RESUMO
Tiopronin, formally 2-mercaptopropionylglycine (MPG), is currently prescribed to treat cystinuria and rheumatoid arthritis, and its antioxidant properties have led to its investigation as a treatment for cataracts, a condition in which oxidative stress is strongly implicated. To study its accumulation in the eye, a reliable, isocratic HPLC method was developed for the determination of MPG and its primary metabolite 2-mercaptopropionic acid (MPA) in plasma and relevant ocular tissues. This method utilizes pre-column derivatization and fluorescence detection. The 3.5 min separation enables high-throughput analysis, and validation experiments demonstrated that this method is suitable for evaluating ocular accumulation of MPG and MPA at concentrations as low as 66 and 33 nm, respectively. Excellent linearity was achieved over the working concentration range with R2 > 0.997. Extraction recovery was reproducible within each matrix and exceeded 97%. Accuracy was within 13.3% relative error, and intra- and inter-day precisions were within 6% CV and 7% CV, respectively. Sample stability was demonstrated under various storage conditions, and the use of an internal standard conferred exceptional ruggedness. This method has been successfully applied for the determination of MPG and MPA in plasma, cornea, lens and retina following intraperitoneal administration of the drug in Wistar rats.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Olho/química , Tiopronina/análise , Animais , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Fluorescência , Tiopronina/sangue , Tiopronina/química , Tiopronina/farmacocinéticaRESUMO
Post-operatory infections in orthopedic surgeries pose a significant risk. The common approach of using antibiotics, both parenterally or embedded in bone cement (when this is employed during surgery) faces the challenge of the rising population of pathogens exhibiting resistance properties against one or more of these compounds; therefore, novel approaches need to be developed. Silver nanoparticles appear to be an exciting prospect because of their antimicrobial activity and safety at the levels used in medical applications. In this paper, a novel type of silver nanoparticles capped with tiopronin is presented. Two ratios of reagents during synthesis were tested and the effect on the nanoparticles investigated through TEM, TGA, and UV-Vis spectroscopy. Once encapsulated in bone cement, only the nanoparticles with the highest amount of inorganic fraction conferred antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA) at concentrations as low as 0.1% w/w. No other characteristics of the bone cement, such as cytotoxicity or mechanical properties, were affected by the presence of the nanoparticles. Our work presents a new type of silver nanoparticles and demonstrates that they can be embedded in bone cement to prevent infections once the synthetic conditions are tailored for such applications.
Assuntos
Antibacterianos/farmacologia , Cimentos Ósseos/química , Nanopartículas Metálicas/química , Prata/farmacologia , Tiopronina/farmacologia , Antibacterianos/química , Antibacterianos/farmacocinética , Cimentos Ósseos/farmacologia , Força Compressiva , Teste de Materiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Tamanho da Partícula , Prata/química , Prata/farmacocinética , Tiopronina/química , Tiopronina/farmacocinéticaRESUMO
Tiopronin-conjugated gold nanoparticles (TPN@GNPs), with glutathione (GSH)-responsive drug release property, were developed for acute liver injury therapy. The TPN@GNPs were prepared using a one-pot synthesis method and characterized by UV-vis and transmission electronic microscopy methods. The TPN@GNPs displayed typical surface plasmon resonance of nanogold with a narrow size distribution (ca. 2 nm). The in vitro drug release profiles of the conjugates indicated that TPN@GNPs were able to release TPN in a sustained fashion for 4 h at a simulated intracellular level of GSH. pH values or ionic strengths of the release media had no obvious influence on TPN release from the surface of nanoparticles. The pharmacokinetic studies in rats showed that the TPN@GNPs had longer MRT (7.71 h) than TPN (3.96 h), indicating sustained release pattern of TPN@GNPs in vivo. The sustained release of TPN at the relative high GSH concentration could ameliorate the instability of TPN and enable the drug release in the target cells. Although the IC50 value of TPN@GNPs with TPN/AuCl4(-) of 3:1 (mol/mol) showed slight increase in comparison with that of the free TPN in HepG2 cells (1.26±1.07 vs. 1.73±1.16 mg/mL), the TPN@GNPs displayed better effects over TPN in the treatment of acute liver injury in vivo. In a liver injury mice model induced by CCl4, the histological analysis showed both the TPN@GNPs and free TPN group could repair the liver injury. In addition, the biochemical parameters showed TPN@GNPs could reduced the aminotransferase to a lower level compared with TPN, which might be due to the sustained drug release and passive liver targeting properties of TPN@GNPs. It demonstrated that gold nanoparticle-based drug delivery system allowed smart functions and superior properties by taking advantages of the unique small size effects and surface chemical properties.