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Residue-level features of bovine pancreatic trypsin inhibitor (BPTI) unfolding on reversed-phase chromatography (RPC) surfaces were investigated using hydrogen-deuterium exchange labeling and NMR. A set of silica-based RPC surfaces was used to examine the influence of alkyl chain length and media pore size on adsorbed BPTI conformation. In all cases there was substantial unfolding in the adsorbed state; however, residual protection from exchange was consistently observed. Particle pore size did not influence conformation substantially for C4-alkyl modified silica; however, 120 A pore C18 media produced more hydrogen exchange than any other surface examined. In this case, the radius of curvature inside the pore approaches the size of the BPTI molecule. Generally, the pattern of hydrogen exchange protection was uniform; however, the beta-sheet region was selectively protected on the large-pore C18 media. The beta-sheet region forms a hydrophobic core that forms early when BPTI folds in solution. This suggests that partially unfolded states possessing a native-like structure play an important role in adsorption and elution in RPC. Finally, increased contact time with the surface before elution fostered unfolding and altered chromatographic behavior considerably., (Copyright 2001 John Wiley & Sons, Inc.)

While reversed-phase chromatography (RPC) may be a powerful method for purification of proteins at the analytical scale, both preparative and analytical applications have been hindered by the complex chromatographic behavior of proteins compared to small molecules. Further, preparative applications have been limited because of poor yields caused by the denaturing conditions involved. One means for modulating both the stability and chromatographic behavior of proteins is through the use of added salt. In this investigation, we show how salt type and ionic strength affect protein conformation on RPC surfaces. Exposure of amide groups of adsorbed BPTI was monitored using nuclear magnetic resonance (NMR) spectroscopy and hydrogen-deuterium isotope exchange. Sodium chloride, sodium acetate, and ammonium sulfate were studied at ionic strengths up to I = 0.375, with adsorption hold times being 5 min and 2 h. We found that increasing ionic strength decreased exposure of the exchange reporter groups in essentially all cases. However, even at the same ionic strength the level and distribution of residue protection varied with salt type and hold time. NaCl does not protect certain reporter groups at all, while those that it does protect to some degree at short hold times can exchange slightly more at longer times. The pattern and level of protection for NaAc at short times is similar to that for NaCl, but at longer times more uniform protection is seen as the reporter groups completely exposed at short times become more protected. For (NH(4))(2)SO(4) the pattern of protection at short hold time is similar to those of the other salts, although it protects all groups much more. This would be expected from the Hofmeister series. However, at longer times the level of protection with (NH(4))(2)SO(4) decreases below that of the other salts, while it uniquely protects all groups to nearly the same level. Such subtle variations in the protein structure would not have been detected without the measurements and analysis used here. Chromatographic retention times and peak shapes were obtained for the above systems. Variations of behavior were seen that could not be correlated with any of the above protection patterns and levels or even with heuristics such as the Hofmeister series. This suggests further conformational changes upon elution may be critical to the retention process. However, an excellent correlation was found between peak width at half-height and the average degree of unfolding, as indicated by the average level of isotopic exchange. Thus, while further studies are needed to definitively determine the connection between protein unfolding and retention, use of this correlation may improve designing and screening for chromatographic conditions that minimize protein unfolding., (Copyright 2001 John Wiley & Sons, Inc.)

Aims: To assess the pharmacodynamics of moxonidine in patients with functional NYHA Class II-III congestive heart failure (CHF)., Methods: A parallel population pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed to assess the effect of moxonidine (0.1, 0.2, 0.3 mg twice daily) and placebo treatment on plasma noradrenaline (NA) levels, standing systolic blood pressure (SBP), and heart rate (HR) over 12 weeks in 97 patients with CHF using a parallel group design with dose escalation. A sequential analysis was also developed, where the relative changes in NA concentration were related to both SBP and HR., Results: In the parallel PD analysis, an effect delay was shown for all three end points (NA, SBP, and HR). An inhibitory Emax model was used to characterize the concentration-effect relationships. For SBP and HR, the EC50 value increased over time. For NA, there was a positive baseline drift over the 12 weeks; this was interpreted as disease progression. Moxonidine delayed this increase by 9.8 weeks. For SBP, there was a circadian pattern at baseline. In the sequential PD analysis, the relationship between the drug response (NA) and SBP or HR was best described by an inhibitory Emax model. No effect delays between the response and effects were found., Conclusions: Effects of moxonidine on NA, SBP, and HR could be quantified by an effect compartment model in the presence of disease progression and circadian variations. Disease progression, as judged by increasing NA levels with time, was delayed by moxonidine. A direct relationship was found between NA and SBP/HR.

