Differences in the serum protein binding of prazosin in man and rat

The serum protein binding of prazosin in man and rat has been studied in vitro by equilibrium dialysis. Prazosin was more extensively bound in human serum than in rat serum with binding ratios (B/F) of 14.3 +/- 3.4 and 4.4 +/- 0.2 (corresponding to 93.4 and 81.4% bound), respectively. This difference in binding between the species was partly due to qualitative differences between human and rat serum albumin, but also to the lower concentration of albumin in rat serum. Rat serum albumin (RSA) apparently showed two different classes of binding sites for prazosin, one with high (KD = 5.78 X 10(-6) M) and one with low (KD = 1.1 X 10(-4) M) affinity; the former is suggested as representing alpha 1-acid glycoprotein (alpha 1-AGP) with one binding site for prazosin per molecule, the latter as representing RSA with 0.28 binding sites per molecule. Human serum albumin (HSA) and human alpha 1-AGP both showed one class of binding sites with KD values of 2.7 X 10(-5) and 1.95 X 10(-6) M, respectively. HSA possessed 0.5 and human alpha 1-AGP 1 binding site for prazosin per molecule. The binding parameters obtained for the isolated serum proteins overestimated to some degree the total serum protein binding of prazosin in man. This was explained by a specific deviation from the law of mass action. HSA was the major binding protein in human serum at therapeutic concentrations, with ca. 60% of the total binding, the remaining 40% being bound to alpha 1-AGP. Anticipating that the high affinity binding site on the RSA preparation represents the binding of prazosin to alpha 1-AGP, then this protein accounts for 70% of the binding in rat serum, while rat serum albumin accounts for approximately 23%. The binding of prazosin to lipoproteins was insignificant in both species. The observed differences between man and rat in the serum protein binding of prazosin implicate differences in the two species with respect to prazosin pharmacokinetics and the pharmacological effect.     

Impact of anti-cancer drugs and other determinants on serum protein binding of morphine 6-glucuronide

BACKGROUND AND THE PURPOSE OF THE STUDY: The aim of the present study was to examine factors that may influence the protein binding of morphine 6-glucuronide (M6G), the most active metabolite of morphine. METHODS: An enzyme-linked immunoabsorbent assay technique was used to measure the M6G concentration in serum of 18 healthy adults, 18 neonatal and 7 children with cancer. Total and free M6G concentrations were measured following equilibrium dialysis for 3 hrs and at physiological pH at 37°C. The influence of vincristine, methotrexate, 6-mercaptopurine, morphine, human albumin, alpha-1-acid glycoprotein, palmitic acid, oleic acid and pH on M6G protein binding was examined. RESULTS: M6G was 66.87±0.73 percent free in human serum at physiological pH and temperature. The percentage free (unbound) was increased significantly by vincristine (4.33%) and methotrexate (9.68%), but 6- mercaptopurine and morphine had no significant effect on it. Free percentages of M6G was reduced by decreasing serum albumin concentration but was unaffected by the presence of alpa-1-acid glycoprotein (AAG) or changes in serum pH. Similar results were obtained in human serum albumin (HAS) solutions. Addition of palmitic acid and oleic acid reduced protein binding significantly by 6.3% and 7.4%, respectively. MAJOR CONCLUSION: Although M6G in this study was not highly bounded, but because of its high analgesic potency, any change in its free concentration due to concurrent medication or disease caused significant changes in its effects. This dearth of evidence has been implicated in the reluctance of professionals to be cautious in prescribing them to children, particularly in the neonatal period.     

Differences in the determinants of eplerenone, spironolactone and aldosterone binding to the mineralocorticoid receptor

