Stereoselective differences in propranolol disposition in female Sprague-Dawley and Dark Agouti rats.

Although genetic constitution is recognized as an important factor in drug elimination, there is still only a modest amount of information about its impact on stereoselective drug disposition. To examine how strain differences in cytochrome P-450 expression may affect stereoselective drug elimination, the disposition of R- and S-propranolol was examined in female Sprague-Dawley (SD) and Dark Agouti (DA) rats. The half-life of R-propranolol was increased and its apparent oral clearance decreased significantly in DA rats relative to the SD strain [85 +/- 41 min (DA) vs. 26 +/- 10 min (SD) and 0.020 +/- 0.014 liter/min/kg (DA) vs. 0.31 +/- 0.14 liter/min/kg (SD)], respectively. Strain differences in S-propranolol half-life and apparent oral clearance also were observed, but were not as marked [97 +/- 36 min (DA) vs. 52 +/- 35 min (SD) and 0.20 +/- 0.13 liter/min/kg (DA) vs. 0.70 +/- 0.68 liter/min/kg (SD)], respectively. Due to a more pronounced effect of rat strain on the disposition of R-propranolol, the stereoselective S-/R- oral clearance ratio for propranolol was also strain-dependent [10 +/- 3 (DA) vs. 2.1 +/- 0.9 (SD)]. These differences could not be accounted for by propranolol stereoselective plasma protein binding or by a difference in the blood/plasma ratio for R- or S-propranolol in these separate strains. This represents one of the few examples where an increase in stereoselective drug disposition has been observed due to strain differences that diminish drug elimination.

Mechanisms of lung polyamine accumulation in chronic hypoxic pulmonary hypertension.

Chronic hypoxia causes polyamine-dependent hypertensive pulmonary vascular remodeling (J. E. Atkinson. J. W. Olson, R. J. Altierre, and M. N. Gillespie, J. Appl. Physiol. 62: 1562-1568, 1987), but mechanisms by which lung polyamine contents are elevated have not been established. This study measured polyamine contents, biosynthetic activities, and transport in lungs of rats exposed to hypobaric hypoxia (simulated altitude: 4,570 m) for 4-14 days. Hypoxia increased lung contents of spermidine and spermine within 40 h and of putrescine within 4 days. These changes preceded hypoxia-induced increases in pulmonary arterial pressure and development of right ventricular hypertrophy. Additional experiments determined whether increased lung polyamine contents could be ascribed to elevated activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in conversion of ornithine to putrescine. Lung ODC activity did not differ from controls at 40 h posthypoxia and was reduced below control levels from 4-14 days of exposure. Putrescine transport kinetics were assessed in isolated, salt solution-perfused lungs. Apparent Km for putrescine uptake was increased from 10.4 microM in control lungs to 16.9 microM in lungs from rats maintained for 7 days in an hypoxic environment. Maximal velocity (Vmax) of lung putrescine transport was increased from 1.67 nmol.g-1.min-1 in controls to 2.65 in hypoxic lungs. Putrescine efflux also was altered by hypoxic exposure; T1/2 for loss of diamine from a slowly effluxing pool was increased from 60.6 min in controls to 91.5 min in hypoxic lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization.

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(-)-propranolol from 100-microliters rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl-L-leucine anhydride to form diastereomeric propranolol-L-leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-microliters samples of whole rat blood (12.5-750 ng/ml; r = 0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(-)-propranolol. Terminal half-lives of R(+)- and S(-)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.

Assessment of potential aluminum chelators in an octanol/aqueous system and in the aluminum-loaded rabbit.

