High-throughput solution-based medicinal library screening against human serum albumin.

High-throughput screening of combinatorial libraries has evolved from studying large diverse libraries to analyzing small, structurally similar, focused libraries. This paradigm shift has generated a need for rapid screening technologies to screen both diverse and focused libraries in a simple, efficient, and inexpensive manner. We have proactively addressed these needs by developing a high-throughput, solution-based method combining size exclusion (SEC), two-dimensional liquid chromatography (2-D LC), and mass spectrometry (MS) for determining the relative binding of drug candidates in small, focused medicinal libraries against human serum albumin (HSA). Two types of libraries were used to evaluate the performance of the system. The first consisted of five diverse ligands with a wide range of hydrophobicities and whose association constants to HSA cover 3 orders of magnitude. A beta-lactam library composed of structurally similar compounds was used to further confirm the validity of the methodology. The ability to distinguish site-specific interactions of drugs competing for individual domains of the HSA receptor is also demonstrated. Comparison of chromatographic profiles of the library components before and after incubation with the receptor using multiple reaction monitoring allowed a ranking of the ligands according to their relative binding affinities. The observed rankings correlate closely with literature values of the association constants between the respective ligands and HSA. This simple, rugged methodology can screen a wide spectrum of chemical entities from combinatorial mixtures in less than 6 min.

Inhibition of N-acetylation of procainamide and renal clearance of N-acetylprocainamide by para-aminobenzoic acid in humans.

Procainamide administration often results in excessively high serum N-acetylprocainamide (NAPA) concentrations and subtherapeutic serum procainamide concentrations. Inhibition of N-acetylation of procainamide may prevent accumulation of excessive NAPA while maintaining therapeutic serum procainamide concentrations. The purpose of this randomized, two-way crossover study was to determine if para-aminobenzoic acid (PABA) inhibits N-acetylation of procainamide in healthy volunteers. Eleven (7 female, 4 male) fast acetylators of caffeine received, in random order, PABA 1.5 g orally every 6 hours for 5 days, with a single intravenous dose of procainamide 750 mg administered over 30 minutes on the third day, or intravenous procainamide alone. Blood samples were collected during a 48-hour period after initiation of the infusion. Urine was collected over a 72-hour period. Serum procainamide and NAPA concentrations were analyzed using fluorescence polarization immunoassay. Urine procainamide and NAPA concentrations were measured with high performance liquid chromatography. PABA did not significantly influence total or renal procainamide clearance, elimination rate constant, AUC0-00, amount of procainamide excreted unchanged in the urine, or volume of distribution. However, concomitant PABA administration with procainamide resulted in increases in NAPA AUC0-00 and t1/2 and reductions in NAPA Ke, procainamide acetylation (NAPA formation) clearance, and NAPA renal clearance. Although PABA inhibits metabolic conversion of procainamide to NAPA, it also impairs the renal clearance of NAPA (but not procainamide) in healthy subjects. Therefore, PABA may not be useful for optimizing the safety of efficacy of procainamide in patients.

Effect of H2-receptor antagonists on rat liver cytosolic acetyl CoA:arylamine N-acetyltransferase activity.

This investigation examined the effect of cimetidine, famotidine, and ranitidine on rat liver acetyl CoA:arylamine N-acetyltransferase (NAT) activity. Studies were conducted using procainamide and p-aminobenzoic acid as substrate probes for NAT isozymes II and I, respectively. At an inhibitor:substrate ratio of 2:1, ranitidine, cimetidine, and famotidine reduced NAT II activity by 9, 48, and 75%, respectively. At this same ratio, none of the H2-receptor antagonists significantly reduced NAT I activity. The inhibition of NAT II activity by cimetidine and famotidine was mixed in nature, with characteristics consistent with predominantly competitive inhibitors. Preincubation of NAT with acetyl CoA did not attenuate the inhibitory effects of famotidine, suggesting this agent does not associate with the sulfhydryl of the critical cysteine residue on NAT. These results indicate the ability of H2-receptor antagonists to inhibit NAT activity with some degree of specificity for the two isozymes and significant differences in inhibitory potency between the antagonists.

Function of rat hepatocyte tight junctions: studies with bile acid infusions.