The importance of mineralocorticoid receptor (MR) antagonists in the treatment of cardiovascular disease has been emphasised by two recent clinical trials, one using spironolactone and the other using a new selective MR antagonist, namely eplerenone. Eplerenone has a very low affinity for the glucocorticoid receptor (GR). Determinants of binding specificity of eplerenone to the MR were investigated using chimeras created between the ligand-binding domains (LBD) of the MR and the GR. These chimeras had been used previously to investigate aldosterone and spironolactone binding specificity to the MR. Eplerenone competed strongly for [(3)H]-dexamethasone binding to a MR/GR chimera containing amino acids 804-874 of the MR and weakly to a chimera containing amino acids 672-803 of the MR. Within the 804-874 region, eplerenone competed for [(3)H]-dexamethasone binding to a chimera containing amino acids 820-844 of the MR, although the calculated affinity was approximately 10-fold lower than for binding to the full-length MR LBD. Similar results were obtained using another MR antagonist, namely spironolactone. Modelling of eplerenone binding to the MR LBD, based on the GR LBD crystal structure, suggests that amino acids 820-844 affect the overall shape of the ligand-binding pocket and that eplerenone acts as an MR antagonist because it fails to stabilize the active conformation of the receptor. In contrast with results with the MR antagonists eplerenone and spironolactone, amino acids 820-844 are sufficient in themselves to confer high-affinity aldosterone binding to the MR, suggesting that the binding determinants of the two antagonists are similar to each other but differ from those of aldosterone.     

Postburn serum drug binding and serum protein concentrations

The free fractions of diazepam, imipramine, lidocaine, meperidine, phenytoin, propranolol, and salicylic acid were determined in the serum of seven burn patients (25% to 80% of their skin surface burned) about one week after the burn and in three of the patients at about four weeks following the injury. Serum protein fractions were measured by electrophoresis, and alpha-1 acid glycoprotein levels were determined. For the drugs that bind predominantly to albumin, the serum free fractions were greater in patients one week after the burn incident than in control subjects (diazepam, 0.055 vs. 0.017; phenytoin, 0.24 vs. 0.16; and salicylic acid, 0.69 vs. 0.32). The increase in free fraction for these drugs was attributed to the postburn decrease in serum albumin levels (2.2 vs. 4.4 g/dL, control). Imipramine, lidocaine, meperidine, and propranolol bind primarily to alpha-1 acid glycoprotein. The free fractions for these drugs decreased one week following the burn (imipramine, 0.074 vs. 0.095; lidocaine, 0.17 vs. 0.35; meperidine, 0.37 vs. 0.48; and propranolol, 0.045 vs. 0.107), presumably in response to the increased alpha-1 acid glycoprotein concentration (222 vs. 83 mg/dL, control). Diazepam, lidocaine, propranolol, and salicylic acid free fractions were still different from control values at four weeks after the accident.     

Comparative binding of two closely related dihydropyridines (isradipine and darodipine) to serum proteins and erythrocytes

The binding of the two drugs isradipine and darodipine, chemically related to dihydropyridines and potent calcium channel blockers, was studied in vitro to isolated plasma proteins, erythrocytes and human serum. The two drugs were strongly bound to serum proteins (up to 97%), mainly to human serum albumin (HSA), alpha 1-glycoprotein (AAG) and lipoproteins (VLDL, LDL and HDL). Their bindings to AAG were saturable with high affinity constants (isradipine 498,000 M-1, darodipine = 155,000 M-1; n = 1). The binding of these drugs to HSA, VLDL and HDL was unsaturable, but it was saturable on LDL. In blood the drugs partitioned in erythrocytes, 16% for isradipine and 14.8% for darodipine.     

Age does not influence the serum protein binding of bupivacaine.

The serum protein binding of bupivacaine was studied in 74 subjects, 39 males and 35 females, aged 20-90 years, without evidence of acute or chronic inflammatory disease or malignancy. Subjects were drug free for at least 1 month. The free fractions of bupivacaine did not change with age in either males or females. This is in keeping with the lack of effect of age on AAG concentrations. Free fractions of bupivacaine were slightly higher in females as compared with males. The previously observed decline in clearance of bupivacaine with age probably reflects a concomitant decline in the metabolic activity of hepatic enzymes.     

Serum protein binding of diazepam in maternal and foetal serum during pregnancy.

1 The serum binding capacity for diazepam was significantly lower in pregnancy and there was a linear correlation with gestational age. 2 The binding of diazepam was not correlated to albumin during pregnancy. 3 In cord sera there was a significantly reduced binding capacity for diazepam with albumin levels of less than 40 g/l.     

High prostaglandin-E1 binding to serum protein in allergic subjects.

