Altered drug-serum protein binding in the genetically obese Zucker rat

Drug-serum protein binding was evaluated in genetically obese Zucker rats, their lean littermates, and lean Sprague-Dawley rats. The free fraction (fp) of phenytoin was significantly higher in the obese rat (fp = 0.177) compared to its lean littermate (fp = 0.136), apparently due to displacement by free fatty acids. Conversely, diazepam and propranolol fp values were decreased in the obese Zucker rat (fp = 0.107 and fp = 0.122, respectively) compared to the lean Zucker rat (fp = 0.140 and fp = 0.174, respectively). Evidence strongly suggests that the increased binding of propranolol was not due to elevations in the serum concentrations of alpha1-acid glycoprotein (as is the case in the human obese population). Rather, the decreased fp for both diazepam and propranolol was a result of increased lipoprotein partitioning. Strain differences between the lean Zucker rat and lean Sprague-Dawley rat were also evident, with serum binding of the Sprague-Dawley rat more closely resembling the obese Zucker rat.     

Intraindividual relationships between serum protein binding of drugs in normal human subjects, patients with impaired renal function, and rats.

The serum protein binding of phenytoin, salicylic acid, sulfisoxazole, and warfarin was determined in normal human adults, in patients with impaired renal function (kidney donor and recipient), and in adult male Sprague--Dawley rats. The free fraction values for salicylate and sulfisoxazole were significantly correlated in all three groups. The other correlations were statistically significant in only one or two of these groups. There was a statistically significant negative correlation between albumin concentration and the free fraction values of salicylic acid and sulfisoxazole (but not of phenytoin and only under special circumstances with warfarin) in normal subjects and of phenytoin, salicylic acid, and sulfisoxazole (but not warfarin) in rats. No such correlation was observed for any of the drugs in patients with impaired renal function. These observations show that no single weakly acidic drug can serve as an index for quantitatively determining the effect of disease or species differences on the serum protein binding of other weakly acidic drugs.     

Protein covalent binding of maxipost through a cytochrome P450-mediated ortho-quinone methide intermediate in rats.

(3S)-(+)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one) (MaxiPost, BMS-204352) is a potent and specific opener for maxi-K channels and has potential to prevent and treat ischemic stroke. Following single intravenous doses of [14C]BMS-204352 to rats, only 10 to 12% of radioactivity was extractable from plasma with organic solvents. The unextractable radioactivity remained associated with the proteins (mostly albumin) after SDS-polyacrylamide gel electrophoresis or dialysis. Following acid hydrolysis in 6 M HCl for 24 h at 110 degrees C from plasma proteins collected from nine rats dosed with [14C]BMS-204352, one major radioactive product was isolated and identified as a lysine-adduct of des-fluoro des-O-methyl BMS-204352 by liquid chromatography/mass spectrometry and NMR analyses as well as by comparison with the synthetic analog, lysine-adduct of des-fluoro BMS-204352 (BMS-349821). The covalent binding of BMS-204352 results from the displacement of the ring-fluorine atom of des-O-methyl BMS-204352 with the epsilon-amino group of a lysine residue. Microsomal incubations of [14C]BMS-204352 resulted in low levels of covalent binding of radioactivity to proteins. This in vitro covalent binding required cytochrome P450-reductase cofactor NADPH and was attenuated by glutathione. P4503A inhibitors ketoconazole and troleadomycin selectively prevented the covalent binding in vitro. Based on these observations, a two-step bioactivation process for the protein covalent binding of BMS-204352 was postulated: 1) P4503A-mediated O-demethylation leading to spontaneous release of HF and the formation of an ortho-quinone methide reactive metabolite and 2) nucleophilic addition of the epsilon-amino group of protein lysine residue(s) in protein to form des-fluoro des-O-methyl BMS-204352 lysine adduct.     

