Catherine A. White

Age, Dose, and Time-Dependency of Plasma and Tissue Distribution of Deltamethrin in Immature Rats

The major objective of this project was to characterize the systemic disposition of the pyrethroid, deltamethrin (DLT), in immature rats, with emphasis on the age dependence of target organ (brain) dosimetry. Postnatal day (PND) 10, 21, and 40 male Sprague-Dawley rats received 0.4, 2, or 10 mg DLT/kg by gavage in glycerol formal. Serial plasma, brain, fat, liver, and skeletal muscle samples were collected for up to 510 h and analyzed for DLT and/or 3-phenoxybenzoic acid (PBA) content by high-performance liquid chromatography. Toxicokinetic data from previous experiments of the same design with young adult (PND 90) rats (Kim, K.-B., Anand, S. S., Kim, H. J., White, C. A., and Bruckner, J. V. [2008]. Toxicokinetics and tissue distribution of deltamethrin in adult Sprague-Dawley rats. Toxicol. Sci. 101, 197-205) were used to compare to immature rat data. Plasma and tissue DLT levels were inversely related to age. Preweanlings and weanlings showed markedly elevated brain concentrations and pronounced salivation, tremors, choreoathetosis, and eventual fatalities. Plasma DLT levels did not reliably reflect brain levels over time. Plasma:brain ratios were time and dose dependent, but apparently not age dependent. Brain levels were better correlated with the magnitude of salivation and tremors than plasma levels. Hepatic intrinsic clearance of DLT progressively increased during maturation, as did the hepatic extraction ratio. Thus, limited capacity to metabolically inactivate DLT appeared primarily responsible for the inordinately high target organ doses and acute neurotoxicity in pups and weanling rats. Hepatic blood flow was not rate limiting in any age group. Limited DLT hydrolysis was manifest in vivo in the pups by relatively low plasma PBA levels. Elevated exposure of the immature brain to a pyrethroid may prove to be of consequence for long-term, as well as short-term neurotoxicity.

Determination of acyclovir in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography.

Acyclovir [9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV] is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C(18) solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C(8) column (150 x 2.1 mm, 5 microm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer-3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid-acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25-100 microg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R(2)>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.

Pharmacokinetics of Intravenous Acyclovir, Zidovudine, and Acyclovir-Zidovudine in Pregnant Rats

ABSTRACT The pharmacokinetics and placental transfer of acyclovir and zidovudine monotherapies and acyclovir-zidovudine combination therapy were compared in the pregnant rat. Timed-pregnancy Sprague-Dawley rats were used for the study. Doses of 60 mg of each drug/kg of body weight in monotherapy and in combination therapy were given by intravenous bolus, and samples of maternal plasma, amniotic fluid, fetal tissue, and placental tissue were collected over a period of 8 h postdose. Concentrations of each drug in the various matrices were measured by high-performance liquid chromatography. All data were analyzed by using WinNonlin. A one-compartment model with first-order elimination was used to fit the AZT plasma data from the combination therapy rats, but the plasma data from the other groups were fit to a two-compartment model. Tissue data were analyzed by noncompartmental analysis to generate area-under-the-concentration-time-curve values. Implementation of the combination therapy altered the pharmacokinetics of each drug compared to its monotherapy pharmacokinetics. The combination of these two drugs may potentiate fetal and amniotic fluid exposures to each drug.

High-performance liquid chromatographic assay of cefazolin in rat tissues.

A rapid, sensitive and reproducible high-performance liquid chromatographic (HPLC) assay for cefazolin in rat tissues was developed. Tissue samples were homogenized in distilled water, acidified with 8.5% phosphoric acid, and centrifuged. Cefazolin was isolated from the supernatant by solid-phase extraction on C18 cartridges. The eluate containing cefazolin and internal standard, cephalexin, was injected onto a reversed-phase C18 column and eluted with a mobile phase of 23% methanol in 0.02 M sodium phosphate monobasic (pH 5.0) and detected with UV absorbance at 270 nm. Recoveries of cefazolin were 33.7 +/- 2.5%, 45.4 +/- 2.1%, and 42.9 +/- 1.0% from liver, spleen and lung, respectively. The calibration curves for cefazolin were established at 0.5-1500 micrograms/g in spleen, 0.1-250 micrograms/g in liver and 0.1-75 micrograms/g in lung. The assay was reproducible with within-day and between-day variations of 1-2 and 1-4%, respectively. Application of the assay for tissue distribution of cefazolin in liposomal targeting study was demonstrated.

