Protein binding of oxazepam and its glucuronide conjugates to human albumin

The binding of oxazepam and its glucuronide conjugates to human serum albumin (HSA), as well as the binding interactions of the drug and its metabolites, were examined by equilibrium dialysis and kinetic probe studies. Oxazepam and its S(+) glucuronide are bound to the HSA molecule with affinity constants of 3.5 X 10(5) M-1 and 5.5 X 10(4) M-1, respectively, which were independent of protein concentration over a range of 0.1 to 5.0 g/dl. The R(-) glucuronide bound weakly to albumin, with the binding parameter, N X K, increasing at lower albumin concentrations. Pre-acetylation of fatty acid free-HSA resulted in decreased binding of all three compounds, probably by altering the conformation of the binding sites. Kinetic probe studies with p-nitrophenyl acetate indicate that oxazepam and its S(+) glucuronide shared a common binding site on HSA, but that the R(-) glucuronide bound at another site. Oxazepam binding was unaffected by the presence of its glucuronide conjugates but was inhibited by fatty acids. The percentage of oxazepam bound to plasma proteins in patients with renal impairment (94%) was lower than in normal volunteers (97%). This lower binding can neither be attributed to lower albumin concentrations because of the large binding capacity of the protein and linearity of N X K nor to displacement by elevated concentrations of glucuronide conjugates, but it may be ascribed partly to increased plasma fatty acids.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

Photoinduced covalent binding of frusemide and frusemide glucuronide to human serum albumin

AIMS: To study reaction of photoactivated frusemide (F) and F glucuronide (Fgnd metabolite) with human serum albumin in order to find a clue to clarify a mechanism of phototoxic blisters from high frusemide dosage. METHODS: F was exposed to light in the presence of human serum albumin (HSA). HSA treated with this method (TR-HSA) was characterized by fluorescence spectroscopic experiment, alkali treatment and reversible binding experiment. RESULTS: Less 4-hydroxyl-N-furfuryl-5-sulphamoylanthranilic acid (4HFSA, a photodegradation product of F) was formed in the presence of HSA than in the absence of HSA. A new fluorescence spectrum excited at 320 nm was observed for TR-HSA. Alkali treatment of TR-HSA released 4HFSA. Quenching of the fluorescence due to the lone tryptophan near the warfarin-binding site of HSA was observed in TR-HSA. The reversible binding of F or naproxen to the warfarin-binding site of TR-HSA was less than to that of native HSA. These results indicate the photoactivated F was covalently bound to the warfarin-binding site of HSA. The covalent binding of Fgnd, which is also reversibly bound to the warfarin-binding site of HSA, was also induced by exposure to sunlight. Fgnd was more photoactive than F, indicating that F could be activated by glucuronidation to become a more photoactive compound. CONCLUSIONS: The reactivity of photoactivated F and Fgnd to HSA and/or to other endogenous compounds may cause the phototoxic blisters that result at high F dosage.     

Effect of ethanol on the binding of warfarin enantiomers to human serum albumin

Ethanol is widely used as a pharmaceutical excipient for the solubilization of many hydrophobic drugs for injections. However, there are only few studies about drug interaction with pharmaceutical excipients in the body after injection. In this study, the effect of ethanol (500 mM) or several alcohols (500 mM) on the stereoselective binding of warfarin enantiomers to fatty acid-free human serum albumin (HSA) or proteins of commercial albumin preparations was investigated. An ultrafiltration method was used for the separation of unbound warfarin enantiomers. By the addition of ethanol or 1-propanol, the unbound fraction of the S-enantiomer was decreased. On the other hand, the unbound fraction of the R-enantiomer was increased by the addition of ethanol or 1-propanol. Unbound fractions of both the S- and R-enantiomer were decreased by 2-propanol. In various commercial albumin preparations, unbound fractions of both the S- and R-enantiomer were increased by ethanol. The different effects of ethanol among fatty acid-free HSA and commercial albumin preparations were observed.     

Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein.