Protein binding to both salicylate and 3H-labelled prostaglandin-E1 ([3H]PGE1) was examined in the sera of 22 allergic and 16 normal individuals. Protein binding to salicylate (P less than 0.001) and to [3H]PGE1 (P less than 0.01) was significantly greater in the allergic than in the normal group. The nature of the binding sites of salicylate and PGE1 was investigated with two fluorescent probes, dansylamide and dansylsarcosine as specific marker ligands for established Sites I and II, found to be specific for anionic drugs. The serum protein of 11 allergic subjects showed a higher binding at Site I (P less than 0.05) and a lower binding at Site II (P less than 0.05) than that of seven normal subjects. Salicylate and [3H]PGE1 bound competitively at the two sites. It was concluded, when comparing allergic to normal subjects, that the high protein binding of allergic individuals to salicylate and PGE1 could be attributed to qualitative and/or quantitative differences in the lipophilic substances which are tightly bound to the albumin of normal sera, causing a reduction in binding ability at Site I.     

Differences between blacks and whites in plasma protein binding of drugs

Objective: Disopyramide and salicylic acid were used as model compounds to characterize racial differences in binding of drugs by alpha₁-acid glycoprotein (AGP) and albumin, respectively. Drug-free plasma was collected from 29 healthy volunteers (15 white, 14 black). Disopyramide and salicylic acid unbound fractions (fu) in plasma were determined by equilibrium dialysis using ¹⁴C-disopyramide and ¹⁴C-salicylic acid. Results: Disopyramide unbound fractions were significantly higher in blacks than whites (0.131 vs 0.113) as were salicylic acid unbound fractions (0.053 vs 0.048). When unbound fractions were corrected for AGP and albumin concentration, racial differences were no longer present. Conclusion: Many drugs which bind to AGP and/or albumin may exhibit racial differences in unbound fractions. However, these differences are likely explained by differences in protein concentrations rather than differences in the number of binding sites on the protein or racial differences in affinity of the protein for drugs. Received: 22 April 1996 / Accepted in revised form: 17 July 1996     

In vitro protein binding studies with BMS-204352: lack of protein binding displacement interaction in human serum.

BMS-204352, a maxi-K channel opener, is currently under development for the treatment of stroke. Protein binding of BMS-204352 was determined in sera from several species, namely, rat, monkey, dog, and human. Data indicated that the compound was shown to be highly protein bound in serum from all species (ca. 99.6%). In order to test for the potential for drug-drug interactions and competitive displacement of BMS-204352 by diazepam, phenytoin, propranolol, and warfarin, in vitro experiments were performed using spiked human serum and ex vivo human plasma samples. Protein binding was determined using equilibrium dialysis for 4 h at maximal therapeutic concentrations for each drug alone or in appropriate combination in spiked serum samples. Ex vivo samples from a clinical BMS-204352 study (0, 1, and 24 h) were dialyzed separately after addition of diazepam, phenytoin, propranolol, or warfarin. Drug content in biological matrices was measured for radioactivity using liquid scintillation counting. Results indicated that (1) addition of diazepam, phenytoin, propranolol, or warfarin did not alter the free fraction of BMS-204352; (2) BMS-204352 did not displace diazepam, phenytoin, propranolol, or warfarin from their protein binding sites, and (3) comparison of ex vivo plasma samples after BMS-204352 dosing indicated no impact of BMS-204352 and/or its metabolites on the free fraction of diazepam, phenytoin, propranolol, or warfarin. In conclusion, the potential for a drug-drug interaction due to alterations in protein binding with BMS-204352 is unlikely.     

The binding protein of erythromycin in human serum

Erythromycin binding to human serum albumin and to alpha 1-acid glycoprotein was measured under conditions of binding equilibrium. At therapeutical concentrations of erythromycin the binding to albumin is not saturable. The fraction of total erythromycin bound to alpha 1-acid glycoprotein is proportionally related to the protein concentration and is bound to a single class of binding sites with an apparent association constant Ka = 0.16 X 10(6) M-1 (38 degrees). About one mole of erythromycin is bound per mole of alpha 1-acid glycoprotein. The binding affinity can be enhanced and vice versa lowered by increasing the concentrations of NaCl and urea, respectively. The semilogarithmic plot of bound/free ratios vs log concentration of NaCl or urea exhibits linear relationships. Erythromycin binding can be competitively inhibited by mersalyl (Ki = 11-16 microM) but not by other SH-reagents or by neuraminidase treatment. A marked reduction of erythromycin binding to alpha 1-acid glycoprotein is seen with dithiothreitol. alpha 1-acid glycoprotein is the main erythromycin binding protein in human serum.     