Bioavailability of a novel midazolam gel after intranasal administration in dogs

OBJECTIVE To compare the pharmacokinetics of a novel bioadhesive gel formulation of midazolam after intranasal (IN) administration with that of midazolam solution after IN, IV, and rectal administration to dogs. ANIMALS 10 (5 males and 5 females) healthy adult Beagles. PROCEDURES Dogs were assigned to 4 treatment groups for a crossover study design. Initially, midazolam solution (5 mg/mL) was administered (0.2 mg/kg) IV to group 1, rectally to group 2, and IN to group 3; a 0.4% hydroxypropyl methylcellulose midazolam gel formulation (50 mg/mL) was administered (0.2 mg/kg, IN) to group 4. Each dog received all 4 treatments; there was a 7-day washout period between subsequent treatments. Blood samples were collected before and after midazolam administration. Plasma concentration of midazolam was determined by use of high-performance liquid chromatography. RESULTS The peak plasma concentration after IN administration of the gel formulation was significantly higher than that after IN and rectal administration of the solution. Mean ± SD time to peak concentration was 11.70 ± 2.63 minutes (gel IN), 17.50 ± 2.64 minutes (solution IN), and 39 ± 14.49 minutes (solution rectally). Mean bioavailability of midazolam was 70.4% (gel IN), 52.0% (solution IN), and 49.0% (solution rectally). Bioavailability after IN administration of the gel formulation was significantly higher than that after IN and rectal administration of the solution. CONCLUSIONS AND CLINICAL RELEVANCE IN administration of midazolam gel was superior to both IN and rectal administration of midazolam solution with respect to peak plasma concentration and bioavailability.

Measurement of plasma protein and lipoprotein binding of pyrethroids.

INTRODUCTION A simple, reliable procedure was developed to measure binding of pyrethroid insecticides to total proteins and lipoproteins of rat and human plasma. METHODS The extent of binding of (14)C-labeled deltamethrin (DLM), cis-permethrin (CIS) and trans-permethrin (TRANS) was quantified by a 3-step organic solvent extraction technique. Rat and human plasma samples, containing NaF to inhibit esterases, were spiked with a range of concentrations of each radiolabeled pyrethroid. Protein binding reached equilibrium within ~1h of incubation at 37°C. The samples were extracted in turn with: isooctane to collect the unbound fraction; 2-octanol to extract the lipoprotein-bound fraction; and acetonitrile to obtain the protein-bound fraction. RESULTS Absolute recoveries of DLM, CIS and TRANS ranged from 86 to 95%. Adherence of these very lipophilic chemicals to glass and plastic was minimized by using silanized glass vials and LoBind® plastic pipettes. The methods ability to distinguish lipoprotein from protein binding was confirmed by experiments with diazepam and cyclosporine, drugs that bind selectively to albumin and lipoproteins, respectively. DISCUSSION This procedure was effectively utilized for studies of the species-dependence of plasma protein and lipoprotein binding of three pyrethroids for inclusion in physiologically-based pharmacokinetic models of pyrethroids for use in health risk assessments of the insecticides in children and adults.

Ontogeny of plasma proteins, albumin and binding of diazepam, cyclosporine, and deltamethrin

Background:To characterize the ontogeny of plasma albumin and total proteins, due to the lack of a comprehensive pediatric database. Secondly, to establish the magnitude and duration of maturational changes in binding of highly-bound drugs/chemicals.Methods:Anonymized plasma samples from 296 donors were pooled in 6 age brackets from birth to adolescence. Total protein and albumin levels were measured in each age group, as was the age-dependency of plasma binding of diazepam (DZP), cyclosporine (CYC), and deltamethrin (DLM), a pyrethroid insecticide.Results:Plasma levels of albumin and total proteins steadily increased for the first 1–3 y of life. Unbound DZP and CYC fractions were elevated three- to fourfold in neonates, but decreased to adult levels after 1 and 3 y, respectively. Unbound DLM levels exceeded those in adults for just 1 mo.Conclusion:Neonates and infants under 1–3 y may be at risk from increased amounts of free drug, when given standard doses of some highly-bound drugs. Pyrethroid insecticides might be anticipated to pose increased risk for 1 mo.

