Factors affecting quinidine protein binding in humans

The free (unbound) concentration of drug in plasma is often an important determinant of pharmacological and toxicological effects. Unfortunately, studies examining the factors influencing the free fraction of quinidine in plasma have yielded inconsistent results. It is probable that differences in the type of blood collection tubes utilized and the analytical procedure employed biased some of these estimates of quinidine binding. The present study was executed in a manner free of factors now known to introduce artifacts into estimates of the free fraction of quinidine. In healthy volunteers, the free fraction of quinidine (1.0 microgram/mL) was 0.129 +/- 0.019 (mean +/- SD) and was constant throughout the therapeutic range. A high-affinity, low-capacity binding site (K = 1.17 X 10(5) M-1; nP = 3.49 X 10(-5) M) and a low-affinity, high-capacity binding site (K = 1.33 X 10(3) M-1; nP = 3.14 X 10(-3) M) were identified. The characteristics of quinidine binding in a 4.5-g/dL solution of human serum albumin (K = 3.05 X 10(3) M-1; nP = 1.36 X 10(-3) M) suggested that the low-affinity, high-capacity binding site was on this quinidine free fraction increased from 0.114 to 0.231. A lidocaine concentration of 250 micrograms/mL caused a similar increase. Patients suffering traumatic injury had a significant increase in alpha 1-acid glycoprotein concentration (197 mg/dL) and a decreased quinidine free fraction (0.075 +/- 0.019). Patients with hyperlipidemia had free fractions similar to those observed in healthy individuals (0.118 +/- 0.019).(ABSTRACT TRUNCATED AT 250 WORDS)     

Theophylline protein binding in humans.

Theophylline protein binding was 58-82% in serum from six normal adults and 42 asthmatic patients, 1-25 years old, who were given 5 mg of theophylline/kg. The binding range was greatest in young patients, but the proportion of protein-bound drug did not correlate with age. Theophylline protein binding was higher than previously reported. The effect of binding should be considered in patients who do not have optimal bronchodilation from theophylline despite total serum theophylline concentrations of 10-20 microgram/ml.     

Factors affecting ceftriaxone plasma protein binding during open heart surgery

Factors most likely contributing to reduced ceftriaxone plasma protein binding in patients undergoing open heart surgery (OHS) were examined. Binding was determined by equilibrium dialysis. It was found that ceftriaxone does not bind significantly to red blood cells, alpha 1-acid glycoprotein, or to protamine, and that the pH of serum did not significantly affect binding. Albumin is the major protein to bind ceftriaxone, and binding decreases with lower albumin concentrations due to fewer binding sites. The binding of ceftriaxone was not affected by the in vitro addition of heparin or methylprednisolone, but high concentrations of methylprednisolone hemisuccinate increased the free fraction of ceftriaxone. Increased concentrations of free fatty acids (FFA) were demonstrated in several patients undergoing OHS. The in vitro addition of palmitic, stearic, linoleic, and oleic acids in high concentrations decreased the binding of ceftriaxone. Ceftriaxone binding in patient samples correlated with the molar ratio of FFA to albumin, but not to either individually. The dual effect of increased FFA and decreased albumin concentrations in OHS patients appears responsible for most of the observed binding alterations.     

Factors affecting drug binding in plasma of elderly patients.

The binding of salicylate, sulphadiazine and phenylbutazone to plasma proteins has been studied in young and elderly subjects. Elderly patients had significantly reduced concentrations of plasma albumin, compared with subjects under 40 years of age. Sifnificant increases in free levels of all three drugs were found in elderly patients receiving multiple drug therapy, and a correlation obtained with the number of drugs being taken. It is suggested that because of their low albumin levels, elderly patients may be more susceptible to the effects of multiple drug therapy on drug binding. The clinical implications of these observations are discussed.     

Factors affecting the tissue binding of nicotine in various species.