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Glutationa/química , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Substâncias Protetoras/administração & dosagem , Tiopronina/administração & dosagem , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ouro/química , Ouro/farmacocinética , Células Hep G2 , Humanos , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos ICR , Substâncias Protetoras/química , Substâncias Protetoras/farmacocinética , Ratos , Tiopronina/química , Tiopronina/farmacocinéticaRESUMO
The present study characterized the in vitro biological response of a comprehensive set of cancer cell lines to gold nanoparticles (2.7 nm) coated with tiopronin (AuNPs-TP). Our findings suggest that upon entering cells, the AuNPs-TP are sequestered in vacuoles such as endosomes and lysosomes, and mostly localize in perinuclear areas. Peak cell accumulation was achieved at 8 hours after incubation. L929 and H520 cells showed more than 75% surviving fraction when treated with 0.5 mg/mL of AuNPs-TP for 24 hours, whereas the surviving fractions were 60% in MCF-7 and 20% in HeLa cells. Reactive oxygen species (ROS) production by the AuNPs-TP was dependent on cell line and exposure time. Antioxidants inhibited ROS generation to various extents, with glutathione and tiopronin being most effective. Overall, exposure time, concentration of the AuNPs-TP, and cell line influenced neoplastic cell response. Furthermore, the mechanism of cytotoxicity of the AuNPs-TP was found to be ROS generation. FROM THE CLINICAL EDITOR: This study describes the basic intracellular characteristics of Tiopronin-Au nanoparticles from the standpoint of their anti-cancer activity in different cancer cell cultures.
Assuntos
Ouro/farmacologia , Nanopartículas/química , Neoplasias/tratamento farmacológico , Tiopronina/farmacologia , Animais , Antioxidantes/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Ouro/química , Ouro/farmacocinética , Humanos , Camundongos , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiopronina/química , Tiopronina/farmacocinéticaRESUMO
Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC-MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4-dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post-treatment samples were analyzed on a C8 column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol-5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0-5000 ng/mL. The intra- and inter-day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC-MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Tiopronina/sangue , Ditiotreitol/farmacologia , Humanos , Masculino , Niacina , Substâncias Redutoras/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tiopronina/farmacocinéticaRESUMO
OBJECTIVE: To evaluate the bioequivalence of tiopronin enteric capsules (testing preparation, T) versus tablets (reference preparation, R). METHODS: A single oral dose of tiopronin enteric capsules or tablets at 200 mg was administered in 2 groups of Chinese healthy volunteers (n=9) in a randomized crossover design at the interval of 2 weeks. The plasma concentrations of tiopronin were measured by HPLC-MS/MS, and the pharmacokinetic parameters were calculated by DAS 2.0 program. The bioequivalence between the two preparations was evaluated. RESULTS: The main pharmacokinetic parameters were as follows: C(max)(microg.ml(-1)) 3.612-/+1.2393 (R), 3.644-/+1.540 (T); t(max) 4.333-/+1.0853 (R), 3.611-/+1.420 (T); t((1/2))(h) 18.245-/+11.270 (R), 23.403-/+10.500 (T); AUC0-t (microg.h.ml(-1)) 18.732-/+6.92318 (R), 18.713-/+6.585 (T); AUC0-infinity (microg.h.ml(-1)) 21.900-/+7.31220 (R), 20.780-/+7.965 (T). The relative bioavailability of tiopronin enteric capsule was 103.712-/+23.956%, with 90% confidential intervals of ln(AUC0-->72), ln(AUC0-infinity) and ln(C(max)) of 91.1%-111.8%, 96.8%-118.3%, and 85.1%-113.0%, respectively. CONCLUSION: The tiopronin enteric capsules were bioequivalent to the tablets.