The effect of the phosphodiesterase inhibitors (tibenelast, theophylline) and placebo on isoproterenol-induced changes in heart rate, cAMP and norepinephrine levels in normal male volunteers was studied. Heart rates in response to isoproterenol dosing were fitted by linear regression, and horizontal shifts in the regression lines were examined between the three treatments. The shift of the regression line after placebo compared to the preplacebo line was to the right by 2.6 +/- 2.2 ng ISO/kg/min, theophylline pretreatment shifted this line to the left by 2.4 +/- 1.8 ng/kg/min (p < 0.05) and tibenelast by 3.7 +/- 1.9 ng/kg/min (p < 0.02). Both tibenelast and theophylline increased the heart rate response to isoproterenol infusion, whereas norepinephrine and cAMP levels were not different in any treatment.

1. The mechanism responsible for ipsilateral renal vasoconstriction resulting from mechanical stimulation of the ureter was examined using the pharmacological antagonists saralasin, SQ20881, indomethacin, atropine and phentolamine. 2. A segment of PE tubing was implanted in the left ureter, patency being well maintained. Three to four days later renal blood flow was measured bilaterally and antagonists administered intravenously. 3. The mechanism responsible for vasoconstriction could not be attributed to either increased intrarenal noradrenaline or angiotensin, nor to a decreased activity of prostaglandins, kinins or acetylcholine. 4. Histological and bacteriological examination also proved negative. 5. Thus, additional evidence of an unidentified intrarenal mechanism causing vasoconstriction is presented.

1. The cardiovascular responses to varying doses of hydrallazine were studied in renal hypertensive rabbits. 2. The depressor effect of hydrallazine was independent of the rate of its i.v. administration (bolus v. a 15 min constant infusion). 3. Intraperitoneally administered hydrallazine was significantly less active than equimolar i.v. doses, and the relative potency (i.p. v. i.v.) did not increase with increasing doses. 4. In spite of inferential evidence to the contrary, we did not find any pharmacodynamic support for non-linear processes being involved in metabolism of hydrallazine.

Baroreflex activity is a determinant of the homeostatic response to alteration in blood pressure. We examined the factors that determine the magnitude of the vasodepressor response to sequential incremental intravenous infusions of sodium nitroprusside (NP), 0.05 to 6.4 micrograms/kg/min, in eight male patients with essential hypertension. Each infusion level was of 10 minutes' duration. Change from control values of mean arterial pressure (delta MAP), heart rate (delta HR) and plasma norepinephrine (delta NE) were obtained at the end of each infusion level. Significant correlations were found between delta MAP vs log dose NP, delta HR vs delta MAP and delta NE vs delta MAP for each patient (p less than 0.05). However, the slopes of these relationships varied widely between subjects and were significantly correlated with the control blood pressure of each patient. In addition, the sympathetic responsiveness, as measured by delta NE vs delta MAP, was inversely correlated with the degree of vasodepressor response seen. Thus, the magnitude of the vasodepressor response was determined by two major factors: 1) the predrug blood pressure, possibly reflecting altered vascular geometry with hypertension; 2) the degree of sympathetic response, which probably acts by mediating the degree of reflex alpha-adrenergic-mediated arteriolar vasoconstriction.

To determine the role of the peripheral sympathetic nervous system in the persistent tachycardia caused by the antihypertensive drug hydralazine, we examined the temporal relationships between the changes in heart rate and plasma norepinephrine concentration and the reduction in blood pressure produced by a range of doses of hydralazine administered intravenously to five hypertensive patients. Significant linear correlations were found between the increases in heart rate and plasma norepinephrine concentration and the reduction in blood pressure at 15 and 30 minutes after injection. However, at 240 minutes after injection, changes in heart rate and plasma norepinephrine were not correlated with changes in blood pressure and were disproportionately elevated relative to the reduction in blood pressure. A significant linear correlation between changes in heart rate and plasma norepinephrine concentration was noted at 15, 30, and 240 minutes after injection. The temporal discordance of the changes of both heart rate and plasma norepinephrine relative to the reduction in blood pressure and the significant linear correlation between the increases in heart rate and plasma norepinephrine concentration suggest that continued activation of the peripheral sympathetic nervous system contributes to the persistent tachycardia seen after the administration of hydralazine.

Previous studies on intravenous hydralazine kinetics have been performed using nonselective analytical techniques that measure not only hydralazine but also certain hydralazine metabolites such as hydralazine pyruvic acid hydrazone (HPH). We studied the time course of hydralazine and HPH in eight hypertensive patients after 0.3 mg/kg intravenous with selective high-pressure liquid chromatographic assays. "Apparent" hydralazine concentrations were also determined using a nonselective gas-liquid chromatographic procedure. Total plasma clearance, CLT[72.9 +/- 4.9 (SEM) ml . min-1 . kg-1], apparent volume of distribution, Vd area (5.83 +/- 0.30 1 . kg-1), steady-state volume of distribution, Vd ss (1.83 +/- 0.17 . kg-1), and terminal half-life, t1/2 (53.7 min, harmonic mean) were independent of acetylator phenotype. The high ClT is compatible with rapid intravascular conversion of hydralazine to HPH and a high hepatic extraction ratio. Peak HPH concentrations occurred 10 to 60 min after dose; mean HPH t1/2 was 239 min. "Apparent" hydralazine concentrations were usually highest in the 2-min plasma sample and declined with a mean t1/2 of 296 min. Reports based on nonselective assay methods have underestimated CLT, Vd ss, and Vd area and have overestimated the t1/2 of hydralazine.

