Power spectrum analysis of EEG in a translational nonhuman primate model after chronic exposure to low levels of the common marine neurotoxin, domoic acid.

Domoic acid (DA), the focus of this research, is a marine algal neurotoxin and epileptogen produced by species in the genus Pseudo-nitzschia. DA is found in finfish and shellfish across the globe. The current regulatory limit for DA consumption (20 ppm in shellfish) was set to protect humans from acute toxic effects, but there is a growing body of evidence suggesting that regular consumption of DA contaminated seafood at or below the regulatory limit may lead to subtle neurological effects in adults. The present research uses a translational nonhuman primate model to assess neurophysiological changes after chronic exposure to DA near the regulatory limit. Sedated electroencephalography (EEG) was used in 20 healthy adult female Macaca fascicularis, orally administered 0.075 and 0.15 mg DA/kg/day for at least 10 months. Paired video and EEG recordings were cleaned and a Fast Fourier Transformation was applied to EEG recordings to assess power differences in frequency bands from 1-20 Hz. When DA exposed animals were compared to controls, power was significantly decreased in the delta band (1-4 Hz, p < 0.005) and significantly increased in the alpha band (5-8 Hz, p < 0.005), theta band (9-12 Hz, p < 0.01), and beta band (13-20 Hz, p < 0.05). The power differences were not dose dependent or related to the duration of DA exposure, or subtle clinical symptoms of DA exposure (intentional tremors). Alterations of power in these bands have been associated with a host of clinical symptoms, such as deficits in memory and neurodegenerative diseases, and ultimately provide new insight into the subclinical toxicity of chronic, low-dose DA exposure on the adult primate brain.

Isotretinoin administration improves sperm production in men with infertility from oligoasthenozoospermia: a pilot study.

There is currently no effective medical therapy for men with infertility due to oligoasthenozoospermia. As men with abnormal sperm production have lower concentrations of 13-cis-retinoic acid in their testes, we hypothesized that men with infertility from oligoasthenozoospermia might have improved sperm counts when treated with isotretinoin (13-cis-retinoic acid). We conducted a single-site, single-arm, pilot study to determine the effect of therapy with isotretinoin on sperm indices in 19 infertile men with oligoasthenozoospermia. Subjects were men between 21 and 60 years of age with infertility for longer than 12 months associated with sperm concentrations below 15 million sperm/mL. All men received isotretinoin 20 mg by mouth twice daily for 20 weeks. Subjects had semen analyses, physical examinations, and laboratory tests every 4 weeks during treatment. Nineteen men enrolled in the study. Median (25th, 75th) sperm concentration increased from 2.5 (0.1, 5.9) million/mL at baseline to 3.8 (2.1, 13.0) million/mL at the end of treatment (p = 0.006). No significant changes in sperm motility were observed. There was a trend toward improved sperm morphology (p = 0.056). Six pregnancies (three spontaneous and three from intracytoplasmic sperm injection) and five births occurred during the study. Four of the births, including all three of the spontaneous pregnancies, were observed in men with improvements in sperm counts with isotretinoin therapy. Treatment was well tolerated. Isotretinoin therapy improves sperm production in some men with oligoasthenozoospermia. Additional studies of isotretinoin in men with infertility from oligoasthenozoospermia are warranted.

Accelerated Skeletal Maturation in Disorders of Retinoic Acid Metabolism: A Case Report and Focused Review of the Literature.