To determine the effects of alteration of biliary paracellular permeability on bile flow and composition, we measured the biliary outputs of compounds highly concentrated in bile, all infused at a constant rate in the isolated rat liver perfused with Krebs-Henseleit buffer in a one-pass fashion. Paracellular permeability was increased by infusing 10(-8) M vasopressin (VP). The cholephilic compounds were three cations of various molecular weights, tributylmethylammonium (TBuMA), N-acetylprocainamide ethobromide (APAEB), and propidium iodide, and two anions, taurocholate (TC), a micelle-forming bile acid, and taurodehydrocholate (TDHC), an nonmicelle former. When TC was infused and paracellular permeability increased with VP, neither bile flow nor TC output changed, whereas outputs of cations fell. When TDHC was infused, TDHC output fell, as did outputs of all cations. The decrements in cation outputs exceeded that of TDHC and were inversely related to the molecular weight of the cation. To document that these changes were not related to reduced uptake of these compounds, we tested the uptakes of TBuMA, APAEB, and TDHC into isolated hepatocytes. In no case did 10(-8) M VP significantly reduce uptake. The data demonstrate that micelle-forming bile acids, with their high effective molecular weights, do not efflux from the biliary tree when permeability is increased with VP, whereas nonmicelle-forming bile acids do. Cations efflux more readily than anions, and within this group efflux rate is inversely related to molecular weight. The data confirm the size and charge selectivity of biliary tree permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

[Simultaneous predictions of disposition kinetics of procainamide and its metabolite N-acetylprocainamide in rat by a physiological pharmacokinetic model].

Disposition kinetics of procainamide (PA) and its metabolite N-acetylprocainamide (NAPA) in rats was simulataneously predicted by a physiological pharmacokinetic model. The parameters, such as clearances in kidney and liver and tissue/blood concentration ratios, which were needed for simulations, were determined. The estimated clearances of PA in rat blood, kidney and liver were 47. 28, 13. 56 and and 33. 71 ml.kg-1.min-1, respectively. Tissue/blood drug concentration ratios were obtained after iv administration according to Gallo's method and demonstrated that heart, liver, kidney, muscle and small intestine have greater affinity for PA than do blood components. The concentrations of PA and NAPA in rat tissues following iv administration of PA.HCl 75 mg/kg were predicted and compared with observed values. The results showed that a good agreement between predictions and observed data was found in most of rat tissues. Concentrations of PA and NAPA in plasma of man, based on scaling-up of kinetics of PA and NAPA from rat to man, was also simulated.

Effect of the immunomodulator tilorone on the in vivo acetylation of procainamide in the rat.

Interferon and interferon inducers have been found to inhibit cytochrome P-450-dependent metabolism in animals and man. The effect of these agents on the acetylation of drugs has not been previously reported. Since these agents stimulate the reticuloendothelial system, together with the abundance of N-acetyltransferase in the reticuloendothelial system, it was hypothesized that these immunomodulators may affect drug acetylation. To test this hypothesis, the effect of tilorone (a synthetic interferon inducer) on the in vivo acetylation of procainamide was examined in the rat. Pretreatment with tilorone hydrochloride (50 mg/kg) 48 hr prior to the administration of procainamide hydrochloride (50 mg/kg) resulted in a 32% increase in the urinary recovery of N-acetylprocainamide and a 35% increase in the metabolic clearance of procainamide to N-acetylprocainamide. These data indicate that interferon inducers increase the N-acetylation of drugs in vivo.

Procainamide N-acetyltransferase: modulation by clofibrate and a microsomal form.

1. Detoxification of procainamide by N-acetyltransferase which occurs primarily in the cytoplasmic fraction of the rat liver can be modulated by clofibrate treatment. 2. When the concentration of one of the substrates, procainamide, is 100 microM while the other, acetyl CoA, is 10 or 100 microM, the specific activity is reduced following clofibrate treatment. However, total activity is unchanged because of a 44% increase in cytoplasmic protein. 3. At more physiological levels of the two substrates (10 microM), total enzyme activity is increased from 9.5 +/- 1.5 to 14.0 +/- 1.8 pmol/mg/min, P less than 0.05. 4. A microsomal form of N-acetyltransferase activity is reported which is unaffected by the concentration of acetyl CoA in contrast to the cytoplasmic form.

Metabolite formation pharmacokinetics: rate and extent of metabolite formation determined by deconvolution.

A two-step analytic procedure to determine the rate and extent of metabolite production following administration of the parent compound is described. The procedure provides the rate and extent of metabolite production as a function of time by application of the general model independent approach of deconvolution. The metabolite unit impulse response function is obtained by implicit deconvolution of the metabolite data with a truncated constant-rate metabolite input function. Then the obtained unit impulse response function is used in an analytic deconvolution with metabolite data following administration of the parent compound to obtain the rate and extent of metabolite production. The input function is also deconvolved with metabolite data to obtain the unit impulse response function appropriate for prediction of metabolite levels given a selected input of parent compound. The expected profile following administration of the consecutive infusions of parent drug is shown for both parent and metabolite. The rationale for selection of deconvolution methods is discussed. The approach is applied to data for procainamide and N-acetylprocainamide from three human subjects. The results indicate that from 27 to 39% of the procainamide was converted to N-acetylprocainamide in these subjects.

