Gopal Chari

Pharmacokinetics of a single dose of morphine in preterm infants during the first week of life

We studied morphine pharmacokinetics after a single intravenous dose of 0.1 mg/kg in 20 newborn infants, who were born at 26 to 40 weeks of gestation and were less than 5 days of age. In the 10 infants whose gestational age was less than or equal to 30 weeks, the mean (+/- SD) distribution half-life was 50 +/- 35 minutes, elimination half-life was 10 +/- 3.7 hours, and clearance was 3.39 +/- 3.28 ml/kg/min; the corresponding values for the three term infants were 19 +/- 8 minutes, 6.7 +/- 4.6 hours, and 15.5 +/- 10 ml/kg/min, respectively. The data suggested a trend of decreasing values for distribution and elimination half-lives with increasing gestation, but a considerable degree of variation was seen. The morphine clearance rate increased as a function of gestational age at a rate of 0.9 ml/kg/min per week of gestation. Between 18% and 22% of the drug was found to be protein bound. Four hours after the dose, the drug level remained greater than or equal to 12 ng/ml in 8 of 10 infants born at greater than or equal to 31 weeks of gestation. In 8 of 10 infants born at less than or equal to 30 weeks of gestation, similar levels were maintained at 8 hours after the initial dose. We conclude that (1) there is a marked degree of variation in morphine pharmacokinetics during the neonatal period, (2) nearly 80% of the intravenously infused drug remains free, which might explain the high sensitivity to morphine in this age group, and (3) during the first week of age, adequate blood levels can be maintained by administration of morphine at 4- to 6-hour intervals in term infants and at less frequent intervals in very premature infants (less than or equal to 30 weeks of gestation).

Morphine metabolism in acutely ill preterm newborn infants

To examine the manner in which morphine is metabolized in acutely ill premature infants, we measured the levels of morphine, morphine-3- and -6-glucuronides, and codeine in timed urine specimens and paired plasma specimens at 4 hours and 24 hours after a single dose of morphine in 16 preterm infants (less than 32 weeks of gestational age). A large amount of unmetabolized morphine was found in the urine in 13 (81.2%) of the 16 infants at 4 hours; in 12 of them, morphine was excreted even at 24 hours. Urinary morphine levels varied greatly; the coefficient of variation was 130% at 4 hours and 118% at 24 hours. Codeine was not found in any of the infants. In 10 (62.5%) of the 16 infants, at least one metabolite was found in either plasma or urine. Plasma and urinary levels of morphine conjugates also varied greatly among these 10 infants (coefficient of variation: 109% to 317%). All six infants (37.5%) who had no metabolites excreted large amounts of unmetabolized morphine in the urine for up to 24 hours. Birth weight, gestational age, postnatal age, systemic blood pressure, and other clinical or physiologic variables did not differ significantly between the 10 infants who had morphine conjugates and the six who did not. We conclude that (1) nearly two thirds of acutely ill preterm infants born at less than 32 weeks of gestational age conjugate morphine; (2) irrespective of their ability to produce morphine conjugates, preterm infants excrete large amounts of morphine unmetabolized, as late as 24 hours after a single dose; (3) morphine handling patterns are highly variable among premature infants, and no obvious factors account for the variability; and (4) such variability in morphine handling in general, and the production of the highly potent morphine-6-glucuronide in particular, could explain the variance in morphine pharmacokinetic measures and in the clinical responses to morphine during the newborn period.

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.

Inhibition of Meconium-Induced Cytokine Expression and Cell Apoptosis by Pretreatment With Captopril

OBJECTIVE. To study whether pretreatment of newborn lungs by captopril inhibits meconium-induced lung injury and inflammatory cytokine expression. DESIGN. Four groups of 2-week-old rabbit pups were used for the study: group 1, saline instilled rabbits; group 2, captopril-pretreated rabbits; group 3, meconium-instilled rabbits; and group 4, captopril-pretreated and then meconium-instilled rabbits. Each group was studied at different time points: 0, 2, 4, 8, and 24 hours after instillation of meconium. Experiments were done at the University of Illinois and Michael Reese Hospital at Chicago. After treatment and instillation of meconium, the right lung was fixed with formalin, and 2-μm slices were obtained for immunohistochemistry. The left lung was used for obtaining of lung lavage and measurement of total proteins (for enzyme-linked immunosorbent assay) and mRNA (for reverse transcription-polymerase chain reaction) purification. RESULTS. We found that meconium induces inflammatory cytokine expression and apoptotic lung cell death. In situ end labeling revealed a dramatic DNA fragmentation in the meconium group, which supports the presence of apoptosis. Using enzyme-linked immunosorbent assay, we demonstrated increase of interleukin 6 and interleukin 8 cytokines in meconium-instilled lungs, which were significantly decreased in captopril-pretreated lungs. Captopril pretreatment also decreased meconium-induced cell death and angiotensinogen expression. We believe this effect is explained by the ability of captopril to decrease processing of ANGEN to angiotensinogen (ANG) I and finally to ANG II. It suggests that captopril inhibits ANG II-induced lung cell apoptosis. CONCLUSION. Our results demonstrate that captopril pretreatment significantly inhibits meconium-induced lung cell death, cytokine, and ANGEN expression in newborn lungs.

High-performance liquid chromatographic determination of morphine and its metabolites in plasma using diode-array detection

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, codeine, normorphine, morphine 3-glucuronide and morphine 6-glucuronide in plasma using a diol column and diode-array detection. Samples were extracted using solid-phase extraction with recoveries in excess of 90%. The limit of determination was 1 ng/ml for morphine, codeine and morphine 3-glucuronide, and 10 ng/ml for normorphine and morphine 6-glucuronide. Inter- and intra-day precision were better than 10%.