Nutritional excess of vitamin A, a precursor for retinoic acid (RA), causes premature epiphyseal fusion, craniosynostosis, and light-dependent retinopathy. Similarly, homozygous loss-of-function mutations in CYP26B1, one of the major RA-metabolizing enzymes, cause advanced bone age, premature epiphyseal fusion, and craniosynostosis. In this paper, a patient with markedly accelerated skeletal and dental development, retinal scarring, and autism-spectrum disease is presented and the role of retinoic acid in longitudinal bone growth and skeletal maturation is reviewed. Genetic studies were carried out using SNP array and exome sequencing. RA isomers were measured in the patient, family members, and in 18 age-matched healthy children using high-performance liquid chromatography coupled to tandem mass spectrometry. A genomic SNP array identified a novel 8.3 megabase microdeletion on chromosome 10q23.2-23.33. The 79 deleted genes included CYP26A1 and C1, both major RA-metabolizing enzymes. Exome sequencing did not detect any variants that were predicted to be deleterious in the remaining alleles of these genes or other known retinoic acid-metabolizing enzymes. The patient exhibited elevated plasma total RA (16.5 vs. 12.6±1.5 nM, mean±SD, subject vs. controls) and 13-cisRA (10.7 nM vs. 6.1±1.1). The findings support the hypothesis that elevated RA concentrations accelerate bone and dental maturation in humans. CYP26A1 and C1 haploinsufficiency may contribute to the elevated retinoic acid concentrations and clinical findings of the patient, although this phenotype has not been reported in other patients with similar deletions, suggesting that other unknown genetic or environmental factors may also contribute.

Chronic Kidney Disease Alters Vitamin A Homeostasis via Effects on Hepatic RBP4 Protein Expression and Metabolic Enzymes.

Vitamin A, via retinoic acid (RA), is a critical micronutrient. Normally, plasma concentrations are tightly regulated. Concentrations of vitamin A metabolites (13cis-RA, atRA) and relationships between RBP4 and retinoids have never been fully evaluated in adult patients with CKD. We measured retinoid and RBP4 concentrations in plasma and urine from 55 adult patients with CKD and 21 matched healthy subjects. RBP4 and retinol levels were increased approximately twofold in patients with CKD, with a negative correlation between plasma retinol and eGFR (p = 0.006) and plasma RBP4 and eGFR (p = 0.0007). RBP4 renal clearance was higher in patients with CKD than healthy subjects but not associated with eGFR. Circulating concentrations of atRA increased and concentrations of 13cis-RA decreased in subjects with CKD with no change in RA-to-retinol ratio. Increases in circulating retinol, RBP4, and atRA may be due to increased hepatic RBP4 synthesis, retinyl ester hydrolysis, and/or hepatic secretion of RBP4-retinol.

Interindividual variability of CYP2C19-catalyzed drug metabolism due to differences in gene diplotypes and cytochrome P450 oxidoreductase content.

Large interindividual variability has been observed in the metabolism of CYP2C19 substrates in vivo. The study aimed to evaluate sources of this variability in CYP2C19 activity, focusing on CYP2C19 diplotypes and the cytochrome P450 oxidoreductase (POR). CYP2C19 gene analysis was carried out on 347 human liver samples. CYP2C19 activity assayed using human liver microsomes confirmed a significant a priori predicted rank order for (S)-mephenytoin hydroxylase activity of CYP2C19*17/*17 > *1B/*17 > *1B/*1B > *2A/*17 > *1B/*2A > *2A/*2A diplotypes. In a multivariate analysis, the CYP2C19*2A allele and POR protein content were associated with CYP2C19 activity. Further analysis indicated a strong effect of the CYP2C19*2A, but not the *17, allele on both metabolic steps in the conversion of clopidogrel to its active metabolite. The present study demonstrates that interindividual variability in CYP2C19 activity is due to differences in both CYP2C19 protein content associated with gene diplotypes and the POR concentration.The Pharmacogenomics Journal advance online publication, 1 September 2015; doi:10.1038/tpj.2015.58.

Fluoxetine- and norfluoxetine-mediated complex drug-drug interactions: in vitro to in vivo correlation of effects on CYP2D6, CYP2C19, and CYP3A4.

