Michael Cwik

Nitric oxide (NO) is an endogenous anticonvulsant but not a mediator of the increase in cerebral blood flow accompanying bicuculline-induced seizures in rats

Neurons synthesize NO, which may act as a retrograde messenger, involved in either potentiating or depressing neuronal excitability. NO may also play a role in the cerebral vasodilatory response to increased neuronal activity (i.e., seizures). In this study, two questions were asked: (1) is NO an endogenous anticonvulsant or proconvulsant substance? and (2) is the cerebral blood flow (CBF) increase accompanying bicuculline (BC)-induced seizures mediated by NO? The experiments were performed in 300-400-g Wistar rats anesthetized with 0.6% halothane and 70% N2O/30% O2. CBF was measured using the intracarotid 133Xe clearance method or laser-Doppler flowmetry. EEG activity was recorded. Chronic treatment (4 days) with nitro-L-arginine (L-NA), a potent NO synthase (NOS) inhibitor (400 mg/kg total), suppressed brain NOS by > 97% and prolonged seizure duration from 6 +/- 1 (saline-treated controls) to 12 +/- 2 min. In the L-NA-treated group, the CBF increase was sustained as long as seizure activity remained, indicating that CBF was still tightly coupled to seizure activity. Interestingly, the supposed inactive enantiomer of L-NA, D-NA, also showed an inhibition of brain NOS activity, ranging from 87 to 100%. The duration of seizures in this group (average 8 +/- 2 min) corresponded directly to the magnitude of reduction in NOS activity (r = 0.83, P < 0.05). Specifically, the D-NA results indicated that NOS inhibition had to exceed 95% before any effect on seizure duration could be seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Saturable transport of gabapentin at the blood-brain barrier

Gabapentin readily crosses the blood-brain barrier and concentrates in brain tissue via an active transport process believed to be system-L. Blood-brain barrier system-L has a low K(m), making it particularly susceptible to substrate saturation. The purpose of this study was to determine whether the fraction of gabapentin crossing the blood-brain barrier remains constant over a broad range of doses. Using a rat model, microdialysis techniques were employed to determine if fluctuations in gabapentin concentrations in the brain extracellular fluid (ECF) coincided with proportional changes in plasma concentrations. Area under the concentration-time curve was calculated for plasma (AUCplasma) and brain extracellular fluid (AUCECF). The ratios of AUFECF to AUCplasma (AUCratio) and brain extracellular fluid to midpoint plasma gabapentin concentration for each collection interval (Cratio) were determined to provide indicators of the relative (i.e. fractional) amount of gabapentin crossing the blood-brain barrier. Analysis of the association between AUCECF and AUCplasma using linear regression analysis revealed a small, but significant relationship (r = 0.62; p < 0.01). Although higher AUCECF values were obtained with higher AUCplasma values, changes in AUCECF were less than proportional to observed changes in AUCplasma. Blood-brain barrier saturation of gabapentin transport was evident as the AUCratio decreased with increased AUCplasma. Collectively, these results support a trend towards saturation at higher plasma concentrations of the carrier-mediated transport mechanism of gabapentin through the blood-brain barrier.

Intraocular penetration of gentamicin after once-daily aminoglycoside dosing.

Background. Intraocular concentrations—particularly intravitreal concentrations—after systemic administration of gentamicin are poor. Once-daily aminoglycoside dosing of intravenous gentamicin achieves peak serum levels up to five times higher than conventional dosing. Whether these increased serum levels of gentamicin improve the aqueous or vitreous concentrations in humans has not been determined. The authors sought to determine if the intraocular penetration of gentamicin would be improved using this method. Methods. Patients undergoing vitrectomy procedures were administered intravenous gentamicin in a dose of 7 mg/kg approximately 1 hour before surgery. An adjustment in dosing was made for anyone more than 20% over his or her ideal body weight. Aqueous, vitreous, and serum samples were collected before any intraocular surgical manipulation. The samples were analyzed by fluorescence polarization immunoassay (TDx system). Results. The average single gentamicin dose was 498 mg (range, 360–700 mg). The aqueous, vitreous, and serum levels averaged 1.14 μg/mL, 0.41 μg/mL, and 22.07 μg/mL, respectively. No correlation between serum level concentrations and time of administration was found for the aqueous and vitreous levels in this study. Conclusion. Although the average peak serum level of gentamicin was five times higher than previously reported, the vitreous levels averaged only 1.5 times higher. The blood-retinal barrier is difficult to penetrate even when higher serum levels are achieved. Due to its poor ocular penetration, gentamicin may not be among the best drugs for prophylaxis of penetrating eye injuries, surgical prophylaxis, or treatment of endophthalmitis.

Determination of paromomycin in human plasma and urine by reversed-phase high-performance liquid chromatography using 2,4-dinitrofluorobenzene derivatization

A sensitive high-performance liquid chromatographic method for the determination of paromomycin in human plasma and urine was developed. Paromomycin was quantitated following pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB). The chromatographic separation was carried out on a C18 column at 50 degrees C using a mobile phase consisting of 64% methanol in water adjusted to pH 3.0 with phosphoric acid. The eluents were monitored by UV detection at 350 nm. The linearity of response for paromomycin was demonstrated at concentrations from 0.5 to 50 microg/ml in plasma and 1 to 50 microg/ml in urine. The relative standard deviation of the assay procedure is less than 5%.