The sensitivity achieved by the described thin-layer chromatographic (TLC) method greatly exceeds that of previously published TLC methods for the determination of cocaine and its principal metabolite, benzoylecgonine, in urine. Sensitivity for cocaine and benzoylecgonine approaches 0.1 and 0.25 mug/ml, respectively, for a 5.0-ml specimen. A simple extraction with a mixed organic solvent provides the basic mechanism for isolating the drugs from biologic specimens. Cocaine and its metabolites are stable in sulfuric acid solutions but labile in aqueous media containing certain other inorganic and organic acids; therefore, an emphasis on the utilization of sulfuric acid solutions is employed throughout the procedure. An evaluation of sensitivities achieved for cocaine and benzoylecgonine by various detection reagents is presented. The technique is applicable to drug screening programs.

The effect of uremia on the binding of diazoxide to plasma proteins was studied. An equilibrium dialysis technique, using diazoxide-minus14C at approximately 30 and 300 mug/ml in the plasma phase, was used to measure diazoxide binding to plasma. Serum albumin concentration (Alb) and serum creatinine (Cr) or blood urea nitrogen (BUN) were negatively correlated. By single regression analysis, per cent free diazoxide (%FD) correlated negatively with Alb and positively with Cr or BUN. When %FD was regressed simultaneously against Alb and Cr or BUN, Alb emerged as the sole determinant of %FD (p less than 0.001), indicating that creatinine or BUN correlated with %FD by their inverse correlation to Alb rather than by an effect on drug protein binding. At the levels of Alb studied, %FD varied over a 2-fold range. In a retrospective study of the influence of uremia on diazoxide effect in hypertensive patients, a relatively low correlation (r, 0.59) was found between BUN and hypotensive effect. Prospective studies involving correlations of drug effect with renal function and %FD are required to assess the clinical importance of decreased binding of diazoxide to uremic plasma.

1. Twelve patients with essential hypertension were treated aggressively with minoxidil in order to achieve blood pressure control as rapidly as possible. 2. After an initial dose of 5 mg, dose increments were administered 6 hourly until a fall in blood pressure was observed. 3. The size of additional doses was determined by the magnitude of and response to the lowest effective dose and the therapeutic objective. 4. Over a time-interval of 24-42 h blood pressure was reduced to normal or near normal in each case. 5. Analysis of the relationship between blood pressure response and cumulative dose indicates that at sub-optimum blood pressure responses it is safe and efficacious to give half the antecedent cumulative dose as a single dose in arriving at the therapeutic objective.

1. Changes in plasma noradrenaline levels and heart rate were used as measures of baroreflex sensitivity in six hypertensive subjects given serial incremental doses of sodium nitroprusside (intravenously) to lower blood pressure. 2. The rises in both heart rate and plasma noradrenaline concentration were linearly related to the decrement in blood pressure and inversely related to the severity of the hypertension. 3. A positive correlation between rise in heart rate and rise in plasma noradrenaline was found for each subject. With increasing severity of hypertension, a greater increase in heart rate occurred for each increment in plasma noradrenaline concentration. 4. Baroreflex sensitivity can be assessed by relating changes in heart rate to change in arterial pressure; however, this method does not distinguish the relative contributions of the vagal and sympathetic components of the autonomic neural response or variations in the chronotropic response to sympathetic stimulation. 5. Changes in plasma noradrenaline levels in response to graded reductions in blood pressure may be a more appropriate measure of baroreflex sensitivity than the methods currently used in clinical investigation.

The rate of hydrolysis of chloroprocaine by human serum was studied in the presence and absence of a number of aminde local anesthetics and their metabolites. Bupivacaine (2.4 microgram/ml) and etidocaine (2.3 microgram/ml) caused 38% and 21% inhibition respectively of the rate of chloroprocaine hydrolysis. Circulating concentrations of these drugs have been reported in this range by several investigators following epidural doses of 150 to 400 mg of either drug. Mepivacaine, lidocaine, and two lidocaine metabolites (glycine xylidide and monoethylglycine xylidide) were only inhibitory at levels much greater than those seen in blood following the usual local anesthetic doses of the parent compounds. Since serum is an important site of chloroprocaine metabolism in man, the probability of chloroprocaine intoxication may be increased when it is administered with local anesthetics such as bupivacaine and etidocaine.