Fluoxetine and its circulating metabolite norfluoxetine comprise a complex multiple-inhibitor system that causes reversible or time-dependent inhibition of the cytochrome P450 (CYP) family members CYP2D6, CYP3A4, and CYP2C19 in vitro. Although significant inhibition of all three enzymes in vivo was predicted, the areas under the concentration-time curve (AUCs) for midazolam and lovastatin were unaffected by 2-week dosing of fluoxetine, whereas the AUCs of dextromethorphan and omeprazole were increased by 27- and 7.1-fold, respectively. This observed discrepancy between in vitro risk assessment and in vivo drug-drug interaction (DDI) profile was rationalized by time-varying dynamic pharmacokinetic models that incorporated circulating concentrations of fluoxetine and norfluoxetine enantiomers, mutual inhibitor-inhibitor interactions, and CYP3A4 induction. The dynamic models predicted all DDIs with less than twofold error. This study demonstrates that complex DDIs that involve multiple mechanisms, pathways, and inhibitors with their metabolites can be predicted and rationalized via characterization of all the inhibitory species in vitro.

Intratesticular 13-cis retinoic acid is lower in men with abnormal semen analyses: a pilot study.

Intratesticular retinoic acid is necessary for spermatogenesis, but the relationship between intratesticular retinoic acid and sperm quality in man has not been studied. We hypothesized that intratesticular concentrations of retinoic acid would be lower in men with abnormal semen analyses compared to men with normal semen analyses. We recruited men requiring scrotal or penile surgery in a pilot observational study examining the relationship between sperm quality and intratesticular and serum retinoic acid. Twenty-four men provided two pre-operative blood and semen samples, and underwent a testicular biopsy during surgery. Serum and tissue all-trans and 13-cis retinoic acid and reproductive hormones were measured by LC/MS/MS and radioimmunoassays, respectively. Seven men had abnormal semen analyses by at least one WHO criteria and 17 men were normal. In men with abnormal semen, the median (25th, 75th percentile) intratesticular 13-cis retinoic acid was 0.14 (0.08, 0.25) pmol/gram tissue compared with 0.26 (0.18, 0.38) pmol/gram tissue in men with normal semen (p = 0.04). There were no significant differences in intratesticular all-trans retinoic acid or serum reproductive hormones between men with normal and abnormal semen analyses. Intratesticular 13-cis retinoic acid is significantly lower in men with abnormal semen analyses compared to men with normal semen analyses. Lower intratesticular 13-cis retinoic acid concentrations may be due to decreased biosynthesis or increased metabolism in the testes. Further investigation of the relationship between intratesticular 13-cis retinoic acid and poor sperm quality is warranted to determine if this association is present in infertile men.

Evidence of CYP3A allosterism in vivo: analysis of interaction between fluconazole and midazolam.

The allosteric effect of fluconazole (effector) on the formation of 1'-hydroxymidazolam (1'-OH-MDZ) and 4-hydroxymidazolam (4-OH-MDZ) from midazolam (MDZ), a substrate of CYP3A4/5--members of the cytochrome P450 superfamily of enzymes--was examined in healthy volunteers. Following pretreatment with fluconazole, the ratio of the areas under the curve (AUCs) for 4-OH-MDZ and MDZ (AUC(4-OH)/AUC(MDZ)) increased by 35-62%, whereas the ratio AUC(1'-OH)/AUC(MDZ) decreased by 5-37%; the ratio AUC(1'-OH)/AUC(4-OH) decreased by 46-58% after fluconazole administration and had no association with the CYP3A5 genotype. The in vitro formation of 1'-OH-MDZ was more susceptible to inhibition by fluconazole than that of 4-OH-MDZ. Fluconazole decreased the intrinsic formation-clearance ratio of 1'-OH-MDZ/4-OH-MDZ to an extent that was quantitatively comparable to in vivo observations. The elimination clearance of MDZ metabolites appeared unaffected by fluconazole. This study demonstrated that fluconazole alters formation of MDZ metabolites, both in vivo and in vitro, in a manner consistent with an allosteric interaction. The 1'-OH-MDZ/4-OH-MDZ ratio may serve as a biomarker of such interactions among MDZ, CYP3A4/5, and other putative effectors.

