High-performance liquid chromatographic analysis, plasma protein binding and red blood cell partitioning of phenprobamate

A new high-performance liquid chromatographic procedure for the analysis of phenprobamate, a skeletal muscle relaxant in biologic fluids was developed. The method used a C18 reverse phase column, a mobile phase of methanol/acetonitrile/water (33:15:52), and UV detection at 215 nm. The assay procedure was applied to the determination of phenprobamate binding to rat and human plasma proteins using the equilibrium dialysis method. In addition, the red blood cell/plasma partitioning was determined in the whole blood of rats and humans. Phenprobamate exhibited a moderate binding to plasma proteins of rat (74.3 +/- 2.2 per cent) and human (80.5 +/- 1.1 per cent). The protein binding was concentration-independent in the range of 10 to 80 micrograms ml-1. Phenprobamate binding to plasma proteins was also determined in the presence of 10 micrograms ml-1 acetaminophen. The protein binding of phenprobamate was not significantly altered by acetaminophen (74.4 +/- 0.6 per cent for rat plasma; 75.7 +/- 1.6 per cent for human plasma). The distribution ratios of phenprobamate between the red blood cells and plasma were greater than unity, 1.86 and 1.59 in rat and human, respectively, indicating a preferential partitioning of the drug in the red blood cells.     

In-vitro red blood cell partitioning of doxycycline

Objective:

In-vitro red blood cell (RBC) partitioning of doxycycline was studied to determine whether doxycycline penetrates RBC and its concentration was assayed keeping in view its high lipophilicity.

Materials and Methods:

Standardization of doxycycline was performed in whole blood and plasma of cattle by microbiological assay using Bacillus subtillis ATCC 6633 as indicator organizm. Actual concentration of the drug was obtained by comparing zone inhibition with standard graph and the extent of partitioning was mathematically calculated.

Results:

The R² value of standard graph for doxycycline was 0.9934 and 0.9727 for plasma and whole blood, respectively. Overall, RBC partitioning of doxycycline was found to be 18.40 ± 1.70%.

Conclusions:

Overall RBC partitioning of doxycycline indicated low penetration into RBC. Plasma is the fluid suggested for pharmacokinetic evaluation of doxycycline.     

Disposition of etofibrate, clofibric and nicotinic acid esters, and their products in dogs

Etofibrate, the ethylene glycol diester of clofibric and nicotinic acids, on intravenous infusion into dogs, has a terminal half-life of 2 min. The intermediate half-esters, the nicotinate and the clofibrate, have respective terminal half-lives of 4.6 and 1.7 min and appear fleetingly when etofibrate is administered. In contrast to the 42-h terminal half-life of clofibric acid, the other final transformation product, nicotinic acid, shows saturable or dose-dependent pharmacokinetics in dogs that conform to the Michaelis-Menten equation with a terminal half-life of 4.4 min at low concentrations (less than 6.9 microM/kg). Three distinct metabolites of nicotinic acid can be identified and assayed chromatographically in the urine. The partition properties were similar to nicotinic acid. Nicotinic acid is excreted 30% unchanged into urine with a renal clearance of 70 mL/min in 27-kg dogs.     

In vitro pharmacokinetic study of the novel anticancer agent E7070: red blood cell and plasma protein binding in human blood

E7070 is a novel sulfonamide anticancer agent that arrests the G(1)/S phase of the cell cycle. Preclinical and phase I studies have demonstrated non-linear pharmacokinetics (PK) of the drug. A population PK analysis revealed that the human plasma concentration-time data were best described by a three-compartment model with non-linear distribution. We have studied the in vitro interaction of 14C-radiolabeled E7070 with red blood cells (RBC) and its binding to plasma proteins in the concentration range where non-linearity in disposition was observed in humans to get more insight into the behavior of the drug. After the addition of E7070 to whole blood at 37 degrees C, the drug is taken up or binds to RBC in a concentration-dependent manner. The addition of sodium azide, however, did not result in a decrease of drug uptake by RBC, indicating passive diffusion processes. A non-linear increase in drug uptake was observed at incubation concentrations above 4 microg/ml E7070 in whole blood. This non-linearity was confirmed by lower partition coefficients between RBC and plasma at higher incubation concentrations (from 2.37 at 4 microg/ml to 0.31 at 200 microg/ml). The plasma protein binding of E7070 was high (98-99%) and linear in the concentration range studied (20-200 microg/ml). In conclusion, E7070 in whole blood is preferentially bound to RBC and exhibits high plasma protein binding. The non-linear distribution of E7070 in humans can be caused, in part at least, by saturable binding of E7070 to RBC.     