Two methods for arriving at optimum, individual phenytoin dosage regimens have been evaluated in 12 patients. (1) Individual Michaelis-Menten pharmacokinetic parameters for phenytoin were estimated from two reliable steady-state phenytoin serum concentrations resulting from different daily doses: The observed steady-state phenytoin serum levels obtained after 3 to 8 wk of compliance with dosage regimens calculated from the individual pharmacokinetic parameters agreed well with predicted levels (r = 0.824, p less than 0.02). The average deviation between observed and predicted levels was 0.04 mug/ml (range, +/- 3.2 mug/ml). (2) A previously published nomogram for making adjustments in phenytoin dosage regimens: The serum phenytoin concentration actually expected from the dose indicated by the nomogram was calculated using individual pharmacokinetic parameters. The daily dose for one patient would have exceeded his estimated maximal rate of metabolism. The correlation between calculated and predicted phenytoin serum levels in the other 11 patients was weak but significant (r= 0.360, p less than 0.05). The average deviation was --3 mug/ml (range, 3.9 to --11.3 mug/ml). It was concluded that the use of individual pharmacokinetic parameters is practical and is also superior to the nomogram.

A 20-year-old woman presented with malignant hypertension, pulmonary edema, anemia, and azotemia. Blood pressure was adequately controlled only after progressively more intensive drug regimens, finally including minoxidil, nadolol, and furosemide. On these drugs, the patient developed progressive left and right heart failure, anasarca, and malnutrition. The control of hypertension, heart failure, and fluid retention, was accomplished by administration of captopril and furosemide. Captopril is a logical alternative to vasodilators in refractory hypertension complicated by congestive heart failure.

Earlier in vivo studies revealed a low concentration of triamterene in the brain of guinea pigs and baboons, and a transfer of the drug from the fetus to the mother. Additional investigations have been performed to characterize further the transport system(s) for triamterene in the central nervous system (CNS), placenta, and kidney. In guinea pigs a very low brain to free plasma concentration ratio (0.1) was achieved 3.5 min after drug administration and was maintained during 180 min of drug infusion. The cerebrospinal fluid (CSF) concentration was similar to the concentration of the drug in the brain. A higher brain to free plasma concentration ratio was gradually reached in dogs studied with nanogram per ml and microgram per ml concentrations of triamterene in CSF. Administration of triamterene to fetal and maternal sheep revealed a placental extraction (E) from fetal plasma to placenta 20 times greater than that from maternal plasma to placenta. The E from fetal plasma to placenta was unaffected by a triamterene concentration in the maternal circulation 10 times that in the fetus. These findings and studies of renal clearance support an active transfer of triamterene by the CNS, placenta, and kidney; the physiologic substrate for these systems is unknown.

The hypothesis that the pharmacologically active hydralazine hydrazones (HH) are endogenously hydrolyzed to parent hydralazine (H) was tested in a series of in vitro and in vivo systems. The stable hydrazones H alpha-ketoglutaric acid hydrazone and H pyruvic acid hydrazone did not hydrolyze to H in vitro (buffer or plasma), were inactive in vivo and did not generate urinary metabolites of parent H. By contrast, the labile HH, H acetaldehyde hydrazone and acetone hydrazone (HAH) generated H in vitro. H acetaldehyde hydrazone produced in vitro effects that were equipotent to the H concentration measured in the dose solutions. When administered to conscious rats and rabbits, the labile hydrazones reduced blood pressure. This effect was more gradual in onset than that of H. The hypotensive effects of HH were significantly greater than predicted by the amount of H contained in the dose solutions. Metabolic studies were conducted with the labile HH, HAH. After administration of HAH to rabbits, the proportional excretion of the urinary H metabolite, H pyruric acid hydrazone, was equal to that observed after the administration of H. We conclude that HH are inactive, except when hydrolyzed to H. The hydrolysis of certain HH, including HAH and H acetaldehyde hydrazone, in vivo may be nearly complete. Differences in the pharmacodynamic properties between labile HH and H may be related to the time course of generation of H, sequestration of hydrolysis in physiologically inactive sites or other unrecognized mechanisms.

The antihypertensive effect of minoxidil was studied in 6 patients with varying degrees of hypertension. Their baseline mean arterial pressure (MAP bi) ranged from 122 to 197 mm Hg. Single oral doses between 2.5 and 25 mg were administered in sequence and the time-course of hypotensive action was followed. We have reported previously that when the peak lowering of MAP is linearly regressed against log dose, both the dose-response slope (M) and threshold dose (Dt) are positively correlated with the MAP bi of individual patients. This investigation focuses on the temporal pattern of effect. It was found that the hypotensive effect of minoxidil declined linearly with time at a rate consistent with an average effective biologic half-life of about one day. The rate of decline of effect was apparently independent of dose but was dependent on MAP bi. Since both response to and duration of effect of minoxidil are functions of MAP bi, there is an abvious need to individualize dosage regimens based on the severity of disease. Using pharmacodynamic parameters, guidelines for loading dose, maintenance dose, and dosing frequency as a function of the degree of hypertension are suggested. Loading dose requirements were found to increase with MAP bi while maintenance doses were largely independent of the severity of the disease. Frequently of dosing was found to range from 3 times a day in very severe hypertension to once a day in moderate hypertension.