Evaluation of 6ß-hydroxycortisol, 6ß-hydroxycortisone, and a combination of the two as endogenous probes for inhibition of CYP3A4 in vivo.

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6ß-hydroxycortisol and 6ß-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6ß-hydroxycortisol and 6ß-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6ß-hydroxycortisone and 6ß-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6ß-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6ß-hydroxycortisone and 6ß-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.

Are circulating metabolites important in drug-drug interactions?: Quantitative analysis of risk prediction and inhibitory potency.

The potential of metabolites to contribute to drug-drug interactions (DDIs) is not well defined. The aim of this study was to determine the quantitative role of circulating metabolites in inhibitory DDIs in vivo. The area under the plasma concentration-time curve (AUC) data related to at least one circulating metabolite was available for 71% of the 102 inhibitor drugs identified. Of the 80 metabolites characterized at steady state, 78% had AUCs >10% of that of the parent drug. A comparison of the inhibitor concentration/inhibition constant ([I]/K(i)) ratios of metabolites and the respective parent drugs showed that 17 of the 21 (80%) reversible inhibitors studied had metabolites that were likely to contribute to in vivo DDIs, with some metabolites predicted to have inhibitory effects greater than those of the parent drug. The in vivo drug interaction risks associated with amiodarone, bupropion, and sertraline could be identified from in vitro data only, when data pertaining to metabolites were included in the predictions. In conclusion, cytochrome P450 (CYP) inhibitors often have circulating metabolites that contribute to clinically observed CYP inhibition.

Accurate prediction of dose-dependent CYP3A4 inhibition by itraconazole and its metabolites from in vitro inhibition data.

Inhibitory drug metabolites may contribute to drug-drug interactions (DDIs). The aim of this study was to determine the importance of inhibitory metabolites of itraconazole (ITZ) in in vivo cytochrome P450 (CYP) 3A4 inhibition. The pharmacokinetics of ITZ and midazolam (MDZ) were determined in six healthy volunteers in four sessions after administration of MDZ with and without oral ITZ. After doses of 50, 200, and 400 mg of ITZ, the clearance of orally administered MDZ decreased by 27, 74, and 83%, respectively. The in vivo half maximal inhibitory concentration (IC(50)) for ITZ ranged from 5 to 132 nmol/l in the six subjects. The metabolites of ITZ were estimated to account for ~50% of the total CYP3A4 inhibition, with the relative contribution increasing with time after ITZ dosing. Of the total of 18 interactions observed, 15 (84%) could be predicted within a twofold error margin, with improved accuracy observed when ITZ metabolites were included in the predictions. This study shows that the metabolites of ITZ contribute to CYP3A4 inhibition and need to be accounted for in quantitative rationalization of ITZ-mediated DDIs.

Contribution of itraconazole metabolites to inhibition of CYP3A4 in vivo.

Itraconazole (ITZ) is metabolized in vitro to three inhibitory metabolites: hydroxy-itraconazole (OH-ITZ), keto-itraconazole (keto-ITZ), and N-desalkyl-itraconazole (ND-ITZ). The goal of this study was to determine the contribution of these metabolites to drug-drug interactions caused by ITZ. Six healthy volunteers received 100 mg ITZ orally for 7 days, and pharmacokinetic analysis was conducted at days 1 and 7 of the study. The extent of CYP3A4 inhibition by ITZ and its metabolites was predicted using this data. ITZ, OH-ITZ, keto-ITZ, and ND-ITZ were detected in plasma samples of all volunteers. A 3.9-fold decrease in the hepatic intrinsic clearance of a CYP3A4 substrate was predicted using the average unbound steady-state concentrations (C(ss,ave,u)) and liver microsomal inhibition constants for ITZ, OH-ITZ, keto-ITZ, and ND-ITZ. Accounting for circulating metabolites of ITZ significantly improved the in vitro to in vivo extrapolation of CYP3A4 inhibition compared to a consideration of ITZ exposure alone.

Influence of CYP3A5 genotype on the pharmacokinetics and pharmacodynamics of the cytochrome P4503A probes alfentanil and midazolam.

