mukker, Amelia N. Deitchman, Stephanie K. Drescher, Hartmut Derendorf* Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, Florida 32610
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 June 2016 Revised 17 July 2016 Accepted 18 July 2016
In typical nonlinear plasma protein binding (PPB) behavior, the free fraction increases with increasing total concentrations. In contrast, when a drug exhibits atypical nonlinear PPB behavior, the free fraction decreases with increasing total concentrations. Tigecycline, a novel glycylcycline, exhibits atypical nonlinear PPB behavior, but the mechanism of such behavior is currently unknown. Because tigecycline can form complexes with metal ions, an interaction between metal ion, tigecycline, and plasma proteins was hypothesized but not further investigated. The current work explores the role of metal ions in the atypical nonlinear PPB behavior of tigecycline and proposes a plausible mechanism of atypical nonlinear PPB behavior. The addition of ethylenediaminetetraacetic acid resulted in 10- to 30-fold higher unbound fractions, and the atypical behavior was nullified. The saturation of ethylenediaminetetraacetic acid chelation, by addition of excessive divalent metal ions, such as calcium and magnesium, led to the return of the atypical nonlinear PPB behavior. Different possible mechanisms were evaluated by simulation, and a plausible mechanism was proposed. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: tigecycline metal-ion complexation atypical nonlinear plasma protein binding mechanism nonlinear plasma protein binding
Introduction Plasma protein binding (PPB) is a reversible and saturable process that can be described by a Langmuir model.1,2 At lower drug concentrations, protein-bound drug (Cb) increases linearly with increasing unbound drug concentrations (Cu), with unbound fraction of drug (fu) remaining constant. However, the saturation of protein at higher drug concentrations causes Cb to increase less than proportionally to increases in Cu; thereby, fu no longer remains constant and fu increases with the increasing total drug concentration. This type of nonlinear PPB behavior is referred to as “typical” nonlinear PPB. In contrast, “atypical” nonlinear PPB is represented by a decrease in unbound fraction with an increase in total drug concentration (Fig. 1). Because the free fraction of the drug affects its pharmacokinetics and pharmacodynamics,3 PPB determination is vital for dosage regimen design. Fortunately, most drugs follow linear PPB in the therapeutic range, which makes estimation of unbound fraction relatively easy. On the other hand, some drugs such as
This article contains supplementary material available from the authors by request or via the Internet at http://dx.doi.org/10.1016/j.xphs.2016.07.013. * Correspondence to: Hartmut Derendorf (Telephone: þ1-352-2737856; Fax: þ1352-3923249). E-mail address: [email protected]fl.edu (H. Derendorf).
disopyramide,4,5 valproic acid,1 and indisulam2 exhibit typical nonlinear PPB at therapeutic doses. This nonlinearity can pose a challenge in dosing regimen design of these drugs. Tigecycline, a novel glycylcycline antibiotic, with efficacy against a variety of Gram-positive and Gram-negative infections and with indications in complicated intraabdominal and complicated skin and skin structure infections,6 is known to have atypical nonlinear PPB. The in vitro PPB at 0.1, 1, and 15 mg/mL were 29, 11, and 4 percent, respectively.7 Muralidharan et al.8 also reported a decrease in unbound fraction with the increase in total concentration of tigecycline, as determined by ultrafiltration and ultracentrifugation methods. Furthermore, Mukker et al.9 confirmed that the atypical nonlinear PPB behavior is not an artifact of protein binding determination methods. In addition to in vitro determinations, the atypical PPB behavior was also observed clinically in diabetic patients with chronic wound infections.10 Other than tigecycline, an upcoming fluorocycline antibiotic, eravacycline, is also known to exhibit atypical nonlinear PPB in 6 different species.11 Such atypical behavior is counterintuitive, and the mechanism of this atypical nonlinear binding behavior is currently unknown. Given the structural similarity of tigecycline with tetracycline, a complex interaction with metal ions was proposed earlier but was not investigated further.8 The aim of this current work is to investigate the role of metal ions in the PPB of tigecycline and propose a plausible mechanism for its atypical nonlinear PPB behavior.
http://dx.doi.org/10.1016/j.xphs.2016.07.013 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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Figure 1. Left panel shows an example of the relationship between bound and unbound concentrations of drugs described by Langmuir model, which can explain linear and typical PPB. Right panel shows the change in fraction unbound with the change in total concentration in case of typical (solid line) and atypical (dashed line) PPB.