Aluminum (Al) solubilization from Al borate and its distribution in an octanol/aqueous system (Do/w) were determined in the absence and presence of 12 potential Al chelators. Citrate, N,N'-bis-(2-hydroxybenzyl)ethylenediamine- N,N'-diacetic acid (HBED), cyclohexane-1,2-diaminotetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), desferrioxamine, and ethylenediamine-N,N'-bis(2-dihydroxyphenylacetic acid) (EDDHA) were 55 to over 100% efficient in solubilizing equimolar amounts of Al. Tetracycline, EDTA, and 2,3-dihydroxybenzoic acid (DHBA) were less than 20% efficient. 1,4-Dioxane and fluoride were ineffective. The Do/w of Al averaged 0.005. The Do/w of the Al.chelator complex was generally less than that of Al, except for HBED and tetracycline (0.04 and 0.96, respectively). The Do/w of DHBA, desferrioxamine, EDDHA, and HBED were not influenced by Al, but tetracycline became more lipophilic. These compounds were tested for their ability to increase urinary Al excretion in Al-loaded rabbits. Chelators were given po weekly beginning 2 weeks after Al loading. Urine was obtained hourly from 3 hr prior to 6 hr after chelator administration and analyzed for Al. Fluoride and tetracycline (450 and 4500 mumol/kg) and citrate, NTA, EDTA, CDTA, DTPA, DHBA, HBED, and 1,4-dioxane (150 and 1500 mumol/kg) were ineffective. Following HBED administration, some of the Al-loaded rabbits died, presumably due to redistribution of Al within the rabbit. Following DTPA administration, some of the Al-loaded rabbits died, presumably due to DTPA. Oral EDDHA (1500 mumol/kg) significantly increased urinary Al excretion. EDDHA and desferrioxamine (150 mumol/kg) were administered by po, sc, and iv routes and were found to have comparable potency. The in vitro results may explain some of the in vivo findings. The in vitro methods may be useful to screen out compounds with no chelation potential. EDDHA-like compounds may have potential as alternatives to desferrioxamine in the prevention or treatment of Al accumulation and Al-induced toxicity.

Phenobarbital in the genetically obese Zucker rat. I. Pharmacokinetics after acute and chronic administration.

The pharmacokinetics of the enzyme inducer, phenobarbital, was evaluated in genetically obese and lean Zucker and lean Sprague-Dawley rats. The volume of distribution of phenobarbital in obese Zucker rats was larger (0.416 liter) than for lean Zucker (0.299 liter) or Sprague-Dawley rats (0.312 liter). Standardizing the volume of distribution for total body weight in the obese and lean Zucker and lean Sprague-Dawley rats resulted in similar volume terms. Intra- and inter-strain differences in phenobarbital clearance were observed between the obese and lean Zucker (11.9 and 14.1 ml/hr, respectively) and lean Sprague-Dawley rats (23.0 ml/hr). Greater differences in phenobarbital clearance were observed when clearance was corrected for body weight. Whether comparing the absolute or standardized pharmacokinetic data, lean and obese Zucker rats will exhibit 2- to 3-fold higher phenobarbital plasma concentrations after administration of a standard 75- to 100-mg/kg enzyme-inducing regimen relative to Sprague-Dawley rats. Pharmacokinetic parameters from the single dose study were used to calculate appropriate phenobarbital doses (21-58 mg/kg/12 hr) to achieve similar steady-state phenobarbital plasma concentrations after chronic oral administration in all three groups of rats. Steady-state phenobarbital clearance values were not significantly different from clearance values after single dose administration in each group of rats. The dramatic intra- and inter-strain alterations in phenobarbital disposition demonstrated in this study explain the high mortality reported in Zucker rats after administration of traditional enzyme induction doses of phenobarbital. Differences in phenobarbital disposition should be considered in enzyme induction studies.

Phenobarbital in the genetically obese Zucker rat. II. In vivo and in vitro assessments of microsomal enzyme induction.

