Evidence for the safety of ascorbic acid administration to the premature infant.

Ascorbic acid (AA), a plasma antioxidant, is maintained at high levels in premature fetal blood and declines rapidly postpartum. The sudden reduction in blood AA levels secondary to premature delivery may increase the risk of oxidant injury, that is, bronchopulmonary dysplasia and intraventricular hemorrhage. There is concern that administration of AA to premature infants, in an effort to increase antioxidant capacity, may cause hemolysis. We felt that the benefits of early AA administration and prevention of the immediate postnatal drop in blood AA levels, might outweigh the risks of erthrocyte damage. Fifty one high-risk premature infants were randomized to receive either normal saline or 100 mg/kg of AA, daily for the first week of life. Double-blind comparisons were made of hemoglobin, hematocrit, erythrocyte morphology, bilirubin, number of blood transfusions and days of phototherapy, renal function tests, the incidence of infection, bronchopulmonary dysplasia, and intraventricular hemorrhage during the first month of life. The administration of AA prevented the immediate postnatal drop in AA and was not associated with evidence of increased hemolysis. No significant differences in renal function, rate of infection, bronchopulmonary dysplasia, or intraventricular hemorrhage were seen between the two groups. This study suggests that AA administration to the premature infant is safe and supports the designing and performance of larger clinical studies of the antioxidant properties of AA.

Intrauterine exposure to cocaine increased plasma ANP (atrial natriuretic peptide) but did not alter hypoxanthine concentrations in the sheep fetus.

To assess the effects of cocaine, administered to the ewe, on the secretion of atrial natriuretic peptide (ANP), Plasma Renin Activity (PRA) and hypoxanthine in the fetus we studied 6 chronically cannulated sheep fetuses late in gestation. The ewe was given an intravenous injection of cocaine (2 mg/kg). Maternal and fetal arterial blood samples were withdrawn prior to the injection and at 2, 5, 10, 15, 45 and 60 min after the injection for the measurement of ANP, PRA and hypoxanthine. Fetal arterial blood pressure (MAP), plasma ANP and protein levels increased and pH and pO2 decreased after cocaine was administered to the ewe. Fetal plasma hypoxanthine and PRA did not change. These results suggest that cocaine administration to the ewe is associated with fetal hypertension, hypoxemia and acidemia all of which may serve as stimuli for the secretion of ANP.

Effects of phenylephrine induced increase in arterial pressure and closure of the ductus arteriosus on the secretion of atrial natriuretic peptide (ANP) and renin in the ovine fetus.

Although atrial natriuretic peptide (ANP) has been detected in the plasma of late gestation fetal sheep, stimuli that control its secretion and its function in regulating fluid volume are unclear. To determine stimuli that increase ANP secretion and to ascertain whether the response is a function of gestation we studied 20 chronically cannulated fetal sheep. The fetuses were divided into 3 groups; Group I (age range 118-129 days), Group II (age range 129-141 days) and Group III (age range 127-137 days) gestation. Arterial pressure was increased to within a physiologic range by infusing phenylephrine at 6 micrograms/min at 0.1 ml/min into the inferior vena cava of the fetus (Groups I and II). Corrected for body weight fetuses in Group I received 2 micrograms/kg/min whereas fetuses in Group II received 1.66 micrograms/kg/min. In the fetuses in Group III preload to the right side of the heart was increased by inflating an occluder cuff was placed around the ductus arteriosus. Systemic mean arterial pressure (MAP) increased significantly in fetuses in Groups I and II. Plasma ANP concentrations increased significantly in all fetuses. Plasma Renin Activity (PRA) decreased significantly in the fetuses in Group II. These results suggest that phenylephrine infusion and closure of the ductus arteriosus in utero increase circulating ANP in fetal sheep.

Enhancement of hepatic microsomal esterase activity following soman pretreatment in guinea pigs.