Factors affecting the milk-to-plasma drug concentration ratio in lactating women: physical interactions with protein and fat

The factors that affect M/P (milk-to-plasma) ratios of several compounds were assessed in vitro using samples collected from four healthy lactating women. Three drugs were studied: diazepam, phenytoin, and propranolol. Serum and skim milk protein binding were determined by equilibrium dialysis at 37 degrees C. Skim-to-whole milk concentration ratios (S/M) were determined after incubation of whole milk at 37 degrees C for 1 h. Free fractions in serum and skim milk, respectively, were 0.013 and 0.334 for diazepam, 0.142 and 0.584 for phenytoin, and 0.107 and 0.699 for propranolol. The S/M values were 0.220, 0.727, and 0.610 for diazepam, phenytoin, and propranolol, respectively. An equation was derived which relates M/P to milk and plasma pH, unbound fractions of drug in skim milk and serum, and S/M. The M/P ratios calculated from in vitro data agreed with published in vivo M/P ratios. The results indicated that milk protein binding and fat partitioning can make a substantial contribution in determining M/P, and predictions of M/P made without considering these factors may be misleading.     

Serum alpha 1-acid glycoprotein and the binding of drugs in obesity

Although clinically relevant, drug-protein interactions in the morbidly obese population have not been studied thoroughly. The objective of this study was to evaluate serum chemistry profiles and the degree of serum protein binding of propranolol, diazepam and phenytoin in the serum of four female, morbidly obese (greater than 190% of ideal body weight) and eight control female subjects. Serum triglyceride concentrations were higher and high-density lipoproteins were lower in the obese subjects than in the control group. Serum albumin and total protein concentrations in the obese were not different from controls. Unexpectedly, alpha 1-acid glycoprotein concentrations were doubled in the obese subjects (mean obese value 121 mg/100 ml vs 62.9 mg/100 ml for the control subjects). Obese subjects had a mean fraction unbound (fu) for propranolol of 0.086, which was significantly different from the controls (fu = 0.123). The binding of diazepam was decreased slightly in the obese subjects. The binding of phenytoin was similar in both groups. The altered serum chemistry of obesity may play a significant role in the drug management of the obese patient by altering drug-protein interactions.     

The interaction of enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of acidic drugs to human serum albumin. An in vitro study

The interaction of the volatile anaesthetics enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of two highly bound acidic drugs (warfarin, phenytoin) to albumin has been studied in vitro by equilibrium dialysis. Trifluoroacetic acid (TFA) inhibited the binding of both drugs to human serum albumin (HSA). Halothane, on the other hand, increased the binding of warfarin to HSA, while enflurane inhibited only the binding of phenytoin. It seems that the binding of the acidic drugs warfarin and phenytoin to HSA is more sensitive to the structures of the gases than for the basic drug diazepam which was previously shown to be equally affected by both gases. Furthermore, it seems that drugs competing for the same binding site (warfarin, phenytoin) may respond differently to conformational changes of the site. It is suggested that drugs bound to the "diazepam site" are more easily affected by the volatile anaesthetics than drugs bound to the "warfarin site".     

Serum protein binding and the role of increased alpha 1-acid glycoprotein in moderately obese male subjects

Serum protein and lipid concentrations as well as the serum protein binding of propranolol, diazepam and phenytoin were measured in normal weight and obese volunteers. Concentrations of alpha 1-acid glycoprotein (AAG) in the obese subjects were double that of the lean controls. Conversely, concentrations of high density lipoproteins (HDL) were decreased in the obese group. The serum binding of propranolol was increased in the obese subjects and correlated with serum AAG concentrations. Diazepam binding was slightly decreased in the obese as a result of lower serum albumin concentrations and elevated free fatty acids. The binding of phenytoin was comparable in all of the volunteers. These findings point out some of the complex pathophysiologic changes associated with obesity which may in turn influence drug disposition and hence drug therapy in the obese patient.     

Metabolism, pharmacokinetics, and protein covalent binding of radiolabeled MaxiPost (BMS-204352) in humans.