In vivo and in vitro alterations in drug metabolism and the extent of enzyme induction of the hepatic microsomal cytochrome P-450 system were evaluated in obese and lean Zucker and lean Sprague-Dawley rats. Phenobarbital enzyme-inducing regimens were administered p.o. to achieve similar steady-state phenobarbital plasma concentrations. Control rats received p.o. placebo solution. No significant intra- or inter-strain differences in antipyrine clearance (milliliters per hour) or apparent volume of distribution (liters) were observed between the placebo-treated lean Sprague-Dawley, lean Zucker and obese Zucker rats. Intra- and inter-strain differences in hepatic microsomal protein and cytochrome P-450 content were observed. Compared to placebo, antipyrine clearance (milliliters per hour) after chronic phenobarbital pretreatment was increased in the Sprague-Dawley (198%) and lean Zucker rats (131%), but not significantly altered in the obese Zucker rats. Similarly, increases in hepatic weight, whole liver microsomal protein and cytochrome P-450 content were also observed in the Sprague-Dawley (34, 124 and 352%, respectively) and the lean Zucker rats (24, 96 and 249%, respectively). However, no significant alterations in these parameters were observed in the obese Zucker rats after phenobarbital treatment. Results from these in vivo and in vitro studies implicate alterations in drug metabolism and genetic differences in cytochrome P-450 content in Zucker rats relative to the Sprague-Dawley strain. Obese Zucker rats failed to exhibit a significant induction response after phenobarbital pretreatment.

Isolation of a cigarette smoke fraction responsible for the inhibition of benzo[a]pyrene metabolism in the isolated perfused rabbit lung.

Among the several thousand components of cigarette smoke is a substance or substances capable of inhibiting pulmonary metabolism of nicotine and altering the metabolite profile of procarcinogens such as benzo[a]pyrene (BP). This substance(s) inhibits BP metabolism in the lung in amounts present in a few puffs of cigarette smoke. By a series of extractions and chromatographic methods an active subfraction containing only 1% of the total cigarette smoke condensate (CSC), was isolated. This fraction demonstrated the same inhibition of BP metabolism in the isolate perfused lung (IPL) as the whole smoke. The inhibitor(s) present in this fraction possess amphoteric characteristics. The acidic function is believed to be a phenolic one.

Pulmonary disposition and pharmacodynamics of verapamil.

Verapamil, a calcium channel blocker, has received recent attention as a potential therapeutic agent in pulmonary hypertension. Accordingly, the pulmonary disposition and pharmacodynamics of verapamil were evaluated in isolated rat lungs perfused at a constant rate with a physiological salt solution. Isolated rat lungs sequestered but did not metabolize verapamil. The efflux of verapamil into drug-free perfusate occurred from two kinetically distinct pools with half-lives of 1.3 and 14.1 min. The theoretical amount of verapamil effluxed at infinite time was less than the amount taken up during the infusion, thereby suggesting that verapamil was also bound in a third "noneffluxable" pool. The time course for decline of verapamil inhibition of pulmonary vasoconstriction was compared with the rate of verapamil efflux. Inhibition of pulmonary vasoconstriction was related to the amount of verapamil in a pool exhibiting mono-exponential efflux of drug with a half-life of 12.6 min. This half-life suggests association of the inhibitory response with the efflux component having a half-life of 14.1 min. These findings indicate that the verapamil persists in the lungs in the form of a noneffluxable pool and that verapamil in the pool with half-life of 14.1 min is responsible for some of the vasoactivity in the pulmonary circulation.

Metabolism of isosorbide dinitrate in the isolated perfused rabbit lung.

The uptake and metabolism of isosorbide dinitrate was investigated in the recirculating isolated perfused rabbit lung and in lung homogenate 9000 X g supernatant. Concentration versus time profiles from the isolated lung experiments indicate rapid metabolism of isosorbide dinitrate and corresponding increases in the metabolites 5-isosorbide mononitrate, 2-isosorbide mononitrate, and isosorbide. The data suggest that the mononitrates formed in the lung tissue were converted to isosorbide at an extraordinarily high rate. Surprisingly, the rate of appearance of completely denitrated isosorbide was greater when isosorbide dinitrate was administered to the lung than when the mononitrate metabolites of isosorbide dinitrate were administered. The results suggest rapid metabolism of a substantial portion of the mononitrates formed endogenously from isosorbide dinitrate before partitioning of mononitrates into the perfusion medium could occur. The metabolism of isosorbide dinitrate in lung homogenate 9000 X g supernatant exhibited a metabolic scheme kinetically different from the intact lung studies, as isosorbide was formed slowly from a mononitrate intermediate and not by a near-simultaneous cleavage of both nitrate ester groups. Intravascular multiple-dose studies did not demonstrate any inhibition between isosorbide dinitrate and the mononitrates.