The formation and disposition of 3-methyltriazolophthalazine (MTP), an acetylated metabolite of hydralazine (H) was studied in rabbits. Following its iv administration MTP was rapidly and almost completely metabolized. The major metabolite in plasma and urine was found to be a hitherto unrecognized glucuronide conjugate, most likely of the carboxylic acid analogue of MTP. The hydralazine-pyruvic acid hydrazone (HPH), which is a major plasma metabolite of H, was recognized as a more potent source for MPT in vivo than was parent H. MTP was also formed in significant amounts in vitro following incubation of HPH in phosphate buffer. We conclude that MTP can arise directly from HPH. Consequently, MTP and the compounds formed during its further biotransformation may not provide any valid measure of the extent to which H is acetylated in vivo.

Metabolism of the vasodilator hydralazine was investigated by in vivo and in vitro studies. Standards to identify metabolic products were synthesized. Determination and quantification of hydralazine and its metabolites were accomplished by gas chromatography-mass spectrometry. A deuterium-labeled internal standard was used for quantification. 14C-labeled internal standards were synthesized and used to demonstrate recoveries from the biological samples.

The clearance of furosemide (F), whose renal tubular transport shares the classical characteristics of the organic acid system, was determined in dogs with varying degrees of azotemia and compared with tetraethylammonium (TEA), an organic base. Two normal and eight azotemic dogs [blood urea nitrogen (BUN), 12-273] were studied. Azotemia was produced by bilateral uretero-venous anastomoses. The left renal vein and ureter were cannulated and renal blood flow (RBF) was measured by electromagnetic flowmeter. Simultaneous left renal clearances (C) of subpharmacological doses of TEA-14C and furosemide-14C were determined at seven 30-minute intervals. Initial loading doses were followed by continuous maintenance infusions. For TEA, clearance (1.5 ml/min-g +/- 0.2 S.E.M.) and extraction (E) (0.83 +/- 0.02) are independent of the degree of azotemia. Renal plasma flow (RPF), calculated as CTEA/ETEA, agreed closely with directly measured RPF (2.0 ml/g-min +/- 0.3). RPF was independent of azotemia. To allow for individual differences in the animals in RPF, the ratio CTEA/CF was used. CF (1.07-0.17 ml/min-g) and EF (0.54-0.06) decreased as a linear function of the increase in uremic serum: (see article). Furosemide and its principle metabolite were greater than or equal to 97% of the furosemide portion of the radioactivity. The metabolite did not increase with time in either plasma or urine. After acute administration of exogenous urea to two dogs (BUN 170 and 253) CTEA/CF was unrelated to BUN. Thus, the CF decreases proportionately with progressive azotemia and is not related to RBF, exogenous urea or metabolite. This suppression of renal tubular secretion of furosemide may partially account for reduced therapeutic efficacy of furosemide in azotemia.

To determine if clinically effective doses of the antihypertensive agent captopril affected the neuronal release of norepinephrine or baroreflex sensitivity, changes in plasma norepinephrine concentration and heart rate were related to the changes in mean arterial pressure seen during the intravenous infusion of stepwise incremental doses of sodium nitroprusside before and during captopril treatment in eight hypertensive men with normal or low plasma renin activity. At all times, significant linear correlations were found between the decrease in mean arterial pressure and the dose of sodium nitroprusside, the increase in heart rate and the decrease in mean arterial pressure, and the increase in plasma norepinephrine concentration and the decrease in mean arterial pressure. When the subjects were treated with captopril (25 mg t.i.d.) for 2 to 4 weeks, supine mean arterial pressure decreased from 130 to 114 mm Hg (-12%; p less than 0.05), heart rate did not change, supine and upright plasma renin activity increased, while supine plasma norepinephrine and epinephrine concentration decreased slightly. Therapy with captopril (25 mg t.i.d.) increased baroreflex sensitivity, as assessed by the slope of the regression line relating the increase in heart rate to the decrease in mean arterial pressure, and increased the responsiveness of the sympathetic nervous system, as assessed by the slope of the regression line relating the increase in plasma norepinephrine concentration to the decrease in mean arterial pressure. These increases were accompanied by a decrease in the slope of the regression line relating the decrease in mean arterial pressure to the dose of sodium nitroprusside and thus were associated with a decreased sensitivity to the vasodepressor effects of sodium nitroprusside.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in mean arterial pressure (MAP), heart rate (HR), and plasma concentrations of norepinephrine (NE) and epinephrine were measured in eight hypertensive patients in a supine position after stepwise infusion of incremental sodium nitroprusside doses and intravenous injection of cumulative labetalol doses. Both drugs induced rises in plasma NE concentration that were linearly related to reductions in MAP. For any reduction in blood pressure (BP), however, the rise in plasma NE concentration induced by labetalol was approximately four times that induced by sodium nitroprusside. The difference can be explained by two effects of labetalol: impairment of neuronal NE uptake and beta-adrenergic-receptor blockade, which are known to reduce NE clearance from plasma. After both drugs there was a correlation between changes in HR and changes in BP and a correlation between changes in HR and changes in plasma NE concentration. Slopes of the regression lines for both relationships were less after labetalol than after sodium nitroprusside, presumably because of the beta-adrenergic-blocking properties of labetalol. Multiple-regression analysis indicated that the plasma NE rise was an important determinant of the vasodepressor response to each drug. The greater plasma NE elevation after labetalol may limit its antihypertensive effect.