Materials and Methods Chemicals, Reagents, and Apparatus The heparinized unfiltered human plasma pooled from both genders (from more than 6 individuals) was obtained from Bioreclamation LLC (Westbury, NY). Ethylenediaminetetraacetic acid (EDTA), methanol, acetonitrile, sodium chloride, and formic acid were obtained from Fisher Scientific (Pittsburgh, PA). The ultrafiltration cartridges were purchased from EMD Millipore (Billerica, MA). Tigecycline was purchased from TSZ CHEM (Framingham, MA). Calcium chloride was obtained from Allied Chemical Corporation (Morristown, NJ), and magnesium chloride was purchased from Sigma-Aldrich Corporation (St. Louis, MO). In Vitro PPB of Tigecycline in the Presence and Absence of EDTA The plasma protein binding of tigecycline in pooled heparinized human plasma was determined in vitro at concentrations of 0.1, 1, and 10 mg/mL in the presence and absence of EDTA. The determinations were done in triplicate. An aliquot of tigecycline stock solutions in water:methanol (50:50) was added to either saline or EDTAcontaining human plasma. To prepare tigecycline-containing plasma samples with EDTA, 50 mL of EDTA (15 mg/mL) was added to 450 mL of plasma-containing tigecycline to achieve final tigecycline concentrations of 0.1, 1, and 10 mg/mL. Similarly, an aliquot of 50 mL of saline was added instead of EDTA solution to prepare tigecycline-containing plasma samples without EDTA. These tigecycline-containing plasma samples (with and without EDTA) were equilibrated at 37 C for 15 min and added to the Millipore Centrifree® cartridges (30 kDa). The cartridges were centrifuged at 1000 g for 15 min, and the filtrates were stored at 80 C until analysis. Effect of Metal Ions on PPB of Tigecycline Divalent metal ions, such as calcium or magnesium, were added to tigecycline solution in plasma (with and without EDTA) in triplicate. The tigecycline-containing pooled human plasma, with and without EDTA, was prepared as described in section In Vitro PPB of Tigecycline in the Presence and Absence of EDTA. The tigecycline-containing plasma was equilibrated for 15 min at 37 C before addition of calcium chloride (equivalent of 10.6-mM Ca2þ) and magnesium chloride (equivalent of 4.2-mM Mg2þ) solutions. After addition of these divalent metal ions, the samples were equilibrated for another
15 min before ultrafiltration using Millipore Centrifree® cartridges. The cartridges with plasma samples were centrifuged at 1000 g at 37 C for 15 min, and the filtrates were stored at 80 C until analysis. Sample Analysis Tigecycline concentrations were quantified by a validated liquid chromatography-mass spectrometry method.9 In brief, an aliquot of collected eluate was mixed with internal standard, and chromatographic separation was achieved using Varian Polaris C18-A (50 3 mm) column with guard cartridge (MetaGuard 2.0-mm Polaris 3 C18-A guard). A gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile:methanol (1:1) over 10 min at the flow rate of 0.8 mL/min was used as mobile phase and the column eluate was split into 1:1 before electrospray ionization. Tigecycline and internal standard, eravacycline, were monitored at 586.3/569.2 and 280.2/84.0, respectively. A linear regression with a 1/x2 weighting factor was used, and the response was linear between 10-1000 ng/mL. Statistical Analysis The percent fractions unbound at different concentrations of tigecycline were compared using a one-way ANOVA with Tukey's post hoc test (using R 3.0.1) for groupwise comparison. A p value of less than 0.05 was considered statistically significant. Model Simulation Different possible mechanisms involving divalent metal ions were proposed (Fig. 2), and simulations were performed using R 3.0.1 to select the model that can explain the experimentally observed characteristics of atypical PPB of tigecycline. The models whose simulations failed to meet all the observed characteristics of atypical nonlinear PPB behavior were rejected. Putative Mechanism-1 This first proposed mechanism assumes the independent binding of tigecycline to plasma protein and metal ions and that the observed unbound concentration is the mixture of true unbound concentration (Cu) and metal ionetigecycline complexes (Cb,mi), as drug bound to metal ion is freely filterable in protein binding experiments. A sequence of unbound concentrations between 0.001 and 100 mg/mL were simulated. The protein-bound
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Figure 2. Proposed mechanisms causing atypical nonlinear PPB behavior of tigecycline. In mechanism 4, the dotted line shows the metal ioneassisted positive cooperativity of tigecycline binding. Cb,pp, the concentration tigecycline bound to plasma protein; Cb,mi, the concentration of tigecycline bound to metal ion; Cb,comp, the concentration of tigecyclineemetal ioneprotein complex; Cu, the unbound concentration of tigecycline; MI, metal ion; PP, plasma protein.