Assuntos
Tiopronina/farmacocinética , Disponibilidade Biológica , Cápsulas , Estudos Cross-Over , Saúde , Humanos , Modelos Lineares , Masculino , Reprodutibilidade dos Testes , Equivalência Terapêutica , Adulto JovemRESUMO
A sensitive, rapid method for determining reduced tiopronin concentration in rat plasma has been developed by using a high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl) maleimide (NPM). The analytes were separated on a Kromasil C18 column (250 mm x 4.6 mm, 5 microm) using 0.2% glacial acetic acid aqueous solution including 0.015 mol x L(-1) KH2PO4 and acetonitrile (56:44) as a mobile phase at a flow-rate of 0.8 mL x min(-1), and fluorescence detection wavelength were set at lamda(e x) = 340 nm and lamda(e m) = 375 nm, the column temperature was 30 degrees C. The calibration curve was found to be linear over a range of 0.1 - 10.0 microg x mL(-1), the limit of quantitation was 0. 1 mg x L(-1). The coefficients of the variation for the within-run and between-run precisions ranged from 5.3% to 10.8% and 7.0% to 10.8%, respectively. The percentage of absolute recovery ranged from 73.7% to 79.7%. The method was used to determine the concentration of tiopronin in rat plasma after a single intragastric administration of 25 mg x kg(-1) tiopronin to 6 healthy male Wistar rats. The pharmacokinetic process was fitted to a two-compartment model. The method has been successfully applied to the determination of tiopronin in rat plasma.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Tiopronina/sangue , Tiopronina/farmacocinética , Animais , Área Sob a Curva , Corantes Fluorescentes/química , Masculino , Maleimidas/química , Ratos , Ratos WistarRESUMO
A quantitative method for the determination of total tiopronin (TP) in human plasma was developed by liquid chromatography with electrospray ionisation (ESI) mass spectrometric detection. After reduction with tris (2-carboxy-ethyl) phosphine (TCEP) and derivatization with methyl acrylate (MA) for the thiol group of TP, plasma samples were processed successively by deproteinization and solid phase extraction. N-acetyl-l-cysteine (NAC) was selected as internal standard undergoing the same treatment as TP. The method was validated that it could meet the need of biological analysis. The lower limit of quantitation (LLOQ) of TP in plasma was 0.02microg/mL. Finally, the method was successfully applied to a pharmacokinetic study in 20 healthy Chinese male volunteers after an oral dose of 200mg TP tablets.
Assuntos
Acrilatos/química , Cromatografia Líquida/métodos , Indicadores e Reagentes/química , Fosfinas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de Sulfidrila/química , Tiopronina/sangue , Humanos , Sensibilidade e Especificidade , Tiopronina/farmacocinéticaRESUMO
A sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of tiopronin (TP) and its metabolites, 2-mercaptopropionic acid (2-mpa) and S-methylated TP (SA13), in rat blood using methyl acrylate (MA) for the stabilization of a thiol group. The thiol groups of TP and 2-mpa in rat blood were immediately derivatized by the addition of MA-acetonitrile solution in 0.1 M Tris HCl (pH 9.1). The purification of the derivatives was accomplished by a simple liquid-liquid extraction procedure involving protein precipitation step. The analysis was performed on a Zorbax SB-C18 analytical column by a gradient elution with methanol-0.05 M acetic acid (15:85 and 7:3, v/v). Negative ion electrospray ionization with selected reaction monitoring was employed for the detection of analytes. Linearity of calibration was observed over the range of 0.5-1000 ng/ml for TP and 2-mpa, and 2-1000 ng/ml for SA13. The intra- and inter-assay variability for all analytes at the limit of quantitation (LOQ) level ranged from 5.47 to 16.75% and 4.95 to 7.23%, respectively. The LOQs estimated for TP, 2-mpa and SA13 were 0.5, 0.5 and 2 ng/ml, respectively. This assay method was successively applied to a pharmacokinetics study after an oral administration of TP (10 mg/kg) to rats.
Assuntos
Tiopronina/sangue , Acrilatos/química , Animais , Biotransformação , Calibragem , Cromatografia Líquida de Alta Pressão , Eletroquímica , Feminino , Espectrometria de Massas , Ratos , Ratos Endogâmicos Lew , Reprodutibilidade dos Testes , Compostos de Sulfidrila/química , Tiopronina/farmacocinéticaRESUMO
In order to determine the absolute bioavailability, both oral and intravenous administrations of a drug are often used. Recently a new method has been proposed to determine absolute bioavailability in the absence of intravenous dose. Following a single oral dose, this method requires oral and renal clearance data from normal subjects and renal failure patients. The bioavailability is calculated from a plot of oral against renal clearance following an oral dose, where the inverse of the slope is equal to absolute bioavailability. This study examines the prediction of absolute bioavailability from the proposed method for eight drugs which have a wide range of oral and renal clearance. From this study, it appears that the proposed method may not be reliable for the prediction of absolute bioavailability and further investigation is needed to test the validity of this method.