MaxiPost [(3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one); BMS-204352] is an investigational maxi-K channel opener to treat ischemic stroke. This study reports the disposition, metabolism, pharmacokinetics, and protein covalent binding of (14)C-labeled MaxiPost in healthy male volunteers as well as in dogs and rats. After each human subject received a single dose of 10 mg (14)C-labeled BMS-204352 (50 microCi) as a 5-ml intravenous infusion lasting 5 min, the plasma radioactivity concentrations showed a unique profile, wherein the concentration appeared to increase initially, followed by a terminal decline. The mean terminal t(1/2) of plasma radioactivity (259 h) was prolonged compared with that of unchanged parent (37 h). Furthermore, the extractability of radioactivity in plasma decreased over time, reaching approximately 20% at 4 h after dosing. The unextractable radioactivity was covalently bound to plasma proteins through a des-fluoro-des-methyl BMS-204352 lysine adduct. Unchanged BMS-204352 and minor metabolites were identified in plasma extract following protein precipitation. The recovery of the radioactive dose in urine and feces was nearly complete in 14-day collections (approximately 37% in urine and 60% in feces). The N-glucuronide of the parent was the prominent metabolite in urine (16.5% of dose), whereas the parent was a major drug-related component in feces (11% of dose). Similar disposition, metabolism, pharmacokinetic, and protein covalent binding properties of (14)C-labeled BMS-204352 were observed in humans, dogs, and rats.     

Plasma protein binding of drugs in pregnancy

The degree of binding to plasma proteins is an important determinant of drug disposition and response. Normal human pregnancy is associated with concentration of plasma proteins, free fatty acids and possibly other endogenous substances interfering with drug binding. The possibility of an associated change in plasma binding capacity therefore needs to be taken into consideration. Experimental studies conducted mostly in vitro have shown that the plasma protein binding of many (but not all) drugs is decreased during pregnancy, particularly during the last trimester. This phenomenon should be taken into account when interpreting serum concentrations of total (free + protein-bound) drug in clinical practice. Notable examples of drugs whose unbound fraction increases during pregnancy include diazepam, valproic acid, phenytoin, phenobarbitone, salicylic acid, pethidine, lignocaine, dexamethasone, sulphafurazole and propranolol. For many drugs, important differences have been demonstrated in the degree of protein binding between maternal and cord plasma. In some cases, this may provide an explanation for the finding of marked differences in total drug concentration between maternal and fetal plasma at the time of delivery.     

Ceftriaxone binding to human serum albumin. Indirect displacement by probenecid and diazepam

In vitro protein binding studies were conducted to examine the interaction between ceftriaxone (CEF), probenecid (PROB) and diazepam (DIAZ). The presence of PROB and DIAZ at concentrations equal to molar albumin concentration caused a decrease in CEF affinity from 3.7 x 10(4) M-1 (control) to 1.1 x 10(4) (PROB) and 2.6 x 10(4) (DIAZ) M-1, but not in binding capacity in pooled human plasma. PROB and DIAZ at five times the molar albumin concentration also caused a decrease in CEF affinity from 4.5 x 10(4) M-1 (control) to 0.45 x 10(4) (PROB) and 3.0 x 10(4) (DIAZ) M-1 in isolated human serum albumin. DIAZ and PROB displaced one another, confirming their common binding site (Site II, the benzodiazepine site) on serum albumin. By contrast, CEF was unable to displace either PROB or DIAZ from defatted albumin. In the presence of elevated free fatty acid concentrations (four times the albumin concentration), CEF decreased the binding of both drugs. CEF free fraction (fp) in isolated human serum albumin (CEF fp = 7.7%) was increased by drugs which bind to Site I: sulfisoxazole (CEF fp = 68.1%), warfarin (CEF fp = 56.0%) and furosemide (CEF fp = 55.0%). At ten times the molar concentration of albumin, CEF displaced both warfarin (warfarin fp from 0.99 to 2.20%) and phenytoin (phenytoin fp from 17.7 to 23.4%) from defatted albumin. CEF appeared to bind to Site I (the warfarin site) on human serum albumin, and was displaced by PROB and DIAZ via a mechanism which did not involve direct competition at a common binding site.     