Stereoselective degradation of fenoprofen (FEN) glucuronides and irreversible binding of FEN enantiomers to human serum albumin via their glucuronides were studied. At different pH values, 37 degrees C, and in the absence of albumin, degradation half-lives were diastereomeric, resulting mainly from a combination of hydrolysis and acyl migration. Lower pH enhanced FEN glucuronide stability and reduced the extent of irreversible binding. The degradation rate of R-FEN glucuronide was greater than that of the S-glucuronide (S-FEN). When human serum albumin was added to the medium, stability was decreased as compared to protein-free buffer. FEN glucuronides were readily hydrolyzed to parent drug, indicating an esterase-like activity of the albumin molecule. In vitro irreversible binding was higher for R-FEN (1.22% +/- 0.36) than for S-FEN glucuronide (0.76% +/- 0.12), when a 0.1 mM concentration of each conjugate enantiomer was incubated under physiological conditions (pH 7.4, 37 degrees C). Incubation with unconjugated FEN did not lead to measurable irreversible binding. Analysis of plasma samples from a clinical study showed that enantioselective irreversible binding of FEN to plasma proteins also occurs in vivo. After administration of a single 600-mg dose of racemic FEN to six healthy volunteers, covalent binding of R- and S-FEN to plasma proteins was measured in all subjects. The percentage of S-FEN protein adduct was greater than that of its R-enantiomer adduct. Total amounts of FEN irreversibly bound to plasma protein in vivo were also very low (1.02 +/- 0.32 and 3.23 +/- 0.85 mol/mol protein x 10(-4) for R- and S-FEN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective binding of a 2,3-benzodiazepine to human serum albumin: Effect of conformation on tofizopam binding

The binding of Tofizopam enantiomers to human serum albumin has been investigated by ultrafiltration and affinity chromatography. In solution, Tofizopam molecules exist in two conformations which slowly interconvert into each other. Both conformers of (R)-Tofizopam have the same binding constant of 4.8 × 10³ M^?1. The binding of the (S)-enantiomer, however, depends on the conformation. The minor and major (S)-conformers were characterized by association constants of 2.3 × 10³ and 15.1 × 10³ M^?1, respectively. Thus, the stereoselectivity of binding differs for the two conformations of the enantiomers. Kinetic parameters for the interconversion of conformations have been determined. Tofizopam displaces both bound diazepam and warfarin.     

Azapropazone binding to human serum albumin

Summary Azapropazone, a new non-steroidal antiinflammatory drug, is strongly bound to human serum albumin. As revealed by Scatchard analysis, one high-affinity binding site with an association constant of about 1.2×10⁶ M^–1 and two low-affinity binding sites with association constants of about 0.05×10⁶ M^–1 were found. While the high-affinity binding site of azapropazone is clearly not identical with the diazepam or digitoxin binding sites of human serum albumin, contradictory evidence was found by optical measurements and displacement studies for the similarity of the azapropazone and the warfarin binding site of human serum albumin. At present, it is suggested that both drugs bind to different areas of the same binding site. Therefore, the pronounced effects of various disease states on the plasma protein binding of azapropazone can not be explained by a binding to an unusual binding site, but seem to be due to an extreme sensitivity of the azapropazone binding area to the putative endogenous binding inhibitors, present in the blood during those disease states.     

Interactions between oxaprozin glucuronide and human serum albumin

1. The first step in the interaction between oxaprozin glucuronide and human serum albumin (HSA) is formation of a reversible complex which then leads to the following reactions; (a) acyl migration of the aglycone from position 1 to positions 2, 3 and 4 of the glucuronic acid moiety; (b) hydrolysis of the glycosidic bond; and (c) covalent binding of oxaprozin to the HSA molecule. The isomers of oxaprozin glucuronide formed in (a) and the covalently bonded drug in (c) are also hydrolyzed to oxaprozin.

2. Oxaprozin and ligands known to bind at Site II as classified by Sudlow et al. (1976), also called the benzodiazepine binding site (Müller and Wollert 1975), inhibit these reactions with oxaprozin glucuronide, while ligands which are known to bind at other sites on HSA do not.

3. Modification of a single tyrosine residue, located within Site II, with tetranitromethane, diisopropylfluorophosphate, and p-nitrophenylacetate causes significant reduction of the covalent binding of oxaprozin to HSA.

4. Tetranitromethane modification of HSA decreases all three reactions, while not inhibiting the formation of the reversible complex, indicating that the tyrosine located in Site II (tyr-411)acts as the nucleophile in these reactions.

5. Chemical modification of lysine residues has only a small effect on the reactions while modification of the lone free sulphhydryl (cys) in HSA has no effect.     

Stereoselective binding of 3-acetoxy-, and 3-hydroxy-1,4-benzodiazepine-2-ones to human serum albumin. Selective allosteric interaction with warfarin enantiomers

Stereoselective binding of oxazepam, lorazepam, temazepam and methyl lorazepam as well as of their acetates to human serum albumin was investigated by different techniques. The 2'-chlorine and the N(1)-methyl substitution exert opposite effects on the antipodes. Enantiomers of oxazepam acetate (OAc) and lorazepam acetate (LAc) displace diazepam. Allosteric interactions with warfarin were manifested by either mutually increased or decreased binding depending on the structure of benzodiazepine and on the configuration of both benzodiazepine and warfarin. The most remarkable effect could be observed in the simultaneous binding of (S)-lorazepam acetate and (S)-warfarin.     