Soman (pinacolyl methylphosphonofluoridate), a highly toxic organophosphate compound, has been found to be a strong inhibitor of hepatic microsomal carboxylesterase in vitro, but an enhancer of carboxylesterase when administered in vivo. In response to this paradoxical observation, the objective of this study was to determine if soman could cause true enhancement of the metabolism of drugs in the guinea pig and, if so, to characterize the enhanced enzyme activity. Following the pretreatment of guinea pigs with 90% LD50 soman, enhancement of microsomal esterase activity was noted 12 and 24 hr after pretreatment. Using Michaelis-Menten enzyme kinetic studies, enhancement was found to occur with liver carboxylesterase and procaine esterase, but not with aniline hydroxylase. Since the soman-enzyme complex was known to undergo aging with the release of pinacolyl alcohol and the subsequent formation of pinacolone, the effects of these metabolites on the activity of liver microsomal enzymes in vitro were explored. Pinacolone and pinacolyl alcohol produced enzyme enhancement in vitro in a manner similar to that produced by soman pretreatment. These effects were compared with those made by acetone in the same incubations, since the enhancing influence of acetone has already been well documented. Similarity was found between the in vitro effects of acetone and the effects of pinacolone and pinacolyl alcohol. Lastly, the in vivo effects of pinacolone on the activities of the same liver microsomal enzymes were studied following pretreatment of the guinea pigs with 90% LDLo (lowest published lethal dose) pinacolone. Pretreating guinea pigs with pinacolone prior to killing them enhanced liver microsomal carboxylesterase and procaine esterase activities, but had no effect on microsomal aniline hydroxylase activity. This pattern of enzyme enhancement was similar to that observed after soman pretreatment. Therefore, soman was found to enhance hepatic microsomal esterase activity in the guinea pig in a manner similar to that seen with its metabolites, as well as acetone. This information may give insight into how the efficacy and toxicity of therapeutic drugs, other xenobiotics, and endogenous materials may be altered in individuals who survive an exposure to soman.

Formation of a beta-glucuronidase-resistant glucuronide conjugate of digitoxin by dog liver microsomes.

The aim of these studies was to characterize the glucuronide conjugates of digitoxin and digitoxigenin monodigitoxoside (DMD) produced by liver microsomes from the dog with respect to hydrolysis by beta-glucuronidase and to behavior on HPLC. These results have been compared with studies of conjugates produced by liver microsomes from the rat. Glucuronidation was similar with both substrates with dog microsomes, whereas rat microsomes formed the glucuronide with DMD but not with digitoxin. The DMD glucuronide from both species was completely hydrolyzed by beta-glucuronidase, but no hydrolysis of digitoxin glucuronide was detected. The digitoxin glucuronide was hydrolyzed by a buffer at pH 1.5 but not at pH 10. After acid hydrolysis, the major products appear to be digitoxigenin and DMD glucuronide. These results suggest that glucuronidation of certain drugs by the dog is quite different from that of other species and that the dog may be the only species that possesses a glucuronosyltransferase capable of forming a glucuronide conjugate with digitoxin. The dog also has a glucuronosyltransferase, similar to that in the rat, which is responsible for glucuronidation of DMD. Whether this represents a single glucuronosyltransferase or two different enzymes remains to be elucidated.

Alteration of hepatic carboxylesterase activity by soman: inhibition in vitro and enhancement in vivo.

1. Hydrolysis of the drug esters procaine, chloramphenicol succinate, and prednisolone succinate was studied. Addition of soman to guinea pig liver microsomes caused a dose-dependent inhibition of hydrolysis of all three substrates; at the highest soman concentration (1 microM), ester hydrolysis was totally abolished. 2. Ester hydrolysis was also measured in liver microsomes from guinea pigs pretreated with soman at a low dose (10% of LD50) or at a high dose (90% of LD50) either 1 h or 12 h before killing. Plasma-cholinesterase activity was decreased in all pretreated animals. Liver carboxylesterase activity, measured with the three drug substrates and by hydrolysis of 4-nitrophenyl acetate was increased by all pretreatments. 3. This enhancing effect varies with the substrate and increases with dose of soman. The 12 h pretreatment produced a greater increase in activity than did the 1 h pretreatment. 4. These studies indicate that soman is a potent inhibitor of carboxylesterase activity in vitro but increases the activity of the liver enzyme when administered in vivo.

Inhibition of hepatic microsomal carboxylesterase activity by paraoxon.