Binedaline binding to plasma proteins and red blood cells in humans

Serum binding of binedaline, a new antidepressant drug, was studied in vitro by equilibrium dialysis. The percent of binding in serum is high, 99.2%, and remains constant within the range of therapeutic concentrations; no saturation to the binding sites was seen. Investigations performed on isolated proteins with a wide range of concentrations showed one site with a high affinity constant (Ka = 2 X 10(6) M-1) for alpha 1-acid glycoprotein and two sites with a low affinity constant (Ka = 3 X 10(4) M-1) for human serum albumin. Binding to lipoproteins was nonsaturable, with a total affinity constant of 1.25 X 10(5) less than nKa less than 2.79 X 10(6) M-1. Over the range of therapeutic concentrations, the ratio of binedaline concentrations in serum and red blood cells remained constant (1%) and was shown to be dependent on the free fraction of binedaline in serum.     

Effect of salicylate on serum protein binding and red blood cell uptake of acetazolamide in vitro

The diffusion of acetazolamide from buffered saline and buffered albumin solutions into human erythrocytes has been characterized. A model was developed for describing the effects of both intra- and extracellular binding on the approach to distributional equilibrium. Unbound acetazolamide entered the cells via an apparent first-order process at a rate that was unaffected by salicylate at a therapeutic concentration of 200 micrograms/mL. Salicylate concentrations ranging from approximately 100 to 400 micrograms/mL, were, however, extremely effective in displacing acetazolamide from its serum protein binding sites. Free fractions of acetazolamide in human serum were found to increase by an order of magnitude as salicylate concentrations approached 400 micrograms/mL, thereby greatly increasing the concentration of unbound drug available for passive diffusion into cells. The results indicate that while competitive binding effects, which may alter unbound drug concentration-time profiles and potentially impact on toxicity, do occur, alterations in red cell membrane permeability, which could adversely affect carbon dioxide transport, are not of significance.     

Stability, blood partition and plasma protein binding of ipriflavone, an isoflavone derivative

It is generally recognized that the partition between plasma and blood cells, the immediate centrifugation of blood samples after collection for the measurement of 'true' in vivo concentrations and free drug concentrations in plasma are important determinants of the pharmacokinetics and/or pharmacodynamics of drugs. Therefore, the stability, blood partition between plasma and blood cells, and factors influencing the binding of ipriflavone to 4% human serum albumin (HSA) using an equilibrium dialysis technique were evaluated. Ipriflavone was unstable in rat liver homogenate and various pH solutions ranging from 1 to 13, except pH 8, rat blood and plasma and human plasma when incubated in a water-bath shaker for 24 h kept at 37 degrees C and at a rate of 50 oscillations/min. The recoveries of spiked amounts of ipriflavone at 24 h pH solutions ranging from 1 to 12 were 67.0, 78.1, 87.9, 89.6, 84.2, 87.4, 85.5, 99.3, 88.0, 76. 6, 79.4 and 81.5%, respectively. Ipriflavone was very unstable in pH 13 solution; only 0.814% of ipriflavone was recovered after 30 min incubation. Ipriflavone was stable for up to 3 h incubation in human gastric juices. Ipriflavone reached equilibrium fast (within 30 s of being mixed manually) between plasma and blood cells and the equilibrium plasma/blood cells partition ratios were independent of the initial rabbit blood concentrations of ipriflavone: 0.2, 2, and 10 microg/mL; the values were in the range of 0.900-2.45. The binding of ipriflavone to 4% HSA was 96.6+/-0.407% at ipriflavone concentrations ranging from 2 to 100 microg/mL, but it was dependent on HSA concentrations (0.5-6%), incubation temperature (4, 22 and 37 degrees C), 'the buffer' pHs (5.8, 6.4, 7.0, 7.4 and 8.0), and addition of salicylic acid (150-300 microg/mL) and sulphisoxazole (100-300 microg/mL). However, the binding was independent of buffers containing various concentrations of chloride ion (0-0.546%), glucose (0 and 5%), alpha-1-acid glycoprotein (0-0.32%) and heparin (0-40 U/mL), and addition of its metabolites (M1 and M5, 5 microg/mL).     

