Stability of pentobarbital in soil.

Intravenous injection of barbiturates, particularly pentobarbital (5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,5-trione), is a widely used method to euthanize large animals such as horses. However, one concern with this method is the fate of pentobarbital after the disposal of the carcass. As tissues decompose, pentobarbital may leach into the soil and from there migrate to groundwater. A method using methanol extraction, solid phase concentration, and liquid chromatography (LC/MS) has been developed to measure pentobarbital in soils. Recovery of pentobarbital from soil averaged approximately 85% from different soil types including topsoil, potting soil, sand, stall sweepings, and loam. The method was capable of detecting pentobarbital levels of 0.1 ppm. A calibration curve was constructed with a linear range of 1 ppm to 100 ppm. The limit of quantification was 0.5 ppm. The rate of degradation of pentobarbital in sand, topsoil, and potting soil was measured over a 17-week period. At the end of week 17, approximately 17% of the pentobarbital remained in the sand, 19% remained in the topsoil, and 10% remained in the potting soil. While there was a significant decrease in the pentobarbital recovered from the soil, there were still detectable amounts of pentobarbital present in the soil after 17 weeks. To determine the importance of bacterial degradation, the three soil types were autoclaved before addition of pentobarbital. After autoclaving, no degradation of pentobarbital was observed in sand and one topsoil sample, while there was no difference in the degradation of pentobarbital in autoclaved potting soil versus potting soil that had not undergone autoclaving.

Application of Hydrogel in Reconstruction Surgery: Hydrogel/Fat Graft Complex Filler for Volume Reconstruction in Critical Sized Muscle Defects.

Autogenic fat graft usually suffers from degeneration and volume shrinkage in volume reconstruction applications. How to maintain graft viability and graft volume is an essential consideration in reconstruction therapies. In this presented investigation, a new fat graft transplantation method was developed aiming to improve long term graft viability and volume reconstruction effect by incorporation of hydrogel. The harvested fat graft is dissociated into small fragments and incorporated into a collagen based hydrogel to form a hydrogel/fat graft complex for volume reconstruction purpose. In vitro results indicate that the collagen based hydrogel can significantly improve the survivability of cells inside isolated graft. In a 6-month investigation on artificial created defect model, this hydrogel/fat graft complex filler has demonstrated the ability of promoting fat pad formation inside the targeted defect area. The newly generated fat pad can cover the whole defect and restore its original dimension in 6-month time point. Compared to simple fat transplantation, this hydrogel/fat graft complex system provides much improvement on long term volume restoration effect against degeneration and volume shrinkage. One notable effect is that there is continuous proliferation of adipose tissue throughout the 6-month period. In summary, the hydrogel/fat graft system presented in this investigation demonstrated a better and more significant effect on volume reconstruction in large sized volume defect than simple fat transplantation.

Calcium and protons affect the interaction of neurotransmitters and anesthetics with anionic lipid membranes.

We study how zwitterionic and anionic biomembrane models interact with neurotransmitters (NTs) and anesthetics (ATs) in the presence of Ca(2+) and different pH conditions. As NTs we used acetylcholine (ACh), γ-aminobutyric acid (GABA), and l-glutamic acid (LGlu). As ATs, tetracaine (TC), and pentobarbital (PB) were employed. By using differential scanning calorimetry (DSC), we analyzed the changes such molecules produce in the thermal properties of the membranes. We found that calcium and pH play important roles in the interactions of NTs and ATs with the anionic lipid membranes. Changes in pH promote deprotonation of the phosphate groups in anionic phospholipids inducing electrostatic interactions between them and NTs; but if Ca(2+) ions are in the system, these act as bridges. Such interactions impact the physical properties of the membranes in a similar manner that anesthetics do. Beyond the usual biochemical approach, we claim that these effects should be taken into account to understand the excitatory-inhibitory orchestrated balance in the nervous system.

Injectable sodium pentobarbital: Stability at room temperature.