A large number of therapeutic agents are esters of carboxylic acids and are thus substrates for microsomal carboxylesterase enzymes. These studies characterized the effects of the organophosphate compound, paraoxon, on the hydrolysis of several drug esters (procaine, chloramphenicol succinate, prednisolone succinate, lidocaine, procainamide and methylparaben) by microsomal preparations from guinea-pigs. These investigations demonstrate that carboxylesterase activity toward several drug esters is present in liver, lung and kidney. The liver is by far the major site of hydrolysis of these ester compounds. Since no hydrolysis was observed with the two amide esters, the hydrolysis of carboxylesters and amide esters appears to be mediated by different enzymes in the guinea-pig. At the substrate concentrations studied, the hydrolysis of methylparaben followed zero-order kinetics. When added to isolated microsomal preparations, paraoxon produced a dose-dependent inhibition of hydrolysis of all substrates. Administration of paraoxon to guinea-pigs prior to isolation of microsomes did not produce consistent effects with any substrate. Inhibition of ester hydrolysis was observed with some pretreatments, while either no change or increased hydrolysis was observed with other pretreatment regimens.

Species differences in the hydrolysis of meperidine and its inhibition by organophosphate compounds.

The hydrolysis of meperidine was assayed in washed, unfortified liver microsomal fractions of guinea pig, rat, mouse, dog, and human, by following substrate disappearance as quantitated by high-performance liquid chromatography. Using the method of Lineweaver-Burk plots, the velocity of the meperidine hydrolysis reaction was not detectable in guinea pig, very low in human, and extremely high in dog. Hydrolysis of p-nitrophenyl acetate was also monitored in liver microsomal preparations from the same animal species, with guinea pig showing greatest hydrolytic activity and rat showing least hydrolytic activity for this substrate. The data in the above two assays suggested that meperidine hydrolysis is mediated by a unique esterase not present in guinea pig and very low in human, but present with high activity in dog liver microsomes. From these comparative studies we concluded that liver microsomes from different species may contain different carboxylesterases having different affinities for meperidine. To further characterize meperidine carboxylesterase of dog and rat liver microsomes, inhibitory studies in vitro with two organophosphate compounds--paraoxon (diethyl-p-nitrophenyl phosphate) and soman (pinacolyl methylphosphonofluoridate)--indicated a varied pattern of enzyme inhibition. These results suggested that liver microsomal carboxylesterases are involved in the metabolism of meperidine and that interference with these enzymes by organophosphate compounds may alter pharmacologic and toxicologic effects of meperidine.

Comparison of digitoxin and its major glucuronidated metabolite: inhibition of Na-K-ATPase and reactivity with the radioimmunoassay.

The metabolism of digitoxin is known to occur through several major pathways including oxidation and glucuronidation. Several studies have compared the behavior of the various unconjugated metabolites with respect to reactivity toward Na-K-ATPase and toward the radioimmunoassay (RIA). Other studies with conjugated products synthesized chemically have also been performed. However, the activity of the conjugated products produced in vivo has not been reported and these metabolites are generally assumed to be inactive. In the present studies we have prepared and purified the glucuronide conjugate of digitoxigenin monodigitoxoside formed by rat liver microsomes and determined the activity of this metabolite as measured by inhibition of Na-K-ATPase and by the RIA. The concentration of the conjugated product needed to cause a fifty per cent inhibition of Na-K-ATPase was 5.40 microM compared to 0.68 for digitoxin and 0.08 for digitoxigenin monodigitoxoside. However, the conjugated product had a two-fold greater affinity for the antibody in the RIA procedure than did either digitoxin or digitoxigenin monodigitoxoside. Although these data cannot be extrapolated to the effects of these drugs on cardiac contractility, the results suggest that the contribution of the glucuronide conjugate to the therapeutic or toxic effect of digitoxin may be minimal. This metabolite may, however, lead to inaccurate estimation of blood levels by the RIA.

Measurement of vitamin E in serum and plasma by high performance liquid chromatography with electrochemical detection.