Influence of chronic cigarette smoke exposure on the uptake and efflux of imipramine in the isolated perfused rat lung.

The pulmonary uptake and efflux of imipramine was determined in lungs removed from cigarette smoke-exposed and nonexposed rats. Using an isolated perfused lung preparation, the lung was perfused for 220 sec with medium containing 2.5 X 10(-7) M imipramine, followed by a 28-min drug-free perfusion. There was no significant difference (p less than 0.05) between either the rate or the amount of imipramine accumulated in the smoke-exposed and nonexposed animals. During the drug-free perfusion, the previously accumulated imipramine was released from two distinct pools (E1 and E2). Calculation of the total amount of effluxable imipramine indicated that in the nonexposed animals approximately 30% of the amount taken up did not efflux at a measurable rate but formed a "noneffluxable" pool. In the smoke-exposed animals, however, all of the accumulated imipramine appeared to be effluxable. These data demonstrate that there may be components of cigarette smoke which are sequestered by lung tissue at the binding sites associated with the noneffluxable pool of imipramine. The presence in cigarette smoke of components possessing such high pulmonary affinity may be a factor in cigarette smoke-mediated lung damage; this possibility is discussed.

Isolated perfused rabbit lung as a model for intravascular and intrabronchial administration of bronchodilator drugs I:Isoproterenol.

The absorption, uptake, and metabolism of isoproterenol was studied following intravascular, intrabronchial, and aerosol administration of the drug to the isolated perfused rabbit lung. Capacity-limited metabolism of isoproterenol was observed following the addition of five doses, ranging from 10(-7) to 10(-5) moles, directly into the circulation of the lung system. A physiologically based perfusion model was developed to describe the disposition of the drug and metabolite in the isolated lung preparation. This model was also used to analyze data collected following intrabronchial and aerosol administration of isoproterenol.

Isolated perfused rabbit lung as a model for intravascular and intrabronchial administration of bronchodilator drugs II:Isoproterenol prodrugs.

The pulmonary disposition of two diester prodrugs of isoproterenol (di-p-toluoylisoproterenol and dipivaloylisoproterenol) was studied in the isolated perfused rabbit lung preparation. High-pressure liquid chromatographic methods were developed to measure diester, monoester, isoproterenol, and 3-O-methylisoproterenol from a single 1-ml perfusate sample. The prodrugs were administered directly into the circulating perfusion medium and by endotracheal instillation. Perfusate concentrations of diester, monoester, isoproterenol, and 3-O-methylisoproterenol were measured for 180 min. The diesters were rapidly eliminated from the perfusate with a subsequent increase in monoester concentrations. Isoproterenol levels were observed within minutes of prodrug administration, peaked at 60-80 min, and declined slowly thereafter. The prodrugs were rapidly absorbed following endotracheal administration with 30-50% of the diester being metabolized during the first pass through the lung.

Reinforcing and other behavioral effects of nicotine.