Increased plasma binding and decreased blood cell binding of quinidine in blood from anuric rats

The blood cell/plasma concentration ratio of quinidine, as influenced by the plasma protein binding, was studied in normal and anuric rats by applying incubation and equilibrium dialysis techniques on blood and plasma, respectively, from normal and anuric rats. The plasma protein binding of quinidine in anuria was increased at concentrations of unbound drug of less than 1.75 X 10(-4) M and decreased above this concentration. At an assumed "therapeutic" quinidine concentration (1 X 10(-5) M), the mean concentration ratio (total quinidine in blood cells)/(total quinidine in plasma) was 1.84 in normals and 0.46 in anuria, and the mean ratio (total quinidine in blood cells)/(unbound in plasma) was 4.45 and 1.81, respectively. As the latter ratios were concentration dependent and greater than could be accounted for by pH-dependent distribution, quinidine is presumably bound in/on the blood cells. Reduced distribution ratio in anuria, even when related to unbound quinidine in plasma, also indicates changed binding in blood cells, a finding confirmed by applying the data to modified Scatchard plot. this may have implication for the use of blood cell/plasma concentration ratio as screening procedure for the altered plasma binding of quinidine in patients.     

A comparison of plasma, white blood cell, red blood cell, and tissue distribution of amiodarone and desethylamiodarone in anesthetized dogs

Desethylamiodarone (DA) is a major metabolite of amiodarone (AM), a Class III antiarrhythmic drug. The plasma pharmacokinetics and tissue distribution of AM and DA (10 mg/kg i.v.) were compared in anesthetized dogs. Plasma, white blood cell (WBC), red blood cell (RBC), liver, and skeletal muscle samples were obtained at frequent intervals up to 6 h after a single i.v. bolus of the two drugs. Drug concentrations in these and other tissues, i.e., lung, kidney, heart (right and left atrium, right and left ventricle, Purkinje fibers, and AV node), and femoral nerve were measured by a highly sensitive and specific high-pressure liquid chromatographic technique developed in our laboratory. Four different patterns of AM and DA uptake and washout could be identified in these experiments. The first pattern is biexponential decline in plasma drug levels with a rapid distribution phase (t1/2 alpha = 5.1 +/- 2.1 min for AM and 5.5 +/- 1.2 min for DA, respectively) and a slower elimination phase (t1/2 beta = 3.7 +/- 1.3 h for AM and 4.96 +/- 0.8 h for DA, respectively). The volume of distribution of DA was significantly larger than that of AM. The second pattern is that both WBCs and RBCs showed an initial uptake within 5 min followed by a biexponential decrease in drug levels, with t1/2 alpha similar to that in plasma but t1/2 beta significantly longer than in plasma. In both these types of cells, the elimination half-life for DA was significantly longer than that of AM. The third pattern is that in the liver there was a rapid uptake of both drugs with peak concentrations at 15 min; the decline in hepatic levels of AM was biexponential, but that of DA appeared to be monoexponential. In addition, in dogs given AM alone, the metabolite (DA) was easily detected in the liver from the earliest time of measurement, suggesting that the parent drug is rapidly metabolized to DA. In the experiments where DA was injected, two new peaks were also identified in the liver suggesting that DA was metabolized further in the liver. The fourth pattern was in the skeletal muscle, where AM uptake was relatively slow, reaching peak concentrations between 1.5-2 h followed by a monoexponential decline; however, DA was rapidly taken up by skeletal muscle, but the rate of decline appeared to be slower as compared to that of AM.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ibuprofen on lithium plasma and red blood cell concentrations

The effect of ibuprofen on steady-state lithium plasma and red blood cell concentrations was studied in 11 normal volunteers. During the seven-day control phase, sustained-release lithium carbonate 450 mg was administered every 12 hours. Lithium plasma and red blood cell concentrations were determined on days 5, 6, and 7. During the treatment phase (days 7-15), ibuprofen 400 mg was administered four times a day concurrently with lithium. Lithium plasma and red blood cell concentrations were obtained on days 14, 15, and 16. Multiple blood samples were obtained over a 12-hour period on days 6 and 15. Urine samples were collected from six subjects. The mean minimum lithium concentration increased 15% when ibuprofen was added. Mean maximum lithium concentration, area under the curve, red blood cell concentrations, and the lithium red blood cell to plasma ratio were significantly higher during the treatment phase. Mean lithium total body and renal clearance values were significantly lower during the treatment with ibuprofen. The administration of ibuprofen can increase steady-state plasma lithium concentrations and decrease lithium clearance.     

Haloperidol plasma and red blood cell levels and clinical antipsychotic response

Two investigators have recently suggested therapeutic ranges for plasma haloperidol in the treatment of schizophrenia. An apparent optimal therapeutic range of red blood cell haloperidol as early as day 7 of the drug trial is described in this article. With continued treatment, an optimal plasma haloperidol range for response could be observed by day 14 of treatment. The previously described correlation between response at day 7 and plasma/red blood cell haloperidol ratio was confirmed but was found not to predict response at day 14 of drug treatment in this cohort of DSM-III schizophrenic patients.     