The binding of [¹⁴C]nicotine to various tissue fractions has been studied in the cat, pigeon and rat using the technique of equilibrium dialysis. Liver homogenates from all three species bound the drug to varying extents though the distribution of radioactivity showed species differences. Cat and rat liver binding appeared to reside predominantly in the microsomal fraction whereas in the pigeon the activity was essentially present in the cytosol. Lung tissue from all three species showed only a small binding capability which was not associated with the microsomes or soluble cell components. Brain homogenates in cat and rat showed a similar small degree of binding but there was a significant degree of binding in pigeon brain homogenate. Cat and pigeon kidney homogenates showed a small degree of tissue binding which appeared to be uniformly distributed in the cell fractions. Rat kidney, however, exhibited an extremely large in vitro binding capability which was apparently associated with the cytosol. The percentage binding was increased with increasing buffer pH over range pH 6–8 corresponding to an increase in the proportion of free base. A Scatchard plot over the concentration range 62–1540 nM gave a linear response, calculations from which indicated a low affinity, high capacity, binding capability for nicotine. In vivo distribution studies in the rat after subcutaneous administration of 0.4 mg/kg [¹⁴C]nicotine revealed a high degree of localisation of radioactivity in the kidney relative to other tissues and subsequent tissue fractionation confirmed the in vitro observations. Plasma from all three species showed no significant binding properties. The nature of the binding entity is not yet known though it can be concentrated by chromatography on Sephadex G-100 and is associated with a fraction of relatively low molecular weight containing little, if any, lipid.     

Factors affecting the measurement of lidocaine protein binding by equilibrium dialysis in human serum

A systematic study was undertaken to assess in vitro factors that influence the value of the lidocaine free fraction obtained by equilibrium dialysis in human serum. These factors include pH readjustment to 7.40 after serum storage; choice of buffers for dialysis; the effect of phosphate buffer ionic strength; temperature of storage for serum samples; the use of untreated versus silanized glassware for storage; and age of serum. It was concluded that the pH of serum that contains lidocaine must be brought back to the original whole blood pH found in the patient before equilibrium dialysis because the protein binding of lidocaine is critically dependent on pH. It was also found that Krebs-Ringer bicarbonate buffer, when used with room air atmosphere in the dialysis cell, is not adequate to control pH even when serum pH is readjusted to the physiological pH of the patient. Isotonic phosphate buffer and 0.10 M phosphate buffer are effective for pH control and give identical values of lidocaine free fraction when the original serum sample is first pH-adjusted. If the pH of the serum is correct and the pH of the buffer remains constant, then freezing, the choice of container, or the age of serum are not important variables affecting the measurement of the lidocaine free fraction.     

Factors influencing the protein binding of vancomycin.

Various factors influencing the protein binding of vancomycin were examined using equilibrium dialysis method. Four per cent human serum albumin (HSA) and/or 0.08 per cent alpha-1-acid glycoprotein (AAG), dissolved in isotonic phosphate buffer, were dialyzed against isotonic phosphate buffer of pH 7.4 using Spectrapor 2 membrane. The protein binding of vancomycin to 0.08 per cent AAG was dependent on vancomycin concentrations; the values ranged from 21.1 per cent at the vancomycin concentration of 20 micrograms ml-1 to 5.30 per cent at 2400 micrograms ml-1. However, binding to 4 per cent HSA was relatively constant, 8.79 +/- 2.43 per cent over a vancomycin concentration range of 20-2400 micrograms ml-1. The values to 4 per cent HSA alone and 0.08 per cent AAG alone did not predict the greater binding of vancomycin in the presence of both proteins, especially at higher concentrations of vancomycin; the values to 4 per cent HSA with 0.08 per cent AAG were constant, 26.3 +/- 3.74 per cent, at the vancomycin concentration range of 20-2400 micrograms ml-1. This suggested an interaction between the proteins, which resulted in enhanced binding of vancomycin. The protein binding of vancomycin to 4 per cent HSA with 0.08 per cent AAG was not influenced by the different incubation temperatures (4 degrees, 22 degrees, and 37 degrees), quantities of heparin (up to 40 units ml-1) or AAG (up to 0.16 per cent), or buffers (isotonic phosphate buffer of pH 7.4, phosphate buffer of pH 7.4 and 0.9 per cent NaCl solution) at the vancomycin concentration of 80 micrograms ml-1. Vancomycin was found to be stable in human serum albumin or in isotonic phosphate buffer of pH 7.4.     

Plasma binding of benzodiazepines in humans.

Plasma binding of chlordiazepoxide, diazepam, loraxepam, and oxazepam was determined by equilibrium dialysis in 20 male, healthy volunteers, 25-86 years old. A wide range of binding was observed, with the free fraction varying twofold for lorazepam, fourfold for chlordiazepoxide and diazepam, and over 20-fold for oxazepam. Statistically significant linear relationships were not observed between the degree of binding and age, serum albumin, or total protein for any of the drugs. There was, however, a correlation between the extent of binding for the four drugs. Because of the importance of unbound benzodiazepine levels in eliciting any pharmacological response and also in disposition, consideration of the wide interindividual variability in plasma binding must be made in interpreting pharmacodynamic and pharmacokinetic data.     

