Olof Borgå

Protein Binding of Diphenylhydantoin and Desmethylimipramine in Plasma from Patients with Poor Renal Function

Abstract The protein binding of diphenylhydantoin (DPH) and desmethylimipramine (DMI) was measured in plasma from azotemic and uremic patients by an Ultrafiltration technic. DPH binding was decreased in uremic plasma. The impairment of binding was strongly correlated (p less than 0.001) to the degree of azotemia and degree of physical disability of the patients but weakly correlated (p less than 0.05) to their concentrations of serum proteins. DMI binding was almost normal. Dialysis of normal and uremic plasma in tap water or hemodialysis solution did not alter the DPH binding. The binding of DPH by plasma proteins from azotemic patients appears to be decreased, probably owing to a change in the binding proteins. The possibility of alteration of drug binding by pathologic states should be considered when total plasma concentrations of protein-bound drugs are measured and the values used to establish or modify drug-dosage regimens.

Increased Plasma Protein Binding of Propranolol and Chlorpromazine Mediated by Disease-Induced Elevations of Plasma α1 Acid Glycoprotein

To assess the importance of disease-induced increases in plasma concentrations of alpha1 acid glycoprotein (an acute-phase plasma protein that binds cationic drugs), we determined binding of propranolol in plasma from 53 patients and 25 healthy volunteers. Binding was increased in 10 patients with Crohns disease (P less than 0.002), nine with inflammatory arthritis (P less than 0.002) and eight with chronic renal failure with superimposed inflammatory disease (P less than 0.01) as compared with healthy controls. The plasma binding of control subjects did not differ from that of 12 patients with chronic hepatic disease (P greater than 0.45) or 14 with uncomplicated renal failure (P greater than 0.80). Chlorpromazine binding, determined in 60 subjects, yielded similar results. Percentage of free drug and alpha1 acid glycoprotein concentration were inversely correlated (r = -0.77 with propranolol, P less than 0.001, and r = -0.69 with chlorpromazine, P less than 0.001). Increases in plasma protein binding in patients with inflammatory disease appear mediated by increases in alpha1 acid glycoprotein concentration, which may influence drug kinetics.

Plasma protein binding of basic drugs

The protein binding of two basic drugs, alprenolol and imipramine, and the acidic drug, naproxen, was determined in plasma obtained from 23 healthy subjects. A 2jold variation was found between individuals in the free fraction of the two bases, while the range was even greater with naproxen. The free fraction correlated with the plasma concentration of ai‐acid glycoprotein for alprenolol (r = ‐0.75, p < 0.001) and imipramine (r = ‐0.78, p < 0.001), while there was no correlation for naproxen (r = 0.24, p > 0.1). This confirms recent experiments which showed that isolated ai‐acid glycoprotein avidly bound both alprenolol and imipramine. Drug binding, however, did not correlate with albumin concentration, although experiments with isolated albumin demonstrated its unusually high affinity for naproxen.

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

Abstract 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.

Plasma protein binding of tricyclic anti-depressants in man

Abstract The binding of various tricyclic antidepressants to human plasma and the effects thereupon of other drugs were studied by an ultrafiltration technique utilizing labeled compounds. At a total concentration of 0·29 μ/ml the percentage of unbound desmethylimipramine(DMI) was found to be 9·5 ± 1·4 in 41 individuals. The unbound fraction of DMI in plasma increased only twofold when the total concentration of the drug was increased a thousand times. The degree of binding over the entire range of therapeutic plasma drug concentration was relatively constant. The binding of various tricyclic antidepressants was compared at a drug concentration of 1·1 μM. The percentage of unbound drug was for nortriptyline (NT) 5·5 ± 0·6, for amitriptyline (AT) 3·6 ± 0·8, for imipramine (I) 4·2 ± 0·8, for protriptyline (PT) 8·0 ± 0·6 (tested at a cone, of 7·7 μM) and for Leo 640, an imipramine analogue, 0·7 ± 0·7. The acetyl derivatives of DMI, NT and PT were much more bound than the parent compounds. The addition of NT, PT or AT in a “therapeutic” concentration of 0·2 μg/ml didnot displace DMI, nor did chlorpromazine in supratherapeutic concentration. Diphenylhydantoin was found to displace DMI, NT, PT, AT and particularly I.

Protein binding of salicylate in uremic and normal plasma.