Determination of capsaicin and zucapsaicin in human serum by high-performance liquid chromatography with fluorescence detection

A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed to analyze capsaicin and zucapsaicin (civamide) in human serum at concentrations from 1 to 100 ng/ml. Human serum specimens were extracted twice with hexane-methyl tert.-butyl ether (1:1). The chromatographic separation was carried out on a C18 column at 40 degrees C using a mobile phase consisting of 40% acetonitrile in water with 5% tetrahydrofuran and 1% acetic acid. The concentration of the eluting compounds was monitored by a fluorescence detector with excitation at 270 nm and an emission cutoff of 300 nm. No interferences were observed from the extract of blank serum. The standard curves were linear in the detection range. The relative standard deviation of the assay was better than 8.4%. The limit of detection was 0.5 ng/ml.

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C18 column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 microg/ml for plasma and 0.03 to 80 microg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 microg/ml for plasma and from 0.02 to 5.0 microg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.

Stability of Fosphenytoin Sodium with Intravenous Solutions in Glass Bottles, Polyvinyl Chloride Bags, and Polypropylene Syringes

OBJECTIVE: To determine the stability of fosphenytoin sodium admixtures with NaCl 0.9% injection and dextrose 5% (D5W) injection when stored in glass or polyvinyl chloride (PVC) containers, to evaluate the compatibility of fosphenytoin with 11 other intravenous solutions, and to determine the stability of fosphenytoin repackaged in polypropylene syringes. METHODS: Dilutions of fosphenytoin sodium 1, 8, and 20 mg phenytoin sodium equivalents (PE)/mL were prepared in NaCl 0.9%, D5W, and 11 other intravenous fluids. Aliquots of each solution in NaCL 0.9% or D5W were transferred to three glass bottles for storage at 25 °C and 21 PVC bags for storage at 25, 4, or −20 °C Aliquots of each admixture with the other intravenous fluids were transferred to three PVC bags and stored at 25 °C for 7 days. In addition, 63 syringes were filled with fosphenytoin sodium 50 mg PE/mL (undiluted) and stored at 25, 4, or −20 °C. Samples of each solution from the three containers were analyzed for visual compatibility, pH, and fosphenytoin concentration initially and at 0.5, 1, 2, 3, 7, 14, and 30 days during storage at 25 and 4 °C and at 1, 7, 14, and 30 days during storage at–20 °C. Following removal of containers from the freezer, additional samples were obtained after 7 days at 4 or 25 °C, and 7 days at 25 °C, and then 7 days at −20 °C. RESULTS: No visible precipitation or change in color or clarity was observed in any of the fosphenytoin solutions during the study. The concentration of fosphenytoin at each sampling time remained within 97–104% of initial concentration, regardless of container, concentration, intravenous admixture, or storage temperature. CONCLUSIONS: Fosphenytoin sodium, either undiluted in polypropylene syringes or diluted with NaCl 0.9% or D5W in PVC bags, remains stable for at least 30 days at room temperature, under refrigeration, or frozen. After removal from the freezer, fosphenytoin can be thawed, kept at 4 or 25 °C for 7 days, and then returned to the freezer for another 7 days. Admixtures of fosphenytoin sodium in various other intravenous fluids are stable for at least 7 days at room temperature.

ALTERATIONS IN THEOPHYLLINE (T) METABOLISM DURING THE FIRST YEAR OF LIFE

Maturational changes in T pharmacokinetics were evaluated in 45 infants; postconceptional age (PCA) 30.9-95.7 wks. After achievement of steady-state on T maintenance therapy, multiple serum and urine samples were obtained over dosing interval and assayed by HPLC for T and metabolites: caffeine (C), 1-methyluric acid (1MU), 3-methylxanthine (3MX), 1,3-dimethyluric acid (1,3MU). Mean(SD) T clearance (Cl) increased significantly (p<0.05; ANOVA) for PCA groups at 30-40, 40-50 and >50 wks from 21.9(6.3) to 26.6(7.7) and 57.7(17.6). ml/hr/kg, respectively. Concomitant decrease (p<0.05) in serum C/T ratio was observed for same PCA groups; 0.43(0.15), 0.22(0.07) and 0.06(0.1), respectively. Significant serum C concentrations (C/T>0.10) were found in some infants up to 55 weeks PCA. Stepwise multiple regression analysis showed urinary excretion of 3MX to be the primary parameter explaining the change in both T Cl (r=0.81, p<0.01) and serum C/T ratio (r=0.66, p<0.01). Urinary excretion of 3MX (demethylated) for the 3 PCA groups was 1.4, 4.2 and 13.1% compared to 23%, 41% and 44% for 1.3MU (oxidative). Disappearance of serum C and maturation of T metabolism is dependent on development of demethylation pathway which does not occur until approximately 55 wks PCA. Prior to that age, serum C concentration should be monitored in patients receiving T.

Quantitative determination of tolazoline in serum and urine

A high-performance liquid chromatographic procedure is described for the analysis of tolazoline in serum and urine. This assay procedure is suitable for the analysis of micro-samples (50 or 100 microliters serum and 100 microliters urine). Samples are extracted in a single step and injected into a reversed-phase high-performance liquid chromatography system for detection at 210 nm. The clinical applicability of this assay is demonstrated by the determination of tolazoline serum and urine concentrations in neonates. In addition, the presence of urine conjugates and the extent of serum protein binding were investigated. This assay procedure has the required sensitivity (0.1 microgram/ml), accuracy and precision for both routine monitoring and pharmacokinetic characterization of tolazoline in neonates and adults.