Factors affecting the binding of tricyclic tranquillizers and antidepressants to human serum albumin.

The linear free energy-related model for structure activity relations developed by Hansch & Fujita (1964) has been used to correlate the binding of tricylic tranquillizers and antidepressants to human serum albumin (HSA) with hydrophobic and electronic parameters. The parameters chosen being the chromatographic parameter (Rm) and the affinity of charge transfer complex formation (kc). The relative importance of these factors has been assessed by linear and multiple linear regression analysis. Results show that the major factor in binding is electronic with only a minor contribution from the hydrophobic parameter.     

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.     

Enhancement of etoposide and methotrexate sensitivity by indomethacin in vitro.

The possibility of increasing the activity of etoposide (VP-16) by combining this anti-cancer agent with indomethacin (Indo) was investigated by treating murine and human cultured tumor cells with a combination of Indo and VP-16 and quantitating VP-16 cytotoxicity by the [3H]thymidine incorporation assay. Non-toxic concentrations of Indo were found to enhance the sensitivity to VP-16 in cultured Lewis lung carcinoma (LLC), YAC-1, P815, CCRF-CEM and K562 cells which were all relatively sensitive to VP-16. With the LLC, the Indo effect was dose dependent and near maximal at an Indo concentration of 0.5 micrograms/ml. Indo also increased the response of LLC cells to methotrexate, but not to bleomycin. Ibuprofen was less effective than Indo in enhancing VP-16 sensitivity in LLC cells. The enhanced sensitivity of VP-16 by Indo was not reversed by the prostaglandins PGE2 and PGD2, the analogs carbocyclic thromboxane A2 and carba-prostacyclin or conditioned medium removed after 24 h or 48 h of culture from near confluent LLC cell monolayers. This finding suggests that Indo is not augmenting VP-16 cytotoxicity by inhibiting cyclo-oxygenase activity and prostaglandin production. The lipoxygenase inhibitor, eicosatetraynoic acid (ETYA), was also ineffective in reversing the Indo augmentation of VP-16 sensitivity. This finding indicates that Indo is not acting by inhibiting cyclo-oxygenase and converting larger amounts of arachidonic acid to lipoxygenase products, such as leukotrienes, that could then interact with VP-16 to increase its sensitivity. In other studies, Indo was found to significantly increase the steady state accumulation of [3H]-VP-16 in all five cell lines studied. With the LLC cells, this increased steady state was achieved within 15 min after the addition of Indo to these cells and this enhanced VP-16 uptake was not reversed by the addition of prostaglandin E2 or prostaglandin D2. Thus, taken together, these studies indicate that Indo most likely enhances the cytotoxicity of VP-16 by increasing the cellular accumulation of VP-16. This newly identified function of Indo may be of potential clinical significance in the treatment of cancers in man.     

Factors affecting protein thiol reactivity and specificity in peroxide reduction

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.     

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases.

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.     

In vitro protein binding behavior of dipyridamole

The in vitro protein binding behavior of dipyridamole in plasma and buffered protein solutions was investigated by equilibrium dialysis. The drug was highly protein bound (approximately 98%) in heparinized human plasma, and the extent of protein binding remained constant for drug concentrations over the range of therapeutic interest of 0.1-10 micrograms/mL. Comparable binding results were obtained with a mixture of 80 mg % of alpha 1-acid glycoprotein and 40 g/L of human serum albumin in pH 7.4 phosphate buffer solution. Pure alpha 1-acid glycoprotein (80-400 mg %) or pure human serum albumin (40 g/L) in phosphate buffer gave significantly (p less than 0.05) lower binding results, indicating that both proteins are responsible for the high binding of dipyridamole in plasma. Addition of alpha 1-acid glycoprotein to heparinized human plasma, to simulate an acute phase increase in the protein, had no effect on the fraction of free drug in plasma. Binding of dipyridamole to heparinized human plasma or human serum albumin in buffer was concentration independent through 40 micrograms/mL. The free fraction of dipyridamole increases with concentrations exceeding 40 micrograms/mL.