Effect kinetics of N-acetylprocainamide-induced QT interval prolongation.

We attempted to correlate clinical response with the effects of N-acetylprocainamide (NAPA) on the QT interval in five patients with stable chronic ventricular arrhythmias. A 15 mg/kg dose of NAPA was administered and a pharmacokinetic-pharmacodynamic model was used to relate plasma NAPA concentrations to changes in corrected QT interval (QTc). NAPA volume of distribution, elimination clearance, and elimination half-life averaged 1.37 +/- 0.19 L/kg, 174 +/- 63 ml/min, and 8.2 +/- 1.4 hours, respectively (mean +/- SD), and NAPA renal clearance averaged 1.9 +/- 0.6 times creatinine clearance. QTc prolongation was characterized by a linear-effect model in the first four patients and averaged 2.4 msec for every microgram per milliliter NAPA in a hypothetic biophase. QTc prolongation in patient 5 was exaggerated and was analyzed with an Emax model. Nonetheless, NAPA did not control this patient's arrhythmia. Conversely, patient 1 subsequently developed torsade de pointes even though QTc prolongation in this patient was comparable to that in patients 2 through 4, who responded satisfactorily to NAPA. We conclude that QT interval changes during initial NAPA administration do not reliably predict subsequent clinical response.

Food-induced gastric retention and absorption of sustained-release procainamide.

The relationship between variations in the gastric residence time and the absorption of procainamide from a waxed matrix, sustained-release tablet was evaluated in a repeated-measures study conducted in eight healthy men. Subjects received sustained-release procainamide together with a Heidelberg capsule, alone and with food. Blood and urine samples were collected for up to 24 hours before and after gastric emptying of the Heidelberg capsule for procainamide and N-acetylprocainamide concentration determinations. The gastric residence time of the Heidelberg capsule was prolonged by food (median 3.5 [range 1.5 to 10.0] vs. 1.0 [range 0.5 to 2.5] hours; P less than 0.02). No significant differences (median [range]; fasting vs. fed) in procainamide lag time (0.5 [0.5 to 1.0] vs. 0.5 [0.5 to 1.5] hours) or time at which peak procainamide plasma concentrations occurred (2.9 [1.0 to 4.3] vs. 2.8 [2.0 to 6.0] hours) were evident with feeding. Slight increases in procainamide AUC and peak concentrations occurred with feeding. No alteration in the extent of urinary excretion of procainamide or N-acetylprocainamide occurred with feeding. Thus food did not influence the absorption of sustained-release procainamide despite apparent prolonged gastric retention.

Pharmacokinetics of N-acetylprocainamide.

Shortly after Dreyfus and his colleagues demonstrated that procainamide was metabolized by acetylation to N-acetylprocainamide (NAPA), Drayer, Reidenberg and Sevy reported that NAPA had antiarrhythmic activity in an animal model. We confirmed these findings and found that plasma levels of NAPA were high enough to warrant consideration in managing patients requiring procainamide therapy. However, the actual impetus for developing NAPA as an antiarrhythmic drug in its own right was provided by the initial studies of NAPA pharmacokinetics in normal subjects. In these studies, we showed that NAPA has an elimination-phase half-life that is more than twice as long as procainamide and suggested that patient compliance and arrhythmia suppression might be improved if NAPA were used to circumvent the inconvenience of the frequent dosing schedule that has been recommended for procainamide. From the standpoint of managing individual patients with NAPA, the pharmacokinetics of this drug continue to provide the scientific basis for designing dose regimens that will have maximal antiarrhythmic efficacy and minimal toxicity. This review summarizes the salient features of NAPA pharmacokinetics and outlines an approach for individualizing therapy with this drug.

Pharmacokinetics of procainamide hydrochloride in dogs.

Pharmacokinetics of procainamide hydrochloride were studied in 2 groups of dogs. In a group of 6 dogs, procainamide was administered IV at a small dose of 8 mg/kg (group 1), and blood samples were obtained for 3.5 hours. In another group of 6 dogs, procainamide was administered IV and orally at an average dose of 25.5 mg/kg (group 2) in a crossover manner. Blood samples were obtained for 48 hours. In 2 dogs (previously used in part II), N-acetylprocainamide (NAPA) was administered IV at a dose of 10 mg/kg. Plasma samples were assayed for procainamide by fluorescence polarization immunoassay, and NAPA samples were assayed by high-performance liquid chromatography. In group 1, the elimination of procainamide was described by a 1-compartment, open pharmacokinetic model. The elimination half-life was 2.43 hours, the apparent volume of distribution was 1.44 L/kg, and the systemic clearance was 0.412 L/kg/hr. In group 2, 2 of the 6 dogs were described by a 1-compartment model, and 4 of the 6 dogs were described with a 2-compartment pharmacokinetic model. The elimination half-life for the IV dosage was 2.85 hours, the apparent volume of distribution was 2.13 L/kg, and the systemic clearance was 0.519 L/kg/hr. For the orally administered dose, the bioavailability was 85%, and the absorption half-life was 0.5 hours. There was no evidence of acetylation of procainamide to NAPA or deacetylation of NAPA to procainamide. The estimated elimination half-life of NAPA was 4.7 hours.