Interactions between oxaprozin glucuronide and human serum albumin

1. The first step in the interaction between oxaprozin glucuronide and human serum albumin (HSA) is formation of a reversible complex which then leads to the following reactions; (a) acyl migration of the aglycone from position 1 to positions 2, 3 and 4 of the glucuronic acid moiety; (b) hydrolysis of the glycosidic bond; and (c) covalent binding of oxaprozin to the HSA molecule. The isomers of oxaprozin glucuronide formed in (a) and the covalently bonded drug in (c) are also hydrolyzed to oxaprozin. 2. Oxaprozin and ligands known to bind at Site II as classified by Sudlow et al. (1976), also called the benzodiazepine binding site (Müller and Wollert 1975), inhibit these reactions with oxaprozin glucuronide, while ligands which are known to bind at other sites on HSA do not. 3. Modification of a single tyrosine residue, located within Site II, with tetranitromethane, diisopropylfluorophosphate, and p-nitrophenylacetate causes significant reduction of the covalent binding of oxaprozin to HSA. 4. Tetranitromethane modification of HSA decreases all three reactions, while not inhibiting the formation of the reversible complex, indicating that the tyrosine located in Site II (tyr-411)acts as the nucleophile in these reactions. 5. Chemical modification of lysine residues has only a small effect on the reactions while modification of the lone free sulphhydryl (cys) in HSA has no effect.     

Stereoselective binding properties of naproxen glucuronide diastereomers to proteins

The stability of naproxen glucuronide (NAP-G) diastereomers was investigated in buffer, 0.3% and 3% human serum albumin (HSA) solutions, and human plasma.R-NAP-G was found to be less stable in phosphate buffer than itsS-diastereomer, whereas incubation media containing protein in general increased the degradation rate of NAP-G but also caused a change of the stereoselective stability where theR-NAP-G was more stable thanS-NAP-G. Reversible binding of NAP-Gs to HSA (0.3%) was investigated and compared with the corresponding properties of naproxen (NAP) enantiomers. NAP-G diastereomers exibited a considerable and stereoselective affinity to HSA, although less than that observed for the NAP enantiomers.In vitro irreversible binding of NAP-Gs to HSA, human and rat plasma proteins was also investigated. Irreversible binding was higher forR-NAP-G (50 μM) than forS-NAP-G (50 μM) in all incubation media. This stereoselective difference was observed with HSA containing medium as well as in rat and human plasma. Incubation with unconjugated NAP did not lead to irreversible binding. Preincubation of HSA with acetylsalicylic acid (≈ 11 mM) and glucuronic acid (50 mM) decreased the extent of irreversible binding suggesting involvement of lysine residues for covalent binding. Preincubation withS-NAP also decreased the irreversible binding yield. This paper is dedicated to Professor Richard Neidlein, Pharmaceutical Chemistry Institute, Heidelberg, in commemoration of his 65th birthday. Supported in part by National Institutes of Health Grants GM 36633 and DK 26307.     

Stereoselective protein binding of verapamil enantiomers

The binding of the (+)- and (-)-enantiomers of verapamil (V) to purified albumin (40 g/L), alpha 1-acid glycoprotein (0.55 g/L) and fresh serum has been studied over a wide range of verapamil concentrations (0.055 to 22 microM). The free fraction of the pharmacologically more potent (-)-V was always greater than that of (+)-V. Similar free fractions were observed in solutions of alpha 1-acid glycoprotein ((+)-V 0.079 +/- 0.016; (-)-V 0.142 +/- 0.020) and fresh serum ((+)-V 0.096 +/- 0.009; (-)-V 0.136 +/- 0.006), however the free fraction was higher in a solution of albumin ((+)-V 0.400 +/- 0.030; (-)-V 0.572 +/- 0.029). Saturation of verapamil binding sites was observed for alpha 1-acid glycoprotein only. Enantioselective verapamil serum binding was also noted in samples collected from five healthy volunteers following oral and intravenous verapamil administration. The free fraction of the individual isomers in vitro when added to predose serum as the pseudoracemic drug ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed for the enantiomers when studied separately in vitro, indicating that the binding of each enantiomer is independent of the other optical isomer. The free fraction ex vivo after intravenous therapy ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed in vitro in that subjects pre-dose serum. The free fraction of both enantiomers, however, was higher after oral drug therapy ((+)-V 0.13 +/- 0.02, (-)-V 0.23 +/- 0.03). The lower binding noted may be a result of competition for serum binding sites by verapamil metabolites, which attain higher concentrations following oral dosing.     