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.     

Serum protein binding of phenytoin and valproic acid in insulin-dependent diabetes mellitus

The serum protein binding of valproic acid (VPA) and phenytoin (PHT) was determined in spiked serum samples collected from 17 patients with insulin-dependent diabetes mellitus and 16 healthy control subjects. The free fraction of VPA was significantly greater in patients than in controls (7.6 +/- 1.6% vs. 6.2 +/- 1.2%, p less than 0.01); for PHT, free fraction values were similar in the two groups (8.2 +/- 1.1% vs. 8.4 +/- 1.2%). The free fraction of VPA in diabetic patients was positively correlated with free fatty acid (FFA) concentration (r = 0.79, p less than 0.01). No significant relationships could be found between free drug fraction and either serum albumin or glycosylated protein concentration.     

Acetaldehyde adducts with serum proteins: effect on diazepam and phenytoin binding

The in vitro effects of acetaldehyde treatment on the binding of phenytoin and diazepam to human serum albumin (HSA) and human serum proteins (HSP) have been investigated. The incorporation of acetaldehyde into proteins following incubation with different concentrations of [1,2-14 C]-acetaldehyde (0.5, 25, 100 mmol/l) was carried out. The proteins were then dialyzed so that only the stable adduct was retained. Binding of phenytoin and diazepam was then studied. Scatchard plot analysis showed a slight decrease (p less than 0.01 for HSP and 25 mmol/l acetaldehyde) in the number of binding sites for phenytoin when the acetaldehyde/protein ratio was increased. The affinity constant was also increased (p less than 0.01) with 100 mmol/l acetaldehyde. No change could be demonstrated in the number of diazepam binding sites on HSA; an increase in the binding capacity of HSP was shown following incubation with 25 mmol/l acetaldehyde. The fraction of drug bound at therapeutic levels has been also calculated for both drugs. An increase for diazepam but no change for phenytoin can be observed before or after treatment of proteins with acetaldehyde.     

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.     

Interpatient and intrapatient variability in phenytoin protein binding

The authors retrospectively assessed the relation between total and free serum concentrations and serum albumin in a sample of hospitalized patients to evaluate how well free serum concentrations can be estimated from total phenytoin serum concentrations. The authors also assessed the interpatient and intrapatient variability of the phenytoin free fraction. Paired serum samples of total and free phenytoin serum concentrations and serum albumin were obtained from 48 hospitalized patients (28 males, 20 females; mean age, 51 y; range, 13-90 y). Concomitant medications were recorded. Phenytoin free fraction and adjusted total phenytoin serum concentrations (adjusted for serum albumin) were calculated. One hundred sixty-three samples were obtained (mean, 3.4 samples per patient; range, 1-16 samples); 28 patients had more than one pair of samples obtained. Mean phenytoin free fraction was 15% +/- 7% (range, 4%-61%) for the 163 samples. The variability for the total, free, and free fractions were 65%, 75.9%, and 45.8%, respectively. There was significant variability in the phenytoin free fraction within individual patients who had more than one pair of serum concentrations obtained. The intraindividual coefficient of variation in phenytoin free fraction was 85% +/- 21.3% (range, 2%-94%). Despite strong overall correlation between the total phenytoin serum and free serum concentrations, there is excessive variability in phenytoin protein binding. Correction for serum albumin was not useful in this patient group. Because of significant interpatient and intrapatient variability in phenytoin serum concentrations, monitoring of total serum concentrations is unreliable and free phenytoin serum concentrations should be considered for monitoring in hospitalized patients.     

In vitro protein binding interaction studies involving cefixime

Cefixime is a new oral cephalosporin currently undergoing clinical trials. Selected agents with the likelihood for coadministration with cefixime in man were examined for their influence on the in vitro binding of cefixime in pooled serum from dog, monkey, and man. Results from these experiments showed no significant change in cerfixime binding in any animal species studied or in man by acetaminophen, heparin, phenytoin, diazepam, ibuprofen or furosemide at their maximum reported therapeutic concentrations. In contrast, both salicylic acid and probenecid resulted in concentration-dependent increases in the free fraction of cefixime (up to 2.5-fold). These findings demonstrate the usefulness of in vitro protein binding screening procedures for studying potential drug interactions that are mediated, at least in part, by changes in the protein binding of a drug.     