The hepatic and first-pass cytochrome P4503A (CYP3A) probe alfentanil (ALF) is also metabolized in vitro by CYP3A5. Human hepatic microsomal ALF metabolism is higher in livers with at least one CYP3A5*1 allele and higher CYP3A5 protein content, compared with CYP3A5*3 homozygotes with little CYP3A5. The influence of CYP3A5 genotype on ALF pharmacokinetics and pharmacodynamics was studied, and compared to midazolam (MDZ), another CYP3A probe. Healthy volunteers (58 men, 41 women) were genotyped for CYP3A5 *1, *3, *6, and *7 alleles. They received intravenous MDZ then ALF, and oral MDZ and ALF the next day. Plasma MDZ and ALF concentrations were determined by mass spectrometry. Dark-adapted pupil diameters were determined coincident with blood sampling. In CYP3A5(*)3/(*)3 (n=62), (*)1/(*)3 (n=28), and (*)1/(*)1 (n=8) genotypes, systemic clearances of ALF were 4.6+/-1.8, 4.8+/-1.7, and 3.9+/-1.7 ml/kg/min and those of MDZ were 7.8+/-2.3, 7.7+/-2.3, and 6.0+/-1.4 ml/kg/min, respectively (not significant), and apparent oral clearances were 11.8+/-7.2, 13.3+/-6.1, and 12.6+/-8.2 ml/kg/min for ALF and 35.2+/-19.0, 36.4+/-15.7, and 29.4+/-9.3 ml/kg/min for MDZ (not significant). Clearances were not different between African Americans (n=25) and Whites (n=68), or between CYP3A5 genotypes within African Americans. ALF pharmacodynamics was not different between CYP3A5 genotypes. There was consistent concordance between ALF and MDZ, in clearances and extraction ratios. Thus, in a relatively large cohort of healthy subjects with constitutive CYP3A activity, CYP3A5 genotype had no effect on the systemic or apparent oral clearances, or pharmacodynamics, of the CYP3A probes ALF and MDZ, despite affecting their hepatic microsomal metabolism.

Pharmacokinetics of gentamicin C1, C1a and C2 in horses after single intravenous dose.

Gentamicin pharmacokinetics has not been studied in horses. Pharmacokinetics of gentamicin C1, C1a and C2 components following i.v. administration of total gentamicin at 6.6 mg/kg bwt to 6 healthy mature horses was determined. Significant differences in clearance, half-life (t 1/2), and mean residence time (MRT) between the gentamicin Cia and the 2 other components were found. The total body clearance (CL) of gentamicin C1a was 1.62 +/- 0.50 ml/min x kg and similar to the glomerular filtration rate (GFR) reported for horses. The CL of gentamicin C1 and C2 were 1.03 +/- 0.08 ml/min x kg and 1.10 +/- 0.15 ml/min x kg, respectively, and significantly slower than that of gentamicin C1a. The values of apparent volume of distribution at steady state were 0.22 +/- 0.05, 0.26 +/- 0.12 and 0.23 +/- 0.05 l/kg for gentamicin C1, C1a and C2, respectively. The MRT values were mean +/- s.d. 3.6 +/- 0.5, 2.7 +/- 0.3 and 3.5 +/- 0.4 h and the t 1/2 values were 3.1 (2.5-4.0), 2.4 (2.0-3.2) and 33 (2.4-4.3) h (harmonic mean and range) for gentamicin C1, C1a and C2, respectively. The MRT and t 1/2 values for gentamicin C1a were significantly shorter than those of gentamicin C1 and C2. It was concluded that the difference in pharmacokinetics between the gentamicin components has potential pharmacological and toxicological implications.

Pharmacokinetics of levetiracetam and its enantiomer (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide in dogs.