Assuntos
Antiarrítmicos/farmacocinética , Anti-Infecciosos/farmacocinética , Disponibilidade Biológica , Diuréticos/farmacocinética , Antagonistas dos Receptores H2 da Histamina/farmacocinética , Rim/metabolismo , Acebutolol/farmacocinética , Administração Oral , Adulto , Idoso , Amilorida/farmacocinética , Antiarrítmicos/urina , Anti-Infecciosos/urina , Área Sob a Curva , Atenolol/farmacocinética , Diuréticos/urina , Enoxacino/farmacocinética , Flecainida/farmacocinética , Antagonistas dos Receptores H2 da Histamina/urina , Humanos , Imidazóis/farmacocinética , Pessoa de Meia-Idade , Ranitidina/farmacocinética , Insuficiência Renal/metabolismo , Insuficiência Renal/urina , Tiopronina/farmacocinéticaRESUMO
Recent studies suggest that the hydroxyl radical (.OH) plays a pathogenetic role in postischemic ventricular dysfunction (myocardial "stunning"). This concept, however, is predicated exclusively on results obtained in anesthetized open-chest preparations, which are subject to the confounding influence of many unphysiological conditions and in which both myocardial stunning and free radical generation are greatly exaggerated. The lack of supporting evidence in more physiological animal models represents a major limitation of the .OH hypothesis of stunning. Furthermore, concern has been raised that myocardial stunning may be a period of "rest" necessary for full recovery, so that attenuation of the early phase of stunning by antioxidant therapy may have subsequent detrimental effects on the resting function and/or on the return of myocardial contractile reserve. To address these issues, in phase 1 of this study conscious unsedated dogs undergoing a 15-minute coronary artery occlusion received an intravenous infusion of normal saline (n = 22), of the .OH scavenger N-2-mercaptopropionyl glycine (MPG, n = 17), or of the iron chelator desferrioxamine (DF, n = 14). Compared with control dogs, the dogs treated with MPG or DF exhibited significantly greater postischemic wall thickening throughout the first 6 hours of reperfusion; the total deficit of wall thickening during this time interval was reduced 50% by MPG and 50% by DF. The magnitude of this beneficial effect was a function of the severity of ischemia, so that the dogs with the lowest collateral flows had the greatest improvement of wall thickening. The accelerated recovery produced by MPG and DF in the first 6 hours was not followed by any deterioration of resting wall thickening at 24 or 48 hours. Furthermore, in dogs treated with MPG or DF, the increase in wall thickening elicited by maximal inotropic stimulation (isoproterenol or dopamine) was similar before stunning and shortly after resting wall thickening had normalized (24 or 48 hours after reflow); thus, despite the fact that most of the early postischemic dysfunction had been eliminated by antioxidant therapy, there was no subsequent impairment of either resting function or contractile reserve. In phase 2, production of free radicals (measured with the spin trap alpha-phenyl N-tert-butyl nitrone) was markedly (> 80%) inhibited by the same doses of MPG and DF that attenuated stunning in phase 1.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Hidróxidos/toxicidade , Isquemia Miocárdica/fisiopatologia , Função Ventricular Esquerda , Animais , Circulação Coronária/efeitos dos fármacos , Desferroxamina/farmacologia , Cães , Feminino , Hemodinâmica/efeitos dos fármacos , Radical Hidroxila , Masculino , Contração Miocárdica/efeitos dos fármacos , Oxigênio/metabolismo , Tiopronina/farmacocinética , Tiopronina/farmacologiaRESUMO
Ten healthy subjects were given 500 mg (3064 mumol) tiopronin, or 2-mercaptopropionylglycine (2-MPG) by mouth. Cmax was reached after 3-6 h, and after a shorter beta-phase a long terminal half-life of 53 h of total tiopronin was found. Tiopronin measured as unbound (non-protein-bound) drug disappeared more rapidly from plasma, with a calculated t1/2 of 1.8 h. Mean residence time was higher (58 h) when calculated as total tiopronin than as unbound tiopronin (6 h), and this was also the case for the volume of distribution (V lambda = 455 l vs V lambda,u = 41 l). The results indicate extensive protein binding in plasma and a deep pool of tissue bound tiopronin after the first absorption and distribution phases. Absolute bioavailability (f) was 63%, and bioavailability calculated from urinary excretion was 47%, which are well correlated with each other. Urinary excretion was mainly confined to the first 6 h (74%) and was almost complete (98%) within 12 h. We conclude that the maximal absorption of the tiopronin was late, protein and tissue binding of the drug were high and its bioavailability varied. The renal excretion of low molecular weight tiopronin occurred early, which implies that the drug should be given in divided doses, at least twice daily, for optimal efficiency in the treatment of cystinuria.