concentration (Cb,pp) and concentration of metal ionetigecycline complexes were calculated using Equations 1 and 2, respectively. Amax,pp, Kd,pp, and g1 in Equation 1 refer to the capacity, affinity, and cooperativity factors, respectively, for the tigecyclineeplasma protein interaction; Amax,mi and Kd,mi are capacity and affinity factors for the tigecyclineemetal ion interaction. Cb,pp, Cb,mi, and Cu sum to the total concentration of tigecycline (CT). The apparent 0 unbound concentration (Cu) is the sum of Cb,mi and Cu, and the 0 unbound fraction (fu) was calculated by dividing Cu by CT. A set of values for the different parameters that showed the atypical behavior were used as default parameter values for local sensitivity analysis. The local sensitivity analysis was performed by perturbing one parameter while keeping other parameters fixed at their default values.
Cb;pp ¼
Cb;mi ¼
Amax;pp Cug1 g1 Kd;pp þ Cug1
Amax;mi Cu Kd;mi þ Cu
(1)
(2)
The effect of EDTA was simulated by setting Amax,mi equal to zero as EDTA will chelate metal ions, making them unavailable for binding to tigecycline. We assume that EDTA has higher binding affinity to divalent metal ions than tigecycline. Putative Mechanism-2 The second proposed mechanism assumes the independent binding of tigecycline to plasma protein, metal ions, and metal ioneplasma protein complex (Fig. 2). The simulations were performed as described in section Putative Mechanism-1 with an additional entity drug bound to metal ioneprotein complex (Cb,comp) calculated using Equation 3, where Amax,comp, Kd,comp, and g2 refer to the capacity, affinity, and cooperativity factors of tigecycline binding to metal ioneplasma protein complex. Cb,comp, Cb,pp, Cb,mi, and Cu sum to the total concentration of tigecycline (CT).
Putative Mechanism-3 The third mechanism also builds on Putative Mechanism-1 but also proposes the binding of tigecycline to metal ion forming a metal ionetigecycline complex. Tigecycline and metal ionetigecycline complex independently bind to plasma protein (Fig. 2). The concentration of metal ionetigecyclineeprotein complex (Cb,comp) was calculated using Equation 4, whereas all other steps in simulations were the same as described in Putative Mechanism-1.
Cb;comp ¼
g2 Amax;comp Cb;mi g2 g2 Kd;comp þ Cb;mi
(4)
The effect of EDTA was simulated by setting Amax,mi and Amax,comp equal to zero with the assumption that EDTA has a higher affinity for metal ions than tigecycline; and thereby, preventing the formation of tigecyclineemetal ion complex and subsequent binding of this complex to plasma protein. Putative Mechanism-4 The fourth mechanism proposes metal ioneassisted positive cooperativity of tigecycline binding to plasma protein (Fig. 2). The unbound concentrations of tigecycline were simulated in the range of 0.001 to 100 mg/mL, and the bound concentration was calculated using Equation 1. To simulate metal ioneassisted positive cooperativity, g1 was considered as 2 for simulations. Based on local sensitivity analyses, g1 must be 1.5 to exhibit atypical nonlinear behavior (Supplementary Fig. 4). The total concentration (CT) was obtained by summing the unbound concentration (Cu) and plasma protein-bound tigecycline concentration (Cb,pp). The fraction unbound was then calculated by dividing Cu by CT. The effect of metal-ion chelation was simulated by assuming loss of metal ioneassisted positive cooperativity factor (by assuming g1 ¼ 1 and a lowered PPB affinity [increase in Kd,pp]). Results In Vitro Plasma Protein Binding of Tigecycline
Cb;comp ¼
Amax;comp Cug2 g2 Kd;comp þ Cug2
(3)
The effect of EDTA was simulated using Amax,mi and Amax,comp equal to zero, with the assumption that EDTA has higher affinity to metal ion and metal ioneplasma protein complexes than tigecycline.