Alterations of phenytoin protein binding with in vivo haemodialysis in dialysis encephalopathy

Summary Protein binding of phenytoin was assessed in one patient with dialysis encephalopathy before and after haemodialysis. Phenytoin concentrations were measured by radioimmunoassay and continuous ultrafiltration was used to assess phenytoin binding. At a serum concentration of 60 µmol.1^–1 the percentage of phenytoin bound to serum albumin was considerably lower in the patient serum (79.95% predialysis; 92.09% postdialysis) than that in three normal sera (97.90±0.17%). Analysis of Scatchard plots indicated two classes of binding sites. In class I both the affinity and capacity for binding phenytoin were reduced in the pre and post-dialysis serum, whereas in class II the capacity of the uraemic serum was increased although the intrinsic association constant was greatly reduced. It was concluded that in vivo haemodialysis is associated with large fluctuations in the protein binding of phenytoin, in which the concentration of endogenous dialysible metabolites are strongly implicated.     

Effect of total drug concentration on the free fraction in uremic sera

The effect of total drug concentration on the free fraction was studied in vitro using sera of uremic and nonuremic subjects. Both therapeutic and toxic concentrations of radiolabeled phenytoin, diazepam, and propranolol were used. The free fraction was separated by a pressure ultrafiltration method at 37 degrees C and determined from 200-microliters aliquots of serum and the ultrafiltrate. Sera from 11 acutely uremic and 10 chronically uremic patients and from 10 healthy control subjects were used. Only slight increases in the free fraction were found over the concentration range studied in both nonuremic and uremic subjects. The concentration-dependent increases in the unbound fraction of phenytoin and diazepam were quite negligible compared with the two- to threefold increases caused by uremia itself. Thus, variation in total concentrations of phenytoin, diazepam, and propranolol does not change the free fractions to a clinically significant degree even in uremic patients with a compromised binding capacity.     

Protein binding of drugs in uremic and normal serum: The role of endogenous binding inhibitors

The protein binding of diazepam, indomethacin, salicylic acid, sulfadimetoxine and warfarin in serum of uremic patients has been studied by equilibrium dialysis and circular dichroism measurements and compared with that in normal serum. Comparisons have also been made with isolated human serum albumin (HSA) from uremic patients and healthy individuals. The binding of diazepam, salicylic acid, sulfadimetoxine and warfarin is impaired in the uremic sera, while the binding of indomethacin is apparently unchanged. The apparent binding constants of salicylic acid and warfarin in both uremic and normal sera are affected by dilution of the sera in buffer. The binding constants obtained with isolated albumins, however, are unaffected by dilution. The albumin isolated from uremic serum shows lower binding affinity for salicylic acid and warfarin than normal HSA, but the affinity was normalized by charcoal treatment at pH 3.0. It is shown that the binding both in normal and uremic sera is impaired compared with isolated defatted serum albumin due to the presence of competitive inhibitors. The inhibition is more pronounced in uremic serum. In addition, the binding to albumin in uremic sera is impaired by strongly bound allosteric inhibitors. It is also emphasized that determinations of association constants have to be related to the dilution of the serum, plasma or blood, respectively.     

Differential effects of valproic acid on the serum protein binding of lorazepam and diazepam

Valproic acid and free fatty acids have been shown to displace diazepam from its plasma binding sites both in vitro and in vivo. Since lorazepam exhibits a substantial degree of binding, but differs from other benzodiazepines in that no increase in its free fraction in serum is observed when free fatty acids are raised, the effect of valproate on the serum protein binding of diazepam and lorazepam was assessed. Sodium valproate produced a marked increase in the free fraction of diazepam, but practically no effect on the percentage of free lorazepam.