Unchanged protein binding and the increase of serum diazepam levels after food intake.

Seven subjects received diazepam 0.3 mg/kg intravenously twice with a 2-week interval between the doses. The subjects ingested a fatty or carbohydrate meal in a cross-over fashion 4 hours after the injection on both experimental days. Venous blood samples were drawn 2, 3, 4, 5 and 6 hours after the injection of diazepam for measurement of the serum levels of total and free (unbound) diazepam, N-desmethyldiazepam, and free fatty acids. Serum levels of diazepam decreased progressively with time until the food intake, after which a significant (P less than 0.01) postprandial increase (average 23%) occurred with both diets as compared to the preprandial levels at 4 hours (average 240 ng/ml). Serum levels of free fatty acids decreased significantly both after a fatty (P less than 0.01) and a carbohydrate (P less than 0.05) meal. Diazepam was extensively (96 to 98%) bound to proteins and no changes in its protein binding was found. It is concluded that the late impairment of psychomotor skills that occurs with an increase in the diazepam serum level after its intravenous administration is due rather to its re-mobilization from a storage site than to variations in its protein binding.     

Variability of the serum protein binding of theophylline in patients with asthma and cystic fibrosis.

1. The serum protein binding of theophylline was studied in 28 asthmatics and 11 patients with cystic fibrosis (CF) who were receiving the drug regularly. Peak theophylline samples were collected at 2 week intervals on four occasions in each asthmatic and on three occasions in each CF patient. The binding was measured using ultrafiltration at 37 degrees C and pH 7.4. The total and free (unbound) theophylline concentrations were measured using high-performance liquid chromatography. 2. The mean free-fractions (+/- s.d.) in asthmatics (0.50 +/- 0.03) and in CF patients (0.51 +/- 0.04) were not significantly different. The intra- and inter-subject variability in the free-fraction (fu) was relatively small in both patient groups. The binding was found to be concentration-independent at serum theophylline concentrations up to 30.9 micrograms ml-1. The effects of age, gender, serum albumin and total serum protein on the free fraction were evaluated. 3. The results indicate that the binding of theophylline is similar in the two disease states. The low degree of variability in serum theophylline binding indicates that measurements of total serum theophylline concentrations will reflect unbound serum concentrations with acceptable accuracy in both patient groups studied.     

血清降钙素原与C反应蛋白在感染性疾病中的诊断应用

目的：探讨血清降钙素原（PCT）、C反应蛋白（CRP）在感染性疾病诊断中的应用价值。方法：采用全定量的电化学发光法测定58例细菌感染性疾病患者及42例非细菌感染性疾病患者的PCT,应用免疫散射比浊法测定患者的CRP,并同时检测患者白细胞总数及分类。结果：58例细菌感染性疾病患者血清PCT、CRP及WBC均显著升高（P〈0.01）。PCT及WBC在两组间的阳性率比较差异具有统计学意义（P〈0.01）,而CRP差异无统计学意义（P〉0.05）。三者对细菌感染诊断的敏感性分别为93.1%、72.4%和63.8%;特异性分别为85.7%、59.5%、71.4%。对细菌感染的诊断PCT比CRP及WBC具有更高的敏感性及特异性（P〈0.01）。结论：血清PCT检测优于血清CRP和WBC检测,可用于感染性疾病细菌感染与病毒感染的鉴别诊断。     

Influence of smoking on serum protein composition and the protein binding of drugs1

The influence of smoking on α₁-acid glycoprotein (α₁-AGP) and serum albumin concentrations and the protein binding of phenytoin and propranolol in healthy volunteers was investigated. α₁-AGP concentrations were found to be statistically different (P < 0·05) in the smokers (mean = 84·3 mg dl⁻¹) versus non-smokers (mean = 62·8 mg dl⁻¹). There was a trend for lower serum albumin concentrations and lower fraction unbound of propranolol in the smokers. Smoking did not affect the protein binding of phenytoin.     