In vitro plasma protein binding was determined in the presence and absence of EDTA to evaluate the effect of metal ions on the protein binding of tigecycline. The percent unbound of tigecycline at total concentrations of 0.1, 1, and 10 mg/mL, when no EDTA was added, were 13.8 ± 2.2, 7.4 ± 6.7, and 2.2 ± 2.2 percent, respectively, whereas in the presence of EDTA, the percent unbound for each
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Figure 3. The percent-free fractions of tigecycline in heparinized human plasma pooled from both genders (n >6) at different concentrations of tigecycline in presence (1.5 mg/mL) of EDTA alone or in combination with calcium (10.6 mM) or magnesium (4.2 mM) ions. Equal volume of saline or water was added instead of EDTA in controls. The p values for comparisons are summarized in Table 1.
total tigecycline concentration were 39.9 ± 9.2, 35.4 ± 8.9, and 33.1 ± 8.4 percent, respectively. Without the addition of EDTA, free fractions of tigecycline at 0.1 and 10 mg/mL were significantly different (p < 0.001). However, in the presence of EDTA, there were no significant differences (p ¼ 0.415) between free fractions at different concentrations. In addition, the free fractions of tigecycline increased significantly (p < 0.001) in the presence of EDTA.
atypical nonlinear PPB behavior for a limited set of parameter values, but in no case did the chelation of metal ions increase the unbound fraction and nullify the atypical behavior. Simulations of mechanism 4, which assumes metal ion-assisted cooperativity, also showed a U-shaped fraction unbound curve of atypical nonlinear PPB. In addition, the chelation of metal ions not only nullified this atypical nonlinear PPB behavior but also increased the unbound fractions of tigecycline (Fig. 4).
Effect of Metal Ions An excess of metal ions, such as calcium and magnesium, were added to saturate EDTA to reverse its effect on PPB (described in section In Vitro Plasma Protein Binding of Tigecycline). Similar to the effect seen in section In Vitro Plasma Protein Binding of Tigecycline, the addition of EDTA resulted in >3-fold higher free fraction and no significant differences (p < 0.001) in free fractions at different concentrations. The addition of calcium and magnesium in addition to EDTA significantly decreased the free fraction and resulted in reversion to the atypical nonlinear binding behavior. Significant differences in unbound fractions were observed for all concentrations when calcium was added, whereas the addition of magnesium resulted in significant decreases at 1 and 10 mg/mL only. Addition of saline did not change the unbound concentrations significantly (Fig. 3). Tukey's comparisons of different groups are presented in Table 1.