Ontogeny of endothelin and its receptors in rat brain

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.

Bile Flow and Composition in Preterm, Term and Infant Baboons

UNLABELLED We studied the maturational changes in bile composition, bile flow and choleretic effects of sodium taurocholate and secretin in preterm (160 +/- 2 days, n = 4, group I), term (184 +/- 2 days, n = 4, group II), 7-day postnatal age (n = 5, group III) and 60-day-old (n = 5, group IV) baboons. The canalicular bile flow was determined by 14C-erythritol clearance. RESULTS Gall bladder volume increased from group I to group III (0.08 +/- 0.06 to 1.06 +/- 0.93 ml). Bile flow increased significantly from group I to IV (0.13 +/- 0.05 to 0.34 +/- 0.07 microliter/min/g liver weight, p < 0.05). This was associated with significant increases in total bile acid excretion (16 +/- 3.6 to 31 +/- 2.5 mEq/l, p < 0.05). The composition of bile also showed maturational changes with increasing postnatal age. Sodium taurocholate and secretin increased the bile flow significantly in all groups. CONCLUSION Data from these studies clearly demonstrate that the bile flow and bile acid excretion is immature in preterm and term baboons when compared to 7- and 60-day-old baboons. The present studies also suggest that baboons can be used as a model to study the postnatal maturation of hepatic excretory function.

Characterization of serine/cysteine protease inhibitor α1‐antitripsin from meconium‐instilled rabbit lungs

We have recently purified from meconium‐instilled rabbit lungs a novel serine proteinase inhibitor, with an apparent molecular mass of 50 kDa, which we assign to be α1‐antitripsin. We hypothesize that serpin may attenuate pulmonary inflammation and improve surfactant function after meconium aspiration. α1‐antitripsin is a member of the proteinase inhibitor (serpin) superfamily and inhibitor of neutrophil elastase, and it can be identified as a member of the family by its amino acid sequence due to the high degree of conserved residues. α1‐antitripsin is synthesized by epithelial cells, macrophages, monocytes, and neutrophils. Deficiency in α1‐antitripsin leads to exposure of lungs to uncontrolled proteolytic attack from neutrophil elastase or other damaging factors culminating in lung destruction and cell apoptosis. We hypothesize that accumulation of α1‐antitripsin in the lungs serves as a predisposed protection against meconium‐induced lung injury. In this paper, we show how this knowledge can lead to the development of novel therapeutic approaches for treatment of MAS.

A high-performance liquid chromatographic procedure for the separation of cocaine and some of its metabolites from acepromazine, ketamine, and atropine from serum

Abstract A high-performance liquid chromatographic procedure for the separation of cocaine and its metabolites from such non-test drugs as acepromazine, ketamine and atropine in serum is described. The method involves the use of a SemiPermeable Surface (SPS) C8 column with a mobile phase constituted of 3.25% tetrahydrofuran and 96.75% 0.0025 M potassium phosphate buffer, v/v, containing 0.0025% triethylamine, the final pH of the mobile phase being 2.75. The flow rate was 0.5 ml/min. A photodiode array detector equipped with a computer software was used to monitor the analyte peaks Retention times for cocaine (COC), benzoylecgonine (BE), benzoylnorecgonine (BN), norcocaine (NOR) and bupivacaine (BV) were 15.5, 7.4, 12.25, 21.0 and 24 minutes, respectively. The non-test drugs ketamine and atropine co-eluted at 10.0 minutes while acepromazine eluted with the solvent front (3.8 min.). The sensitivity of this assay for each of COC, BE, BN and NOR, at a signal to noise (S/N) ratio of greater than 6.0, was 1.0 n...

Cocaine permeability and metabolism in colonic T-84 epithelial cell line.

Abstract We studied the uptake, transport and metabolism of cocaine in human intestine using the colonic T-84 monolayers as a model. T-84 cells were grown in DMEM/Hams F-12 medium containing 6 % newborn calf serum at 37 ° C on 1.0 μm collagen inserts. The cells develop into a polarized monolayer with the apical surface facing the upper chamber and the basolateral surface facing the lower. The monolayers develop a transepithelial resistance of ≥ 600 Ω cm 2 in 7 days. Varying concentrations of cocaine HCl was added in a serum free medium to the luminal side only, and after 30 min and 60 min samples from the luminal and serosal aspect were removed for analysis. Cocaine and its metabolites were measured by G C/MS. Cocaine transport across T-84 monolayers increased linearly with increasing concentration of cocaine, with no significant difference between 30 min and 60 min of exposure. Transepithelial resistance did not change even at 800 ng of cocaine suggesting no effect on monolayer viability. The metabolites, benzoylecgonine (BE) and ecgonine methyl ester (EME) but not norcocaine were detected in both luminal and serosal side. The concentrations of BE and EME in the luminal side were significantly higher than in the serosal. Combined recovery of cocaine, BE and EME from luminal and serosal sides were 52 – 80 % of total added cocaine. While fresh medium did not metabolize cocaine, media previously exposed to the monolayer (cell-free medium) caused a significant breakdown into BE and EME, suggesting that esterases may be released into the medium. These results indicate transfer of cocaine across this model of intestinal epithelial cell line is by simple diffusion and is concentration dependent. These studies imply that cocaine in swallowed amniotic fluid can be absorbed by the fetal gastrointestinal tract.