A deficiency of vitamin E has been associated with a wide variety of pathological conditions, many of them involving neonates. Previously published methods for the quantitation of vitamin E require large sample volumes as well as extensive extraction and concentration procedures. The present work describes a method for the quantitation of vitamin E in small volumes (less than or equal to 50 microliters) of serum or plasma without the need for extraction or concentration of the sample. With further optimization, even smaller volumes (less than or equal to 10 microliters) can be employed. Sample preparation consisted of precipitation of plasma proteins with absolute ethanol. After centrifugation, a small volume (10 to 50 microliters) of the supernatant was injected directly onto the high performance liquid chromatography (HPLC) column. A dual channel electrochemical detector was used for quantitation of the vitamin E. Pooled serum or plasma was used to determine within-day (8.43 +/- 0.19 mg/L) and between-day (8.49 +/- 0.34 mg/L) precision. Analytical recovery of added vitamin E (4-16 mg/L) averaged 102 +/- 7.0%. No interfering peaks were observed. Linearity was demonstrated over 0.5 to 800 ng. Vitamin E acetate is not detected by this method. HPLC with electrochemical detection is a highly sensitive and specific method for the quantitation of vitamin E in plasma or serum. This procedure is rapid and simple, and can be performed with very small sample volumes.

Continuous intravenous infusion of vinca alkaloid using a subcutaneously implanted pump in a canine model.

A major drawback of infusions of the vinca alkaloids is the lengthy period of hospitalization which is often required for this novel technique of cancer therapy. A potentially useful system to deliver outpatient therapy has been investigated in a preclinical study. A self-contained infusion pump powered by a self-charging fluorocarbon system has been implanted SC in three dogs. The performance of two pumps which had been factory-calibrated to deliver 2.5 and 4.5 ml/day, respectively, was evaluated during 22 infusions of the vinca alkaloids (vincristine, 7; vinblastine, 7; and vindesine, 8). Infusions were given over a 5- to 7-day period and were repeated at 3-week intervals. No malfunctioning of the pumps occurred in over 500 cumulative days of use. The flow rates of the pumps were quite stable except in one animal whose increased flow rate was probably a consequence of fever due to self-induced inflammation about the pump pocket. No local or distant tissue reactions to the pump were observed. Decomposition of vincristine and vinblastine in the infusate at the end of 5- or 7-day infusions was minimal as determined by high-pressure liquid chromatography. The amount of decomposition of vindesine in the infusate was variable. Steady-state concentrations of vincristine during infusion were always greater than 10(-9) M, and were similar to those previously determined in our clinical infusion trials using a dosage of 0.5 mg/m2/day. Clinical evaluation of this system for prolonged infusions of vincristine and other vinca alkaloids appears to be warranted.

Intravenous vincristine infusion: phase I trial.

In an attempt to sustain potentially cytotoxic concentrations of vincristine in man, a five-day continuous infusion of vincristine after an initial intravenous bolus injection was administered to 30 patients with refractory malignancies. Three dosage levels were explored (0.5 mg/m2, 0.75 mg/m2, and 1.0 mg/m2 daily for five days). Neurologic and hematologic toxicity were severe at the high dose level, whereas mild to moderate toxicity occurred at the 0.5 and 0.75 mg/m2 dose levels. Objective responses were noted in 11 patients (37%) with the following malignancies: non-Hodgkin's lymphoma (4), acute non-lymphoblastic leukemia (2), chronic granulocytic leukemia in blast crisis (1), carcinoma of the breast (3), and small cell carcinoma of the lung (1). Responses were observed at each infusion dose level. Nine of the 11 responders had previously progressed while receiving conventional intravenous bolus injection of vincristine. These data suggest that the clinical usefulness of vincristine may be enhanced by the use of infusion techniques. A maximum daily dose of 0.5 mg/m2 given for five days is recommended for future trials of intravenous vincristine infusion.

Pharmacokinetics of vincristine infusion.