Effect of acute renal failure on the disposition and elimination of [3H]N-acetyl procainamide ethobromide in the rat.

The effect of glycerol-induced acute renal failure (ARF) on the disposition and elimination of the organic cation [3H]N-acetyl procainamide ethobromide (APAEB) was investigated in the rat. In rats with ARF the plasma clearance, rate constant for the terminal portion of the plasma concentration-time curve and apparent volume of distribution were all decreased (P less than 0.01). Furthermore, the renal clearance of APAEB and the percentage dose excreted in urine were reduced by 85 and 74%, respectively. Decreased renal excretion probably accounted for the altered kinetics of APAEB in ARF because ligation of the renal pedicles of control rats produced changes in the kinetics of APAEB that were similar to those seen in animals with ARF. No change in either the hepatic content of APAEB or its biliary excretion were detected in rats with ARF. Similarly, the hepatic handling of ouabain and taurocholic acid was previously found to be unaltered in ARF; but by contrast, the hepatic uptake and initial biliary excretion of bromosulphophthalein and indocyanine green were decreased (Bowmer & Yates 1984, Br. J. Pharmacol. 83: 773-782). Together these studies indicate that there is a selective impairment of hepato-biliary transport in ARF.

Cardiovascular actions and pharmacokinetics of desethyl-N-acetylprocainamide in the dog.

The pharmacokinetic profile and the cardiovascular actions of desethyl-N-acetylprocainamide (NAPADE) were studied in chloralose-urethane anesthetized dogs. NAPADE was given as a 15-min i.v. infusion in doses of 12 and 60 mg/kg. In all cases, the plasma concentration vs. time curve could be resolved into two exponential components, and the distribution and elimination of NAPADE were analyzed with a two-compartmental model. Total apparent volume of distribution was 0.4153 +/- 0.0301 liters/kg (mean +/- S.E.M.) for the 12-mg/kg group and 0.4946 +/- 0.0691 liters/kg for the 60-mg/kg group (P greater than .05). Elimination clearance was 0.0061 +/- 0.0006 liters/min/kg for the 12-mg/kg group and 0.0086 +/- 0.0013 liters/min/kg for the 60-mg/kg group (P greater than .05). The elimination phase half-life (T1/2 beta) was 57.0 +/- 4.1 and 53.1 +/- 3.2 min for the 12- and 60-mg/kg groups, respectively (P greater than .05). Thus, NAPADE exhibited first-order kinetics of distribution and elimination in the dose range studied. Renal clearance of unchanged NAPADE amounted to 45.9 +/- 4% of total plasma clearance. NAPADE had a dose- and concentration-related positive inotropic effect, as measured with a Walton-Brodie gauge sutured to the right ventricle. A 12-mg/kg infusion caused a peak increased in myocardial force of 18.4 +/- 3.6% over base line, whereas a 60-mg/kg infusion caused a peak increase in myocardial force of 48.9 +/- 10% over base line. The positive inotropic effect of NAPADE was sustained for 50 to 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Lethal accumulation of procainamide metabolite in severe renal insufficiency.

Four patients, 64-80 years of age, with severe renal dysfunction and heart disease received conventional doses of procainamide as treatment for cardiac arrhythmias. Serum procainamide concentrations at these times ranged from 6.2 to 13.3 micrograms/ml and were within the recently expanded therapeutic range for resistant ventricular arrhythmias. All 4 patients demonstrated marked and delayed accumulation of the active metabolite N-acetylprocainamide, with highest observed serum concentrations ranging from 42.0 to 59.4 micrograms/ml. Cardiotoxicity associated with these levels included progressive widening of the QRS and corrected Q-T intervals, induction of polymorphic non-sustained ventricular tachycardia (torsades de pointes), and severe depression of left ventricular function which appeared to be important factors in the deaths of these patients. The use of lower procainamide doses and careful anticipatory monitoring of serum concentrations of procainamide and N-acetylprocainamide are essential in this high-risk group.