Binding of racemic indoprofen and its enantiomers to human serum albumin

Binding of Racemic Indoprofen and its Enantiomers to Human Serum Albumin. The binding to 2% and 4% human serum albumin (HSA) of (+/-)-indoprofen, (-)-indoprofen, and (+)-indoprofen was examined applying a modified ultrafiltration process. The binding properties to HSA were characterized by determining the unbound portions of the drug (a), the overall binding constants (K1), the free reaction energy (delta F degrees), the apparent binding constants (k*), and slope (m). The following results were obtained: 1. Racemic indoprofen and its enantiomers show a high affinity to human serum albumin (a = 1-2%). 2. At higher HSA concentrations the free portion (a) diminishes. 3. The affinity to HSA is reduced in the order of levo-indoprofen racemic indoprofen, and dex-indoprofen.     

A quantitative circular dichroic investigation of the binding of the enantiomers of ibuprofen and naproxen to human serum albumin

The binding constants for racemic, R and S naproxen and ibuprofen to human serum albumin have been determined by a circular dichroic technique. The ibuprofens and naproxens show no measurable extrinsic optical activity on interaction with the protein, and so the extrinsic Cotton effect shown following the diazepam–albumin interaction is used as a probe. The presence of the drugs reduce the amount of diazepam bound as shown by the reduced size of the induced ellipticity. The calculated primary binding constants show that the S form of both drugs bind to the albumin more tightly than the R form and that the racemic forms bind less tightly than either enantiomer.     

Interaction of human serum albumin with furosemide glucuronide: a role of albumin in isomerization, hydrolysis, reversible binding and irreversible binding of a 1-O-acyl glucuronide metabolite

Furosemide 1-O-acyl glucuronide (Fgnd) was reversibly bound to a single class of binding sites on human serum albumin (HSA), and the binding of Fgnd decreased with increasing F concentrations, suggesting that Fgnd binds to the same warfarin binding sites on HSA as F binds. The rate of Fgnd degradation (hydrolysis and acyl migration) decreased in the presence of HSA. Although the formation of acyl migration isomers of Fgnd was slower in the presence of HSA than in its absence, hydrolysis of Fgnd to F was faster in the presence of HSA. Rapid minor irreversible binding of Fgnd to HSA within 30 min was followed by slow major irreversible binding. Slow irreversible binding of Fgnd to HSA was decreased by F, though not significantly. This suggests that major irreversible binding may proceed via reversible binding. It has been reported that acyl migration is a prerequisite for irreversible binding. Therefore, these results indicate that HSA decreases irreversible binding of Fgnd to protein by suppressing acyl migration. Furthermore, these results suggest that HSA may prevent irreversible binding of Fgnd to other proteins in the body by decreasing the concentration of reactive Fgnd in the unbound form. HSA eliminates reactive Fgnd by hydrolysis to F. Therefore, it is concluded that HSA works as a scavenger to decrease reactive compounds by reversible binding or eliminates reactive compounds by irreversible binding.     

The binding of physostigmine to human serum albumin

The binding of [3H]physostigmine to crystallized human serum albumin (HSA) has been investigated using equilibrium dialysis. The percentage bound to 1% (w/v) HSA decreased from 18 to 4% as the total concentration of physostigmine increased from 3.3 nM to 2.7 microM (0.9 to 750 ng mL-1). A single class of specific binding sites with a large affinity constant, K = 8 x 10(7) L mol-1, was identified. The concentration of binding sites was approximately 3 nM. The Michaelis constants for human serum cholinesterase and albumin were the same; an explanation for these results is that the drug is binding to a trace cholinesterase, in the albumin.     

Stereoselective binding of propranolol enantiomers to human alpha 1-acid glycoprotein and human plasma.

The binding of propranolol enantiomers to human albumin (ALB), alpha 1-acid glycoprotein (alpha 1-AGP) and plasma was studied. (-) propranolol is more bound than (+)propranolol to alpha 1-AGP (P less than 0.001) and to plasma (P less than 0.05). In solutions containing ALB at a constant concentration (580 mumol/l) and alpha 1-AGP at increasing concentrations, the binding of both isomers increases but the stereo selectivity is evident throughout the alpha 1-AGP concentration range examined (25-100 mumol/l).