Six male hypertensive patients, three rapid and three slow acetylators, each received four different intravenous hydralazine doses by constant infusion over 100 sec. Two to four days elapsed between doses. Plasma or whole-blood hydralazine concentrations were measured by HPLC after each dose. There was no influence of acetylator phenotype on hydralazine kinetics after intravenous dosing. There also was no consistent effect of dose size on hydralazine clearance or volume of distribution at doses up to 0.45 mg (2.3 mumol/kg). One subject, who received doses up to 0.6 mg/kg (3.05 mumol), had an apparent decrease in clearance at the higher doses. These findings are consistent with the fact that hydralazine is converted intravascularly to hydralazine pyruvic acid hydrazone and the fact that potentially saturable hepatic metabolic pathways play only a modest role in systemic clearance.

The hydrazone of hydralazine and pyruvic acid (HPH) has been recognized as a quantitatively important metabolite of hydralazine in human plasma. We evaluated the disposition of [14C] HPH after its i.v. administration to normal, anephric and probenecid-pretreated rabbits. Renal clearance of HPH in normal rabbits exceeded the glomerular filtration rate by a factor of 3 to 4 and accounted for 80 to 90% of the total body clearance. Active tubular secretion was established by the effect of probenecid pretreatment to reduce the renal clearance of HPH by 80%. Total body clearance of HPH in anephric rabbits was 10% of that of normal animals, emphasizing the minor importance of metabolic conversion for the overall disposition of HPH. HPH in a maximum dose of 50 mumol/kg i.v. had no hypotensive effect in renal hypertensive rabbits and did not interfere with the subsequent hypotensive response to hydralazine. This HPH dose produced plasma levels at least 50 times in excess of those reported in humans after administration of therapeutic doses of parent hydralazine. HPH is consequently of negligible clinical significance, despite the relatively high plasma concentration of this metabolite which occurs after administration of parent hydralazine.

Twelve patients with essential hypertension were treated aggressively with oral minoxidil in order to achieve blood pressure control as expeditiously as possible. Pretreatment blood pressure ranged from 159/109 to 238/161 mm Hg. Following an initial dose of 5 mg, subsequent dosage increments were administered every 6 hr until a fall in blood pressure was observed. The size of additional doses was determined by the magnitude of, and response to, the lowest effective dose and the therapeutic objective. Over a time interval of 24 to 42 hr, the blood pressure was reduced to normal or near normal in each case. Untoward hypotension was uniformly avoided. Analysis of the relationship between blood pressure response and cumulative dose indicates that at suboptimal blood pressure responses, it is safe and efficacious to give half the antecedent cumulative dose as a single dose in arriving at the therapeutic objective. The use of such a regimen should be considered as an alternative to parenteral drug administration in severe hypertension when immediate control of blood pressure is not required.

Hydralazine and sodium nitroprusside induce different effects on systemic hemodynamics, but their effects on sympathetic neuronal activity have not been compared. Five hypertensive subjects receiving only hydrochlorothiazide were studied during two sessions. During one session, four doses of hydralazine, 0.1 to 0.6 mg/kg, were given intravenously at least 3 days apart, and during the other session, sodium nitroprusside was infused in stepwise doses, 0.05 to 4.8 micrograms/kg/min for 10 min per dose. Mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine (NE) and epinephrine concentrations were determined before and after dosing. The following correlated linearly for hydralazine and sodium nitroprusside: delta HR/delta MAP, delta NE/delta MAP, and delta HR/delta NE. Comparison of these relationships, however, indicated significant differences between the sympathetic neuronal and hemodynamic responses to hydralazine and sodium nitroprusside. Increase in HR relative to decrease in MAP was greater for hydralazine than for sodium nitroprusside. There were greater increases in plasma NE concentration relative to falls in MAP with sodium nitroprusside than with hydralazine, but increases in HR relative to increases in plasma NE concentration were smaller for sodium nitroprusside than for hydralazine. Such responses may reflect differential effects of hydralazine and sodium nitroprusside on the systemic clearance of NE or of the activity of cardiopulmonary baroreceptors.