Effect of plasma cholesterol on red blood cell oxygen transport

1. Oxygen (O2) transfer from the blood to tissues is a function of the red blood cell (RBC) O2 saturation (SO2), the plasma O2 content being negligible. Under conditions of increased tissue O2 demand, the SO2 of arterial blood does not change appreciably (97%); however, the SO2 of mixed venous blood, equal to that of the perfused tissues, can go as low as 20%. 2. Tissue O2 availability is limited by the exposure time to a RBC, which decreases under conditions of maximum stress (< 1 s). If the O2 unloading time was to increase significantly, because of a decrease in the RBC diffusion constant or an increase in the RBC membrane thickness, the RBC O2 unloading time would exceed tissue (e.g. cardiac) transit time and O2 transfer would be impaired. 3. Cholesterol constitutes the non-polar, hydrophobic lipid of the enveloping layer of the RBC membrane. As the cholesterol content of the RBC increases, the fluidity of the membrane decreases and the lipid shell stiffens. 4. Early studies demonstrated that high blood cholesterol concentrations were associated with reduced blood O2 transport; in essence, the haemoglobin dissociation curve was shifted to the left. 5. Current investigations have shown that the cholesterol RBC membrane barrier to O2 diffusion delayed O2 entry into the RBC during saturation and delayed O2 release from the RBC during desaturation. In an analysis of 93 patients divided by their cholesterol concentration into five groups, the percentage change in blood O2 diffusion was inversely proportional to the cholesterol concentration. 6. The RBC membrane cholesterol is in equilibrium with the plasma cholesterol concentration. It stands to reason that as the plasma cholesterol increases, the RBC membrane becomes impaired and O2 transport is reduced. 7. The implications of this new perspective on O2 transport include the ability to increase tissue oxygenation by lowering plasma cholesterol.     

Evaluation of oral pharmacokinetics,in vitro metabolism,blood partitioning and plasma protein binding of novel antidiabetic agent,S009‐0629 in rats

S009‐0629 [methyl‐8‐(methylthio)‐2‐phenyl‐6‐p‐tolyl‐4,5‐dihydro‐2H‐benzo[e]indazole‐9‐carboxylate] is a novel antidiabetic agent with PTP1B inhibitory activity. In this study, we have investigated the in vitro metabolic stability, plasma protein binding, blood partitioning, and oral pharmacokinetic study of S009‐0629 in rats. The plasma protein binding, blood partitioning, and metabolic stability were determined by HPLC method. The oral pharmacokinetic study was analyzed by liquid chromatography coupled mass spectrometry (LC‐MS/MS) method. The plasma protein binding of S009‐0629 using modified charcoal adsorption method at 5 and 10 µg/mL was 80.58 ± 1.04% and 81.95 ± 1.15%, respectively. The K_RBC/PL of S009‐0629 was independent of concentration and time. The in‐vitro half‐life of S009‐0629 at 5 and 10 µM using rat liver microsomes was determined as 273 ± 24.46 and 281.67 ± 26.53 min, respectively. After oral administration, S009‐0629 exhibited C_max 55.51 ± 1.18 ng/mL was observed at 18 hr (t_max). S009‐0629 was found to have the large apparent volume of distribution (1,894.93 ± 363.67 L/kg). Oral in‐vivo t_1/2 of S009‐0629 was found to be 41.23 ± 5.96 hr. A rapid and highly sensitive LC‐MS/MS method was validated for S009‐0629 in rat plasma. S009‐0629 has high plasma protein binding and low hepatic extraction. S009‐0629 has no affinity with human P‐gp and BCRP in ATPase assay. After oral dosing, S009‐0629 has slow absorption and elimination in rats.     

Cytotoxicities of ginseng saponins and their degradation products against some cancer cell lines

In order to elucidate the cytotoxicity-structure correlation of ginseng-derived components, several prosapogenins and sapogenins were prepared from Korean red ginseng (Panax ginseng) saponins by acid hydrolysis or alkaline cleveage, and their chemical structures were identified by a combination of spectral and physical methods. Some of these degradation products showed the cytotoxic activities against various cancer cell lines, A549, SK-OV-3, SK-Mel-2, P388, L 1210 and K562. The significant difference in cytotoxicity between stereoisomers was not found and the activity was inversely proportional to the number of sugars linked to sapogenins. Diol-type prosapogenins and sapogenins showed higher cytotoxicity than triol-type ones.     