HPLC DETERMINATION OF ACYCLOVIR AND ZIDOVUDINE IN MATERNAL PLASMA, AMNIOTIC FLUID, FETAL, AND PLACENTAL TISSUES USING ULTRA-VIOLET DETECTION

ABSTRACT A sensitive and reproducible HPLC assay has been developed and validated for the separation and analysis of acyclovir and zidovudine from plasma, amniotic fluid, placental homogenate, and fetal homogenate. Acyclovir (9-[(2-hydroxyethoxy)-methyl]-guanosine, ACV) is the oldest and most widely used compound to treat episodes of genital herpes (herpes simplex virus-2, HSV-2). Zidovudine (3-azido-3′deoxythymidine, AZT) is the premier reverse transcriptase inhibitor released for the treatment of human immunodeficiency virus (HIV). Both of these drugs have been used in pregnant women to prevent the vertical (mother-to-child) transmission of their respective viruses. This gradient HPLC assay aids in the quantitation of these drugs from the matrices associated with pregnancy (maternal plasma, amniotic fluid, fetal tissue, and placenta). The mobile phase consists of 30 mM acetate/ citrate buffer ( pH 3) and methanol. The plasma and amniotic fluid samples are prepared using a combination of protein precipitation and filtration, while the more complex tissues are prepared with the use of a solid-phase extraction (SPE). The method was validated in the calibration range of 0.1–100 μg/mL and showed precision (%Relative Standard Deviation; %RSD) and accuracy (%Error) of less than 15% for all matrices over three days. The assay was applied to a pharmacokinetic study involving the co-administration of ACV and AZT in the pregnant rat.

Chronokinetics of Active Biliary Ampicillin Secretion in Rats

The existence of temporal variation in biliary excretion has been demonstrated for dibromosulfophthalein and ampicillin (AMP). This study was performed to investigate if the 24 h rhythm of active AMP biliary secretion could be attributed to circadian rhythms in the capacity and/or binding affinity of the active secretion mechanism. In this study, 12 Sprague-Dawley rats, housed under a 12 h light/12 h dark environment, were used. Each rat received four 1 h infusions of incremental doses of AMP during either the active (24:00 group) or rest phase (12:00 group) under pentobarbital anesthesia. High doses of AMP were administered to saturate the biliary secretion of AMP via the anion carrier system. Bile and plasma were collected at steady state for each infusion and analyzed by a microbiological assay. The systemic clearance of AMP was increased approximately twofold during the active phase (24:00 group) compared to the resting phase (12:00 group). Plots of bile excretion rate versus plasma concentration indicated saturation of the anion carrier system. Analysis of the data using the Michaelis-Menten model revealed no significant difference in the binding affinity (1/Km) of the biliary anion carrier system between the 12:00 and 24:00 groups. However, the maximum AMP excretion rate attained in the bile (maximum transport or Vmax) showed a 50% increase during the active phase, thus implicating a day-night variation in transport capacity of the anionic pathway. Therefore, temporal variation in the capacity of the secretory mechanisms is a determinant contributor to the proposed circadian rhythm observed in the biliary elimination of AMP.

PHARMACOKINETIC EVALUATION OF LIPOSOMAL ENCAPSULATED AMPICILLIN IN MALE AND FEMALE RATS

The dispositions of free and liposomal entrapped ampicillin were compared in male and female rats after i.v. administration. Serial blood samples were collected for 2 h in the free drug study and 12 h for the liposomal formulation. Pharmacokinetic parameters obtained with free drug were not significantly different between genders. However, gender significantly influenced the disposition of liposomal encapsulated ampicillin. While no difference was observed in distribution t1/2 between genders, female rats had a shorter MRT, smaller Vss and Vt, and faster clearance as compared to male rats. In a second study, spleen, liver, kidney, heart, and lung were harvested post-injection of free and liposomal entrapped ampicillin. Free ampicillin did not distribute extensively into the tissue compartment and no gender difference was noted. In contrast, liposomal encapsulation resulted in a substantial tissue uptake. In general, female rats had higher concentrations in the spleen and lung as compared to male rats. In vitro plasma stability was not significantly different, suggesting that destabilization of the liposomes does not play a large role in the dispositional differences observed in these studies. However, in vivo interaction of liposomes and plasma lipoproteins may influence the disposition of encapsulated drug.