Protein binding of salicylate was studied by equilibrium dialysis at 37° C in plasma from uremic patients and healthy subjects. The protein binding was considerably lower in the uremic plasma at all salicylate concentrations studied (14 to 1,400 μg/ml). Scatchard plots of the data were computer‐analyzed assuming binding to two classes of binding sites. According to this analysis, the binding to the class of primary binding sites was considerably decreased in the uremic plasma. In addition to the effect of the uremic state, the binding was considerably decreased at high therapeutic plasma levels and at low albumin levels. The combined effect of two or three of these factors may lead to unexpectedly high unbound fractions of salicylate, which should be considered in the monitoring of plasma salicylate levels in patients.

Plasma disappearance of transplacentally transferred diphenylhydantoin in the newborn studied by mass fragmentography

With the intention of studying the fate of transplacentally transferred diphenylhydantoin (DPH, phenytoin) in the newborn infant, a mass fragmentographic analytic method has been developed, in which deuterium‐labeled DPH is used as internal standard. The analytic procedure permits precise determinations of DPH in 100 µl plasma samples in concentrations down to 0.01 µg per milliliter. Seven newborn infants of epileptic mothers treated with DPH throughout the pregnancy were studied during the first 4 to 9 days of life. The rate of plasma disappearance of DPH in the newborn infants was of the same order of magnitude as previously reported in adults. The initial parts of the plasma concentration curves indicated zero‐order disappearance of DPH. Nursing did not seem to affect the rapid plasma level decline in the babies until extremely low concentrations (0.1 p.g per milliliter) were reached. The arr,wunts of unchanged drug excreted in the urine were inSignificant. It is therefore concluded that babies born by mothers treated chronically with DPH are able to metabolize this drug effectively immediately after birth.

Impaired plasma protein binding of phenytoin in uremia and displacement effect of salicylic acid

The plasma protein binding of phenytoin (DPH) was studied by equilibrium dialysis at 37° C in plasma from uremic patients and healthy subjects. Scatchard plot analyses demonstrated a decreased association constant Kafor the DPH‐albumin interaction in the uremic plasma (mean 1.76 . 103 M−1 ± SD 0.12 and a mean 4.10 103 M−1 ± SD 0.24 in normal plasma). Studies on separated fractions of serum did not indicate any significant binding of DPH to proteins other than albumin. The nonlinear mathematical relationship between bound DPH and serum albumin could be linearized at low drug concentrations by plotting the ratio of bound/unbound DPH against albumin concentration. The displacement effect of salicylic acid at a concentration of 276 μg/ml (2mM) on DPH was considerable in plasma from normal subjects. In uremic plasma the effect was of a much smaller magnitude.

The role of plasma protein binding in the inhibitory effect of nortriptyline on the neuronal uptake of norepinephrine

The inhibitory effect of nortriptyline (NT) on the neuronal uptake of radio labeled norepinephrine (NE) was studied with the use of the rat iris preparation in Krebs‐Ringer solution or human plasma. When added to the incubation medium, NT inhibited the uptake of NE approximately ten times as effectively in buffer as in human plasma within the tested concentration range of NT (10−8 to 10−6M). This result is in good agreement with the 94 per cent binding (at NT concentration of 1.1 10−6M) obtained by the ultrafiltration method. Plasma from patients treated with NT also inhibited the uptake of NE. Correlation between the inhibitory effect and the “endogenous” plasma level of NT in the 14 patients studied was significant (p < 0.001). When NT was added in different concentrations to control plasma the inhibitory effect observed was close to that obtained with patient plasma containing the same “endogenous” concentration of NT.

Plasma levels and renal excretion of phenytoin and its metabolites in patients with renal failure

Phenytoin (DPH) and its two major metabolites, conjugated and unconjugated 5‐(4‐hydroxyphenyl)‐5‐phenylhydantoin (4‐OH‐DPH), have been studied in plasma and urine in 4 healthy subjects and 3 uremic patients during two weeks on DPH, 0.1 gm daily. Only 0.4% to 1.2% of the dose was excreted as unchanged DPH. The DPH concentrations in urine were in the same range as calculated unbound levels of DPH in plasma in the normal subjects; 1% to 2% of the dose was excreted as unconjugated 4‐OH‐DPH in the normal subjects. In the uremic patients, renal clearance of this metabolite was reduced to one‐sixth that percentage. Plasma concentrations rose to values twice as high as normal, indicating increased rate of glucuronidation. Urinary recovery of conjugated 4‐OH‐DPH in healthy subjects was 52% to 94%. Its renal clearance was close to glomerular filtration rate when corrected for protein binding, suggesting elimination by glomerular filtration only. Plasma concentrations of conjugated 4‐OH‐DPH reached plateau levels around day 4 in normal subjects. In the uremic patients, plasma concentrations of this metabolite accumulated to levels 10 times normal, and after 15 days of medication plateau levels did not seem to have been reached.