Using a sensitive radioimmunoassay, serial blood concentrations of vincristine were measured in 11 patients with refractory malignancies receiving infusions of vincristine at three dose levels: 0.5, 0.75, and 1.0 mg/m2 daily for 5 days. The pharmacokinetics of vincristine infusion were compared to pharmacologic data obtained from four patients who received conventional iv bolus injections of 2 mg of vincristine. Whereas rapid decline of blood levels was seen following iv bolus injection, with concentrations of vincristine approaching 10(-9) M by 48-72 hours, vincristine infusions of 0.5, 0.75, and 1.0 mg/m2 daily for 5 days consistently resulted in blood concentrations greater than 10(-9) M during the treatment period. Similarly, areas under the concentration curve were greater in patients receiving infusions compared to bolus injections of vincristine. Antitumor responses in patients receiving vincristine infusions were observed after failure to respond to iv bolus injections. These data demonstrate the ability of infusion therapy to sustain blood concentrations of vincristine in man beyond that seen with conventional administration and suggest the possibility of improved therapeutic efficacy with this agent by use of infusion techniques.

Pharmacokinetics of vincristine in the cerebrospinal fluid of subhuman primates.

Adult rhesus monkeys were given 2-mg/sq m i.v. bolus injections of radiochemically pure tritiated vincristine (VCR). Simultaneous specimens of blood and cerebrospinal fluid (CSF) were sampled from 5 min to 72 hr following injection. CSF was obtained from Ommaya reservoirs which had been implanted s.c. in each monkey. VCR and its metabolic and/or decomposition products rapidly entered the CSF producing low concentrations in this fluid; 5 min following injection, the concentration of VCR was 2.3 nM, whereas approximately a 100-fold greater concentration (203.8 nM) was attained in the plasma. Throughout a 3-day period of observation, constant, low levels of VCR and its metabolic and/or decomposition products (2.3 to 5.7 nM) were maintained in the CSF. The principal form of VCR present in the CSF 1 hr or more after i.v. injection was a water-soluble metabolite and/or decomposition product(s). Although the levels achieved in the CSF are unlikely to be lethal to tumor cells, the finding of persistent low concentrations of VCR and its metabolic and/or decomposition products in the CSF indicates prolonged exposure of neural tissue which might result in neurotoxicity, particularly cumulative neurotoxicity following multiple doses of VCR.

Biliary excretion of vincristine.

A patient with pancreatic carcinoma and a choledochal T tube was given tritiated vincristine sulfate ([3H]-VCR) intravenously. Peak biliary excretion occurred in 2 to 4 hr and this sample contained 9.7% of the injected radioactivity. The first 24-hr bile sample contained 21.7% of the dose and 76.4% of the cumulative 72-hr biliary excretion. During a 3-day period of observation, 4.2%, 45.6%, and 49.6% of the excreted radiolabel was present in the feces, urine, and bile, respectively. Products of VCR metabolism and decomposition appeared in the bile rapidly; only 46.5% of the drug was present in the parent form in the 2-hr collection. Since significant amounts of these products were also identified in control specimens of bile, blood, plasma, and buffer alone after brief periods of incubation, the origin and nature of the species appearing in the bile remain unclear. Observing the fecal route to be the major source of elimination of radiolabel following intravenous injection of [3H]-VCR in our patients previously, we now conclude the biliary system to be the principal route of excretion of VCR and its products. Hepatic dysfunction might therefore alter elimination kinetics and increase the exposure to VCR and its products which might augment toxicity and require dose modification.

The pharmacokinetics of [3H]-vincristine in man.

The pharmacokinetics, metabolism, and excretion of aromatically labeled tritiated vincristine (VCR) was examined in 4 patients. Clearance of radioactivity from the blood was triphasic with half-life t1/2 values of 0.85, 7.4, and 164 min. The initial phases probably represent distribution and binding to formed blood elements which exceeded 50% of the administered dose by 20 min. Excretion of radioactivity was principally fecal, with 33% recovered in the feces by 24 hr and 69% by 72 hr. Considerably less radioactivity (12%) was excreted in the urine over the 72-hr period. Approximately 40% of fecally excreted and 46% of urinary excreted radiolabel represented metabolites, which suggests that at least 34% of the VCR dose was excreted as metabolies. Plasma metabolites represented from less than 1% to 30% or more of radioactivity in plasma. Ultraviolet spectral analysis of all metabolites revealed preservation of the intact VCR dimer, which suggests that metabolism involves alteration of side groups.