Comparative protein binding, stability and degradation of satraplatin, JM118 and cisplatin in human plasma in vitro

1. Satraplatin is an investigational orally administered platinum-based antitumour drug. The present study compared the plasma protein binding, stability and degradation of satraplatin with that of its active metabolite JM118 and cisplatin. 2. The platinum complexes were incubated in human plasma for up to 2 h at 37 degrees C and quantified in plasma fractions by inductively coupled plasma-mass spectrometry on- or off-line to high-performance liquid chromatography. 3. All three platinum drugs became irreversibly bound to plasma proteins and showed negligible reversible protein binding. They were also unstable in plasma and generated one or more platinum-containing degradation products during their incubation. However, the three platinum complexes differed in the kinetics of their instability and protein binding, as well as in the number of degradation products formed during their incubation. 4. In conclusion, the plasma protein binding, instability and degradation of satraplatin and its active metabolite JM118 are qualitatively similar to that of cisplatin and other clinically approved platinum-based drugs. Quantitative differences in their irreversible protein binding and degradation were related to their respective physiochemical properties and bioactivation mechanisms.     

Population analysis of the non-linear red blood cell partitioning of draflazine following various infusion durations

Objective: The pharmacokinetics and non-linear red blood cell partitioning of the nucleoside transport inhibitor draflazine were investigated in 19 healthy male and female subjects (age range 22–55 years) after a 15-min i.v. infusion of 1 mg, immediately followed by infusions of variable rates (0.25, 0.5 and 1 mg · h⁻¹) and variable duration (2–24 h). Methods: The parameters describing the capacity-limited specific binding of draflazine to the nucleoside transporters located on erythrocytes were determined by NONMEM analysis. The red blood cell nucleoside transporter occupancy of draflazine (RBC occupancy) was evaluated as a pharmacodynamic endpoint. Results: The population typical value for the dissociation constant K _d (%CV) was 0.648 (12) ng · ml⁻¹ plasma, expressing the very high affinity of draflazine for the erythrocytes. The typical value of the specific maximal binding capacity B_max (%CV) was 155 (2) ng · ml⁻¹ RBC. The interindividual variability (%CV) was moderate for K _d (38.9%) and low for B_max (7.8%). As a consequence, the variability in RBC occupancy of draflazine was relatively low, allowing the justification of only one infusion scheme for all subjects. The specific binding of draflazine to the red blood cells was a source of non-linearity in draflazine pharmacokinetics. Steady-state plasma concentrations of draflazine virtually increased dose-proportionally and steady state was reached at about 18 h after the start of the continuous infusion. The t_1/2βaveraged 11.0–30.5 h and the mean CL from the plasma was 327 to 465 ml · min⁻¹. The disposition of draflazine in whole blood was different from that in plasma. The mean t_1/2β was 30.2 to 42.2 h and the blood CL averaged 17.4–35.6 ml · min⁻¹. Conclusion: Although the pharmacokinetics of draflazine were non-linear, the data of the present study demonstrate that draflazine might be administered as a continuous infusion over a longer time period (e.g., 24 h). During a 15-min i.v. infusion of 1 mg, followed by an infusion of 1 mg · h⁻¹, the RBC occupancy of draflazine was 96% or more. As the favored RBC occupancy should be almost complete, this dose regimen could be justified in patients. Received: 6 February 1997 / Accepted in revised form: 12 May 1997     

Preclinical investigation of the pharmacokinetics,metabolism, and protein and red blood cell binding of DRDE-07: a prophylactic agent against sulphur mustard

DRDE-07, a newly synthesized amifostine analog currently under clinical investigation in a phase I trial, is a potent antidote against sulfur mustard toxicity. The purpose of this research was to evaluate the pharmacokinetic profile of DRDE-07 in female Swiss Albino mice after a single oral dose of 400 or 600 mg/kg. The physicochemical properties of DRDE-07, including solubility, pK_a, Log P, plasma protein binding and plasma/blood partitioning, were determined to support the pharmacokinetic characterization. DRDE-07 concentration was determined by an HPLC-UV method. The profile of plasma concentration versus time was analyzed using a non-compartmental model. Plasma protein binding was assessed using ultrafiltration. DRDE-07 appeared rapidly in plasma after oral administration with peak plasma levels (C_max) observed in less than 15 min. There was a rapid decline in the plasma levels followed by a smaller second peak about 90 min after dosing. The plasma protein binding of DRDE-07 was found to be less than 25% at all concentrations studied. Plasma clearance of DRDE-07 is expected to be ~1.5 fold higher than the blood clearance of DRDE-07. The probable metabolite of DRDE-07 was identified as phenyl-S-ethyl amine.Key Words: DRDE-07, Sulfur mustard, Blood–plasma partitioning, Protein binding, Pharmacokinetics