Discussion The unbound fractions of drugs are expected to increase nonlinearly at higher drug concentrations as drug-binding sites on plasma proteins are saturated. Contrary to this expected behavior, tigecycline shows atypical nonlinear protein binding behavior in which the fraction unbound decreases with the increase in total concentration over a certain concentration range. Evaluation of plasma protein binding by several methods, including ultrafiltration, ultracentrifugation, and microdialysis,9 showed that the atypical behavior of tigecycline is not an artifact. In addition, the atypical behavior has also been observed in vivo10 in diabetic patients, but the mechanism of such counterintuitive behavior is currently unknown. Given the structural similarity of tigecycline with tetracycline, earlier investigations8 indicated that a complex interaction
Model Simulation Simulations were performed to investigate the underlying mechanism of atypical nonlinear PPB behavior; if correct, the simulation should be able to satisfy the experimentally observed properties of the atypical nonlinear PPB of tigecycline. The 3 experimentally observed properties of the atypical nonlinear PPB of tigecycline were (a) the unbound fraction decreases with increases in the total concentration, and the unbound fraction increases at very high total concentrations (“U” shape), (b) the chelation of metal ions nullifies this atypical PPB effect, and (c) the chelation of metal ions increases the unbound fraction. Four different possible mechanisms involving metal ions were proposed, and local sensitivity analyses of the models were performed. All 4 mechanisms exhibited a decrease in unbound concentration with increasing total concentrations of tigecycline for a set of parameters. Simulations of putative mechanisms based on different sets of parameters showed varying profiles (Supplementary Figs. 1-4). Local sensitivity analyses of mechanisms 1, 2, and 3 exhibited
Table 1 Pairwise Comparison of Unbound Fractions Between Different Groups Using Tukey's Test to Show the Effect of Metal-Ion Chelation on Tigecycline's Plasma Protein Binding Groups Comparisonsa
p Values at Total Concentration (mg/mL) 0.1 mg/mL
1 mg/mL
10 mg/mL
EDTA þ Ca versus EDTA EDTA þ Mg versus EDTA Saline versus EDTA Water versus EDTA Saline versus EDTA þ Ca Water versus EDTA þ Ca Saline versus EDTA þ Mg Water versus EDTA þ Mg Water versus saline
<0.001 0.533 <0.001 <0.001 0.132 0.061 <0.001 <0.001 0.985
<0.001 0.007 <0.001 <0.001 0.683 0.732 <0.001 <0.001 1.000
<0.001 <0.001 <0.001 <0.001 1.000 1.000 <0.001 <0.001 1.000
a EDTA ¼ 1.5 mg/mL of EDTA; EDTA þ Ca ¼ 1.5 mg/mL of EDTA þ 10.6-mM calcium ion; EDTA þ Mg ¼ 1.5 mg/mL of EDTA þ 4.2-mM magnesium ion; saline and water ¼ volume equivalent to EDTA solution was replaced with saline and water, respectively.
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Figure 4. Simulation of model representing mechanism 4 in absence (left panel, parameter values: Amax,pp ¼ 1000, Kd,pp ¼ 10, g ¼ 2) and presence (right panel, parameter values: Amax,pp ¼ 1000, Kd,pp ¼ 500, g ¼ 1) of metal-ion (MI) chelating agent.
between drug, metal ion, and plasma protein could possibly be responsible for the observed atypical PPB effect, but no investigations were performed. Earlier reports showed a decreasing unbound fraction of tigecycline with increasing total concentrations, but these PPB determinations were only determined up to a maximum concentration of 15 mg/mL. However, the unbound fraction increased at very high concentrations (100 mg/mL) of tigecycline, thus giving a “U-shape” curve overall.9 This U-shaped unbound fraction curve was also observed for doxycycline and minocycline in the plasma of rats, mice, and monkeys.12 Other than these 2 characteristics of atypical nonlinear PPB, nothing about this behavior was known. The current work aimed to study the interactions between tigecycline, metal ions, and plasma proteins. In the first set of experiments, the metal ions present in the plasma were chelated using EDTA. The fraction unbound increased several fold, and there were no significant differences between plasma protein binding at different concentrations, indicating that the divalent metal ions present in the plasma play an important role in the atypical PPB behavior of tigecycline. This increase was in concurrence with the results obtained by Chen et al.,13 who observed higher unbound fractions of tigecycline when EDTA was used as anticoagulant in comparison to heparin. Because EDTA is capable of chelating several metal ions present in the plasma, the increased unbound fraction shows that metal ions play a vital role in atypical nonlinear PPB behavior. By saturating EDTA's chelating capacity, one can determine if the EDTA effect, the nullification of atypical nonlinear PPB behavior, is reversible. Excess divalent metal ions (Ca2þ and Mg2þ) were added to saturate the EDTA added to plasma samples, and the atypical behavior was