We examined the visceral blood flow distribution during infusion of three vasodilators at doses that produced similar depression of systemic arterial pressure. The studies were performed in pentobarbital-anesthetized dogs using the radioactive microspheres technique. Minoxidil did not alter renal, total visceral, or visceral organ flow distribution with the exception of a modest increase in relative stomach blood flow. Nitroprusside increased the percentage of total visceral flow to the spleen and the hepatic artery. Dopamine increased blood flow to the stomach, intestine, and kidney. After phenoxybenzamine, the augmentation of stomach blood flow by dopamine was greatly increased, while blood flow to the splenic, pancreatic, and hepatic arteriolar vascular beds decreased. The decreases in blood flows may be due to decreased perfusion pressure in the absence of active vasodilation or to myogenic or metabolic autoregulation. Thus, at equivalent hypotensive responses, the vasodilator compounds that we studied produced markedly different patterns of visceral blood flow.

The effect on blood pressure of giving hydralazine orally, 300 mg per day divided into 2, 3, and 4 doses, was studied in 4 hypertensive patients. There was no significant difference in average mean arterial pressure for the 3 regimens. Fluctuations of mean arterial pressure with time were not significantly different for the regimens as indicated by coefficient of variation. The mean coefficient of variation for the 2 days prior to initiation of hydralazine dosing was 4.0% +/- 0.3 (SEM) and for the 3 dosing regimens ranged from 4.1% to 4.8%. Heart rate was used as an index of vasodilator-induced increase in sympathetic tone and therefore as a measure of possible side effects. Fluctuations in heart rate were small and were not associated with symptoms. After discontinuation of hydralazine, the time for blood pressure to return halfway between initial placebo value and end treatment value varied from 30 to 140 hr. A tracer dose of hydralazine 14C. HCl was administered intravenously to the patients. The urinary excretion of 14C showed a multiexponential pattern of elimination with a previously undescribed prolonged terminal phase. Under the conditions of the present study, a daily dose of 300 mg of hydralazine was as effective and free of side effects given in 2 as in 4 divided doses.

Vasodilators are highly efficacious in hypertension and the ideal one(s) have not yet been developed. Key information is obtained from initial studies of the effects of sequentially larger single oral doses. Blood pressure and heart rate responses measured in the recumbent and standing positions can be analyzed in terms of onset, dose response and offset of effect. These data, which can be modeled similarly to plasma concentration data, will determine the optimal dosing interval. The adequacy of blood pressure control should be confirmed by multiple dose administration. Orthostatic effect and intensity of compensatory responses can be evaluated by hemodynamic and neuroendocrine studies, the latter to include plasma catecholamines and plasma renin activity. Baroreflex sensitivity, as a determinant of autonomic response to vasodepression, appears to modify the response to intravenous nitroprusside, in part by increased sympathetic tone. The influence of baseline arterial pressure on the response to vasodilators may be partially determined by its inverse relationship to baroreflex sensitivity.

The mechanism of action and relative potency of hydralazine (H) and tow hydrazone derivatives were investigated using isolated rabbit aortic strips. H, hydralazine acetone hydrazone (HA) and hydralazine butanone hydrazone (HBH) relaxed established K+ and norepinephrine (NE) contractures, and inhibited the development of contractures to these two agents on preincubation. H, HA and HBH increased the threshold to Ca++ and decreased the maximum tension responses during K+-Ca++-contractures (HA greater than H, P less than .05; HBH greater than H P less than .01). The Ca++-dependent and Ca++-independent components of NE contractures were both inhibited by H, HA and HBH. NE contractures were more sensitive to the effects of H than K+ contractures. These results are consistent with the conclusion that H and hydrazone derivatives produce effects on vascular muscle both by interactions with the fluxes of Ca++ from the extracellular space and effects on release from cell stores. However, other possibilities need to be assessed experimentally.

The apparent steady state blood concentration of lidocaine in dogs depends on the animal's lidocaine treatment history. For example, an infusion of 50 microgram/Kg/min X 240 min followed immediately by an infusion of 125 microgram/Kg/min X 240 min yields steady state lidocaine levels which are consistent with a linear drug dispositional system. However, when the sequence of these infusions is reversed the apparent steady state level during the low infusion rate step is much greater than those predicted from the steady state level observed during the high infusion rate (assuming a linear dispositional model). These data suggest that high dose lidocaine infusion treatment alters the dog's ability to clear lidocaine during a subsequent low infusion rate treatment period.