Kinetic determinants of hepatic clearance: plasma protein binding and hepatic uptake

Hepatic clearance prediction, using scaled data obtained from hepatocytes and microsomes, often under-predicts the eventual observed clearance. This occurs commonly where the compound is highly bound and/or a sinusoidal transporter substrate. The authors' own laboratory observations and those reported in the literature indicate that consideration of transporter effects in vitro is not sufficient to provide a direct, quantitative estimate of hepatic clearance in vivo. The physiology of contributing processes has been reviewed and the processes were compiled into a kinetic model of compound disposition for a hepatocyte compartment. The model has variables describing the kinetic effects of plasma protein binding, sinusoidal uptake, passive permeability, and cellular disposition. Parameters were determined experimentally requiring, in some instances, assays less familiar or routine to Drug metabolism and pharmacokinetics (DMPK) laboratories. The model accurately fitted data for the hepatic disposition of a UCB-proprietary compound that did not undergo metabolism but was a substrate for protein binding and sinusoidal uptake. On addition of bovine serum albumin to the assay, the uptake kinetics approximated neither those for the free fraction nor the total concentration. However, the model accurately predicted the intermediate observed kinetics and illustrated the kinetic effects of plasma protein binding and the complex interplay between various competing and collaborating processes. Through the use of simulations the model illustrated the influence of different combinations and influences of hepatic kinetic processes. Possible causes behind the extraction of highly bound compounds were identified as (1) low compound permeability facilitating active uptake and (2) high permeability facilitating the role of intracellular metabolism. In almost all circumstances k(off) is not low enough (<1 s(-1)) to limit the extraction of plasma protein bound compounds; however, k(on) still exerts an effect through competition with the uptake process. Crucially the model indicated the complex nature of the combined processes, whose broad range of effects on hepatic disposition could only be accurately determined with a kinetic model.     

Kinetic determinants of hepatic clearance: Plasma protein binding and hepatic uptake

Hepatic clearance prediction, using scaled data obtained from hepatocytes and microsomes, often under-predicts the eventual observed clearance. This occurs commonly where the compound is highly bound and/or a sinusoidal transporter substrate. The authors’ own laboratory observations and those reported in the literature indicate that consideration of transporter effects in vitro is not sufficient to provide a direct, quantitative estimate of hepatic clearance in vivo. The physiology of contributing processes has been reviewed and the processes were compiled into a kinetic model of compound disposition for a hepatocyte compartment. The model has variables describing the kinetic effects of plasma protein binding, sinusoidal uptake, passive permeability, and cellular disposition. Parameters were determined experimentally requiring, in some instances, assays less familiar or routine to Drug metabolism and pharmacokinetics (DMPK) laboratories. The model accurately fitted data for the hepatic disposition of a UCB-proprietary compound that did not undergo metabolism but was a substrate for protein binding and sinusoidal uptake. On addition of bovine serum albumin to the assay, the uptake kinetics approximated neither those for the free fraction nor the total concentration. However, the model accurately predicted the intermediate observed kinetics and illustrated the kinetic effects of plasma protein binding and the complex interplay between various competing and collaborating processes. Through the use of simulations the model illustrated the influence of different combinations and influences of hepatic kinetic processes. Possible causes behind the extraction of highly bound compounds were identified as (1) low compound permeability facilitating active uptake and (2) high permeability facilitating the role of intracellular metabolism. In almost all circumstances k_off is not low enough (<1?s^–1) to limit the extraction of plasma protein bound compounds; however, k_on still exerts an effect through competition with the uptake process. Crucially the model indicated the complex nature of the combined processes, whose broad range of effects on hepatic disposition could only be accurately determined with a kinetic model.     

血清视黄醇结合蛋白测定在营养评定中的意义

目的评价血清视黄醇结合蛋白在营养评定中的意义。方法18例普外危重患者为观察组 ,20例普外轻病情患者为对照组 ,测定两组入院48小时内及观察组7天后的血清视黄醇结合蛋白浓度 ,同时检测白蛋白指标。结果入院48小时内血清视黄醇结合蛋白观察组显著低于对照组(P<0.05) ,而血清白蛋白两组无显著差异 ,7天后才显著下降(P<0.05)。结论视黄醇结合白蛋白比白蛋白更灵敏地反映患者营养状态的改变。