restored. This showed that the effect of EDTA is reversible and further supported the involvement of divalent metal ions in the atypical PPB behavior of tigecycline. To understand the interplay of metal ions, tigecycline, and plasma proteins, different possible interactions were proposed and simulated to determine if the observed characteristics of tigecycline's atypical nonlinear PPB were present. Based on our experimental results, the following characteristics of atypical nonlinear PPB behavior of tigecycline can be delineated: (a) the unbound fraction shows a “U” shaped behavior with increasing total concentrations of tigecycline; (b) the presence of metal ions is required for this atypical nonlinear PPB behavior and chelation of metal ions nullifies this atypical nonlinear behavior; and (c) the chelation of metal ions increases the unbound fraction. Different possible
mechanisms were proposed (Fig. 2), simulated, and local sensitivity analyses were performed to evaluate these models based on the known characteristics of atypical PPB. Local sensitivity analyses showed that out of 4 different proposed mechanisms, mechanisms 1-3 were able to show the “U” shaped curve with a limited range of parameters, but the unbound fraction did not increase when metal ions were removed from the system. Mechanism 4, which assumed metal ioneassisted cooperativity in tigecycline binding, was able to satisfy all the observed characteristics of atypical nonlinear binding and cooperativity (g 1.5) was required for the atypical nonlinear behavior. Cooperativity of metal ions may influence the protein binding characteristics of ligands (drugs). Brannvall et al. observed that the cooperativity between different divalent metal ions controlled the ribozyme cleavage rate and cleavage site recognition.14 Stojanovic et al. observed higher binding constants for tigecycline with human serum albumin in the presence of Ca2þ, Cu2þ, and Fe3þ using fluorescence, UV-Vis spectroscopic, and molecular docking methods.15 Thus, we propose metal ion cooperativity with tigecycline as a possible reason for atypical nonlinear PPB behavior. However, direct experimental evidence of cooperativity between metal ions and tigecycline will be required to confirm this hypothesis. The current work has a few limitations: the proposed mechanisms assume interactions between a single plasma protein with metal ions and tigecycline. As plasma protein is a mixture of several proteins, more complex interactions between different proteins may also be possible. In addition, the change in unbound fraction was used as indirect evidence of the interactions between tigecycline, divalent metal ions, and plasma proteins; a study of interactions at the molecular level will be confirmatory evidence in the elucidation of the mechanism of atypical nonlinear PPB.
Conclusion The interactions between metal ions and tigecycline with plasma proteins were investigated. Divalent metal ions are necessary for the atypical nonlinear PPB behavior of tigecycline, and the removal of metal ions nullifies the atypical nonlinear behavior and increases the unbound fraction. This work identified a plausible mechanism of atypical nonlinear binding behavior of tigecycline. The metal ioneassisted positive cooperativity in tigecycline's binding with plasma protein is likely an explanation for the atypical
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nonlinear PPB behavior of tigecycline. However, further direct evidence is needed for confirmation of this mechanism. References 1. Kumar S, Wong H, Yeung SA, Riggs KW, Abbott FS, Rurak DW. Disposition of valproic acid in maternal, fetal, and newborn sheep. II: metabolism and renal elimination. Drug Metab Dispos. 2000;28(7):857-864. 2. Zandvliet AS, Copalu W, Schellens JH, Beijnen JH, Huitema AD. Saturable binding of indisulam to plasma proteins and distribution to human erythrocytes. Drug Metab Dispos. 2006;34(6):1041-1046. 3. Schmidt S, Gonzalez D, Derendorf H. Significance of protein binding in pharmacokinetics and pharmacodynamics. J Pharm Sci. 2010;99(3):1107-1122. 4. Lima JJ, Boudoulas H, Blanford M. Concentration-dependence of disopyramide binding to plasma protein and its influence on kinetics and dynamics. J Pharmacol Exp Ther. 1981;219(3):741-747. 5. Meffin PJ, Robert EW, Winkle RA, Harapat S, Peters FA, Harrison DC. Role of concentration-dependent plasma protein binding in disopyramide disposition. J Pharmacokinet Biopharm. 1979;7(1):29-46. 6. U.S. Food and Drug Administration. Tygacil (Tigecycline) label. Available at: http://www.accessdata.fda.gov/drugsatfda_docs/label/2016/021821s042lbl. pdf. Accessed August 10, 2016. 7. U.S. Food and Drug Administration. Tygacil (tigecycline) dclinical pharmacology and biopharmaceutics review(s). Washington, DC: FDA; 2005. Available at: http://www.accessdata.fda.gov/drugsatfda_docs/nda/2005/021821Orig1 s000ClinPharmR.pdf. Accessed August 10, 2016.
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