Published findings of intravenous nicotine self-injection indicate that the reinforcing properties of nicotine are weak when the drug is made available according to continuous reinforcement (CRF) or fixed-ratio (FR) schedules. CRF self-injection rates are generally only 2-3 times saline control levels and self-injection frequency is largely insensitive to changes in unit dose. In contrast, drugs of the psychomotor stimulant, opiate, and sedative-hypnotic classes, with similar pharmacokinetic parameters, maintain much higher self-injection rates and show systematic changes in rate with unit dose variations. Recent studies using interval and second-order schedules of nicotine presentation have been more successful in maintaining higher rates of self-administration behavior. Systematic dose-response functions have also been found under these conditions. Food-deprivation, species and strain differences, circadian rhythms, and duration of exposure to the drug also appear to be important variables in determining self-injection rate. Finally, the rapid development of tolerance to the effects of nicotine may account for changes in the pattern of self-administration within daily sessions and the differential sensitivity of those patterns to nicotine pretreatment.

Synthesis of 1-O-(2'-acetoxy)benzoyl-alpha-D-2-deoxyglucopyranose, a novel aspirin prodrug.

The synthesis and characterization of 1-O-(2'-acetoxy)benzoyl-alpha-D-2-deoxyglucopyranose, a novel aspirin prodrug, are described. 3,4,6-Tri-O-benzyl-alpha-D-2-deoxyglucopyranose was synthesized by methylating the anomeric hydroxyl group of 2-deoxyglucose, benzylating the 3-, 4-, and 6-hydroxy functional grups, and cleaving hydrolytically the anomeric methyl group. Reaction of the tribenzylated sugar with the acid chloride of aspirin and subsequent hydrogenolysis of the benzyl groups resulted in the prodrug, mp 128 degrees. The compound was further characterized by elemental analysis and PMR and 13C-NMR spectroscopy. In vitro, the compound cleaved to aspirin with a half-life of 7 min at 37 degrees. Prodrug cleavage was independent of pH over the pH 3--9 range.

Kinetics and mechanism of hydrolysis of 1-(2'-acetoxybenzoyl)-2-deoxy-alpha-D-glucopyranose, a novel aspirin prodrug.

The formation rate of aspirin from the prodrug was determined as a function of the pH, temperature, and dielectric constant of the solvent spectrophotometrically and was confirmed by high-pressure liquid chromatography. Aspirin formation was first order with respect to the prodrug and zero order with respect to the hydroxide-ion concentrations. The hydrolysis rate was independent of buffer concentration but very sensitive to the dielectric constant of the solvents. The half-life for the formation of aspirin at 37 degrees was 7 min. The activation energy for the hydrolysis was 23.7 kcal/mole. The results suggest that the hydrolysis of the prodrug to aspirin proceeds by an SN1-type mechanism.

Oral phenytoin loading in adults: rapid achievement of therapeutic plasma levels.

Oral loading doses of phenytoin were estimated from a simplified pharmacokinetic model. Twenty patients were administered a calculated average dose of 19.1 mg per kilogram of phenytoin divided into two to four increments given over 3 to 21 hours (10.55 +/- 4.74, mean +/- SD). Plasma samples drawn 2 to 22 hours (10.75 +/- 4.32, mean +/- SD) after completion of the loading dose resulted in therapeutic levels ranging from 8.1 to 18.0 microgram per milliliter (11.42-11.37 +/- 2.41, mean +/- SD). Thus, this regimen in which no increment of the loading dose exceeds 600 mg is sufficient to achieve and maintain therapeutic plasma concentrations 18 to 24 hours after initiation of the loading dose.

The effect of some cigarette smoke constituents and other compounds on the metabolism of benzo(a)pyrene in rabbit lung 9000 g supernatant.

1. The inhibitory effects, of a number of compounds, on the formation of two groups of benzo(a)pyrene metabolites in rabbit lung homogenates (9000 g supernatant) have been investigated. 2. The inhibitors comprised two groups: those inhibiting primarily benzo(a)pyrene hydroxylase activity, and those inhibiting the activity of the epoxide-metabolizing enzymes as well as hydroxylase activity. Phenol, 1-naphthol, nicotine and acetone belong to the former group; naphthalene, anthracene and chlorpromazine to the latter group. 3. The most active inhibitors also caused a significant decrease in the amount of benzo(a)pyrene bound covalently to tissue macromolecules.