PURPOSE: The new antiepileptic drug, levetiracetam (LEV, ucb LO59), is a chiral molecule with one asymmetric carbon atom whose anticonvulsant activity is highly enantioselective. The purpose of this study was to evaluate and compare the pharmacokinetics (PK) of LEV [(S)-alpha-ethyl-2-oxo-pyrrolidine acetamide] and its enantiomer (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide (REV) after i.v. administration to dogs. This is the first time that the pharmacokinetics of both enantiomers has been evaluated. METHODS: Optically pure LEV and REV were synthesized, and 20 mg/kg of individual enantiomers was administered intravenously to six dogs. Plasma and urine samples were collected until 24 h, and the concentrations of LEV and REV were determined by an enantioselective assay. The levels of 2-pyrrolidone-N-butyric acid, an acid metabolite of LEV and REV, were determined by high-performance liquid chromatography (HPLC). The data were used for PK analysis of LEV and REV. RESULTS: LEV and REV had similar mean +/- SD values for clearance; 1.5 +/- 0.3 ml/min/kg and volume of distribution; 0.5 +/- 0.1 L/kg. The half-life (t1/2) and mean residence time (MRT) of REV (t1/2, 4.3 +/- 0.8 h, and MRT, 6.0 +/- 1.1 h) were, however, significantly longer than those of LEV (t1/2, 3.6 +/- 0.8 h, and MRT, 5.0 +/- 1.2 h). The renal clearance and fraction excreted unchanged for LEV and REV were significantly different. CONCLUSIONS: In addition to the enantioselective pharmacodynamics, alpha-ethyl-2-oxo-pyrrolidine acetamide has enantioselective PK. The enantioselectivity was observed in renal clearance. Because REV has more favorable PK in dogs than LEV, the higher antiepileptic potency of LEV is more likely due to intrinsic pharmacodynamic activity rather than to enantioselective PK.

Anticonvulsant profile of valrocemide (TV1901): a new antiepileptic drug.

PURPOSE: We sought to investigate the anticonvulsant activity of the new antiepileptic drug (AED), valrocemide or TV1901 (VGD) in various animal (rodent) models of human epilepsy to determine its anticonvulsant profile and safety margin. METHODS: VGD was administered intraperitoneally to CF no. 1 mice and orally or intraperitoneally to Sprague-Dawley rats. The anticonvulsant activity of VGD was examined in nine different animal models of epilepsy for its ability to block electrically, chemically, or sensorily induced seizures. RESULTS: In mice VGD afforded complete protection against maximal electroshock (MES)-, pentylenetetrazole-, picrotoxin-, and bicuculline-induced seizures and 6-Hz "psychomotor" seizures with median effective dose (ED50) values of 151, 132, 275, 248, and 237 mg/kg, respectively. VGD was also effective in preventing sound-induced seizures in Frings audiogenic-seizure susceptible mice (ED50, 52 mg/kg). The median neurotoxic dose in mice was 332 mg/kg. After oral administration to rats, VGD was active in the MES test, with an ED50 of 73 mg/kg, and the median neurotoxic dose was 1,000 mg/kg. Intraperitoneal administration of 300 mg/kg of VGD to hippocampal kindled Sprague-Dawley rats blocked generalized seizures and shortened the afterdischarge duration significantly. VGD also provided complete protection from focal seizures in the corneally kindled rats (ED50,161 mg/kg). CONCLUSIONS: The results obtained in this study suggest that VGD has a broad spectrum of anticonvulsant activity and promising potential as a new AED.

Enantioselective analysis of levetiracetam and its enantiomer R-alpha-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection.

A gas chromatographic-mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 microM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide in a dog.

Determination of gentamicins C(1), C(1a), and C(2) in plasma and urine by HPLC.