Assuntos
Tiopronina/farmacocinética , Administração Oral , Adulto , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Feminino , Meia-Vida , Humanos , Masculino , Tiopronina/administração & dosagem , Tiopronina/sangueRESUMO
We have studied the pharmacokinetics of tiopronin and its principal metabolite, 2-mercaptopropionic acid (2-MPA) in healthy volunteers after the oral administration of 500 mg (2 Acadione tablets), followed by simultaneous assay of the two compounds in plasma over a period of 48 h using a new method (emission of fluorescence after HPLC and post-column derivatization by pyrene-maleimide). The absorption of tiopronin was slow (tmax between 4 and 6 h) and the plasma concentrations subsequently fell biexponentially. The principal metabolite 2-MPA appeared later in the plasma (tmax between 10 and 12 h after a lag-time of 3 h) then disappeared monoexponentially. About 15% of the tiopronin was metabolized to 2-MPA.
Assuntos
Compostos de Sulfidrila/sangue , Tiopronina/farmacocinética , Administração Oral , Adulto , Cromatografia Líquida de Alta Pressão , Humanos , Masculino , Fatores de Tempo , Tiopronina/administração & dosagem , Tiopronina/sangueRESUMO
The pharmacokinetic disposition of 2-mercaptopropionylglycine (2-MPG) given as a single intravenous injection and/or as a single oral dose was studied in 9 normal and 13 cystinuric dogs. After intravenous injection of approximately 10 or 20 mg/kg body weight the pharmacokinetics were best described by a three-exponential function. The first phase involved a distribution process apparently including establishment of drug-plasma protein and drug-tissue binding. The second phase involved rapid renal elimination and 60% of the drug was excreted within 3 h of administration. There was also a slow terminal third phase with a long half-life after both intravenous (t1/2 = 23 h) and oral (t1/2 = 22 h) administration. No dose dependency was observed. A deep pool of reversibly tissue-bound 2-MPG was indicated by a Vss of 3.3 +/- 0.9 l/kg body weight and the long terminal elimination phase. Total clearance was estimated as 4.1 +/- 0.9 ml/min/kg body weight. 2-MPG was eliminated mainly by renal excretion, but there was a difference in recovery of dose between normal and cystinuric dogs. During the first 24 h after intravenous and oral administration, 69% and 54%, respectively, of the drug was recovered in the urine of normal dogs. The corresponding figures in cystinuric dogs were 44% and 29%, respectively. The absolute bioavailability (FAUC) was 88 +/- 20% in normal dogs.
Assuntos
Cães/metabolismo , Tiopronina/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Cistinúria/metabolismo , Cistinúria/veterinária , Doenças do Cão/metabolismo , Meia-Vida , Injeções Intravenosas/veterinária , Ligação Proteica , Tiopronina/administração & dosagem , Tiopronina/urina , Distribuição TecidualRESUMO
A simple and fast high-performance liquid chromatographic method was developed for the simultaneous measurement of 2-mercaptopropionylglycine (Tiopronine) and its metabolite (2-mercaptopropionic acid) in human plasma after the administration of a pharmaceutical dosage form (Acadione). The sample treatment before high-performance liquid chromatographic analysis consisted of the reduction of the corresponding disulphides by tri-n-butylphosphine and protein precipitation with ethanol. Separation was achieved by ion-pair high-performance liquid chromatography on a reversed-phase column (LiChrospher RP 18e) with cetrimonium bromide as counter ion and detection by fluorimetry after post-column derivatization with a selective thiol reagent, i.e. pyrenemaleimide. The high frequency of the analyzed samples and validation results make the method suitable for pharmacokinetic studies, and this was demonstrated by the first results obtained after the administration of an oral dose of 500 mg of Tiopronine to two healthy subjects.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Compostos de Sulfidrila/sangue , Tiopronina/sangue , Precipitação Química , Etanol , Humanos , Oxirredução , Fosfinas , Tiopronina/farmacocinéticaRESUMO
The pharmacokinetics of 2-mercaptopropionylglycine (2-MPG) was studied in ten healthy volunteers after a single i.v. injection of 250 mg (1532 mumol). The total and non-protein-bound concentrations versus time curves were best described by a three-exponential function with terminal half-lives of 55 and 59 h respectively. Body clearance based upon the total concentration was estimated to be 105 and 231 ml/min based on the non-protein-bound 2-MPG. The corresponding values for Vss were 99 l and Vss,n 173 l, and for V gamma 485 l and V gamma,n 1121 l respectively. 75% of the dose was excreted in the urine, mainly during the first 6 h after injection. The proportion of non-protein-bound 2-MPG diminished exponentially during the first 15 h and then levelled off at about 30%. There was a nonlinear increase in the non-protein-bound fraction of 2-MPG as the total plasma concentration of the drug increased.