The relationship of natriuretic effect and furosemide excretion was studied in normal and azotemic dogs. Graded azotemia was produced in dogs by bilateral uretero-venous shunts of varying duration. The shunts were subsequently opened and urine and blood samples were taken to measure inulin, furosemide and sodium concentrations. Renal blood flow was measured by an electromagnetic flow probe. Two groups of dogs, control and experimental, were studied. The experimental group received a loading dose followed by a constant infusion of furosemide. This dose produced a natriuresis in nonazotemic normal dogs. The magnitude of this natriuresis correlated with furosemide excretion rate (P less than .005) and not with the plasma concentration of the drug. Furosemide clearance and extraction were inversely correlated with blood urea nitrogen. In the furosemide-treated group the augmentation of sodium excretion was not impaired except at blood urea nitrogen concentrations of greater than 200 mg/dl (two dogs). Thus the reduced clearance of furosemide may account in part for the high dose necessary. Further studies appear to be in order to clarify the relationship of the natriuretic response to furosemide to the rate of urinary excretion and plasma concentration of the drug.

A study was made of the possible mechanism(s) underlying minoxidil-induced increase in plasma renin activity (PRA). 10 patients with essential hypertension were treated with minoxidil and subsequently with a combination of minoxidil plus propranolol. Minoxidil lowered mean arterial pressure 31.6 plus or minus 3.3 mm Hg, mean plus or minus SEM. There was an associated increase in both PRA, 6.26 plus or minus 2.43 NG/ML/H, and heart rate, 21.4 plus or minus 2.7 beats/min. The changes in PRA and heart rate were positively correlated, r, 0.79. Addition of propranolol reduced mean arterial pressure by a further 10.1 plus or minus 1.5 mm Hg and returned heart rate to control levels. Propranolol reduced PRA significantly but not to control levels. Control PRA positively correlated with PRA on minoxidil, r, 0.97, and with PRA on minoxidil plus propranolol, r, 0.98. We conclude that control PRA is a major determinant of change in PRA with minoxidil. Minoxidil increased PRA by at least two mechanisms: (a) an adrenergic mechanism closely related to change in heart rate and blocked by propranolol, and (b) a mechanism(s) not sensitive to propranolol and possibly related to decrease in renal perfusion pressure.

Hydralazine is an antihypertensive vasodilator agent. Lack of specific assay techniques for its measurement have delayed elucidation of its pharmacokinetic profile. This study compares the plasma profiles of hydralazine, measured both by a specific and by a previously published nonspecific assay and of a major plasma metabolite, hydralazine pyruvic acid hydrazone. After po and iv administration of hydralazine, peak hydralazine levels were lower (7-33%) and plasma half lives were shorter (15-31%) when measured by the specific technique. The mean plasma half life of the pyruvic acid hydrazone was 156 min and mean urinary clearance, 28 ml/min. The plasma profile of hydralazine and of the major metabolite, the pyruvic acid hydrazone, do not appear to correspond to the duration of antihypertensive effect of administered hydralazine.

A study was made of the possible mechanisms underlying bupicomide- and hydralazine-induced increase of plasma renin activity. Six patients with mild to moderate hypertension were treated with both bupicomide and hydralazine on separate occasions in random order. Bupicomide lowered mean arterial pressure from 124.2 +/- 3.7 mm Hg (mean +/- SE) to 107.2 +/- 3.9 mm Hg (P less than 0.001). The associated increase in plasma renin activity was 1.27 ng/ml per hour and the increase in heart rate was 16.5 beats/min. Hydralazine reduced mean arterial pressure from 124.2 +/- 3.7 mm Hg to 107.0 +/- 2.0 mm Hg (P less than 0.01). The associated increase in plasma renin activity was 2.20 ng/ml per hour and the increase in heart rate was 22.4 beats/min. Plasma renin activity during bupicomide and hydralazine administration correlated positively with control plasma renin activity (r = 0.98, P less than 0.001). The log of plasma renin activity correlated positively with heart rate (r = 0.51, P less than 0.02) and negatively with mean arterial pressure (r = -0.62, P less than 0.005). We conclude that control plasma renin activity is a major determinant of change in plasma renin activity during administration of bupicomide or hydralazine. Both an increase in sympathetic activity and a decrease in perfusion pressure may contribute to the bupicomide- and hydralazine-induced increase in plasma renin activity, possibly by a baroreceptor-mediated increase in adrenergic tone.

A specific, high-performance liquid chromatographic technique for the measurement of hydralazine pyruvic acid hydrazone is described. This method utilized reversed-phase chromatography for the separation of this hydrophilic metabolite of hydralazine from other fluid constituents present in serum, plasma, or urine of human volunteers and rabbits receiving hydralazine. Detection of the compound of interest is accomplished spectrophotometrically at 250 nm.