BACKGROUND: Gentamicin is an aminoglycoside antibiotic complex containing gentamicins C(1), C(1a), and C(2). Few methods have been described for analysis of the three gentamicin components separately in biological fluids, and none has been used in pharmacokinetic studies. Determination of the three gentamicins separately may have pharmacokinetic and toxicological implications. The present study describes development of an HPLC method for the analysis of gentamicin C(1), C(1a), and C(2) components in plasma and urine. METHODS: The three components were isolated by preparative chromatography and their identities verified by thin-layer chromatography, HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, and melting point determination. The gentamicins were extracted from the biological matrix by use of Tris buffer and polymer phase solid-phase extraction. Derivatization was carried out in the solid-phase extraction cartridge with 1-fluoro-2, 4-dinitrobenzene. The 2,4-dinitrophenyl derivatives were separated with reversed-phase HPLC and quantified by the ultraviolet absorbance at 365 nm. RESULTS: The detector response was linear from the limit of quantification to 50 mg/L for the individual components. The limit of quantification was 0.07 mg/L for gentamicin C(1) and 0. 1 mg/L for gentamicins C(2) and C(1a). The recovery of the gentamicin components was 72% from plasma and 98% from urine. The method was validated for human and dog plasma and urine. CONCLUSIONS: The method was repeatable and enabled the analysis of gentamicins C(1), C(1a), and C(2) in plasma and urine in concentrations covering the therapeutic range of the drug, thus being suitable for therapeutic drug monitoring and pharmacokinetic studies.

Pharmacokinetics of gentamicin C(1), C(1a), and C(2) in beagles after a single intravenous dose.

The pharmacokinetics of gentamicin C(1), C(2), and C(1a) were studied in six beagles after administration of gentamicin at 4 mg/kg of body weight as a single intravenous bolus dose. Plasma concentrations of the gentamicin components were analyzed with a novel high-performance liquid chromatography method capable of identifying and quantifying each of the components. The pharmacokinetic analysis of the plasma concentration-versus-time data was performed using the noncompartmental approach. The results indicated significant differences in the pharmacokinetic characteristics between the gentamicin components C(1), C(1a), and C(2). The mean residence times of gentamicin C(1), C(1a), and C(2) were 81+/-13, 84+/-12, and 79+/-13 min (mean +/- standard deviation), respectively. The half-lives of the respective components were 64+/-12, 66+/-12 and 63+/-12 min. Clearance (CL) of gentamicin C(1), 4.62+/-0.71 ml min(-1) kg(-1), was significantly higher (P = 0.0156) than CL of gentamicin C(1a), 1.81+/-0.26 ml min(-1) kg(-1), and C(2), 1.82+/-0.25 ml min(-1) kg(-1). Similarly, the volume of distribution at steady state (V(ss)) of gentamicin C(1), 0.36+/-0.04 liter kg(-1), was significantly higher (P = 0.0156) than the V(ss) of gentamicin C(1a), 0.14+/-0.01 liter kg(-1), and C(2), 0.15+/-0.02 liter kg(-1). Tissue binding was considered the most likely cause for the difference. The difference may have clinical and toxicological significance.

Chromatographic methods for analysis of aminoglycoside antibiotics.

Aminoglycosides are antimicrobial agents used frequently in treatment of human and animal diseases caused by aerobic, gram-negative bacteria. Because of the toxicity of these compounds, considerable effort has been attributed to analysis of aminoglycoside content in drug preparations, in serum and urine specimen in therapeutic drug monitoring, and in edible animal tissues in residue control. The present review emphasizes the analytical problems associated with aminoglycoside analysis. Screening methods based on microbiological and immunological procedures were briefly discussed. Gas chromatography and especially high-performance liquid chromatography appeared the most widely used chemical methods for the analysis of these compounds. Due to lack of volatility, chromophore, and hydrophility of aminoglycosides, most methods applied derivatization for enhancement of their chromatographic characteristics. The applicability and advantages of the various derivatization procedures were discussed in detail. A wide variety of detection methods, including mass spectrometry have been used. Packed column separation was generally used for gas chromatographic separation. In liquid chromatography, reversed phase, ion pair, ion exchange, and normal phase separation has been employed. Mass spectrometry, as a detection method, was discussed in detail. Extraction procedures from body fluids and tissues were emphasized. The performance and the operational conditions of the methods were described and detailed information of the data was provided also in table format.