INTRODUCTION: Sodium pentobarbital (Nembutal) is a barbiturate used in research as an anesthetic in many animal models. The injectable form of this drug has lately become difficult to procure and prohibitively expensive. Due to this lack of availability, researchers have begun to compound injectable sodium pentobarbital from so-called "nonpharmaceutical" pentobarbital. Some oversight agencies have objected to this practice, claiming a lack of quality control and degradation of the drug. We sought with this study to establish both: 1) a protocol for the preparation of injectable sodium pentobarbital, and 2) standard operating procedures to monitor the quality of the preparation and degradation of the drug over time. METHODS: Our preparation consists of a mixture of sodium pentobarbital in alkaline aqueous solution, propylene glycol, and ethanol. Pentobarbital content in this preparation was assayed by high-pressure liquid chromatography (HPLC). We also assayed pentobarbital content over time in preparations of various ages up to 6 years old. RESULTS: We determined that the drug degraded at a maximum of 0.5% per year in our preparation (alkaline water/propylene glycol/ethanol) when stored in the dark at room temperature. A yellow discoloration developed after about 2 years, which we have arbitrarily determined disqualifies the preparation from use as an anesthetic. Attempts to spectroscopically assay this discoloration were not successful. CHEMICALS: Pentobarbital sodium (CID: 14075609).

Hemodynamic responses elicited by systemic injections of isotonic and hypertonic saline in hemorrhaged rats.

PURPOSE: The objectives of this study were (i) to characterize the hemodynamic responses caused by controlled hemorrhage (HEM) in pentobarbital-anesthetized rats, and (ii) to determine the responses elicited by systemic bolus injections of isotonic saline (0.15M) or hypertonic saline (3M) given 5min after completion of HEM. RESULTS: Controlled HEM (4.3±0.2ml/rat at 1.5ml/min) resulted in a pronounced and sustained fall in mean arterial blood pressure (MAP) to about 40mmHg. The fall in MAP was associated with a reduction in hindquarter vascular resistance (HQR) but no changes in renal (RR) or mesenteric (MR) vascular resistances. Systemic injections of isotonic saline (96-212µmol/kg i.v., in 250-550µl) did not produce immediate responses but promoted the recovery of MAP to levels below pre-HEM values. Systemic injections of hypertonic saline (750-3000µmol/kg, i.v., in 250-550µl) produced immediate and pronounced falls in MAP, RR, MR and especially HQR of 30-120s in duration. However, hypertonic saline prompted a full recovery of MAP, HQR and RR to pre-HEM levels and an increase in MR to levels above pre-HEM values. CONCLUSIONS: This study demonstrates that (i) HEM induced a pronounced fall in MAP which likely involved a fall in cardiac output and HQR, (ii) isotonic saline did not fully normalize MAP, and (iii) hypertonic saline produced dramatic initial responses, and promoted normalization of MAP probably by restoring blood volume and cardiac output through sequestration of fluid from intracellular compartments.

Application of dual cyclodextrin systems in capillary electrophoresis enantioseparations.

The enantioseparation of acidic and neutral compounds can be successfully achieved in capillary electrophoresis (CE) using dual cyclodextrin (CD) systems. This chapter describes how to separate the enantiomers of acidic or neutral substances using dual CD systems made up of a negatively charged CD derivative, i.e., sulfobutyl-ß-CD (SB-ß-CD) or carboxymethyl-ß-CD (CM-ß-CD), in combination with a neutral one, namely, heptakis(2,3,6-tri-O-methyl)-ß-CD (TM-ß-CD). An acidic compound (carprofen) and a weakly acidic drug (pentobarbital) were selected as model compounds.

Structural link between γ-aminobutyric acid type A (GABAA) receptor agonist binding site and inner ß-sheet governs channel activation and allosteric drug modulation.

Rapid opening and closing of pentameric ligand-gated ion channels (pLGICs) regulate information flow throughout the brain. For pLGICs, it is postulated that neurotransmitter-induced movements in the extracellular inner ß-sheet trigger channel activation. Homology modeling reveals that the ß4-ß5 linker physically connects the neurotransmitter binding site to the inner ß-sheet. Inserting 1, 2, 4, and 8 glycines in this region of the GABA(A) receptor ß-subunit progressively decreases GABA activation and converts the competitive antagonist SR-95531 into a partial agonist, demonstrating that this linker is a key element whose length and flexibility are optimized for efficient signal propagation. Insertions in the α- and γ-subunits have little effect on GABA or SR-95531 actions, suggesting that asymmetric motions in the extracellular domain power pLGIC gating. The effects of insertions on allosteric modulator actions, pentobarbital, and benzodiazepines, have different subunit dependences, indicating that modulator-induced signaling is distinct from agonist gating.

Surface hydrophilic modification with a sugar moiety for a uniform-sized polymer molecularly imprinted for phenobarbital in serum.

A uniform-sized polymer molecularly imprinted for phenobarbital, which is surface modified by a sugar moiety, has been prepared through a two-step swelling polymerization method using polystyrene beads as seeds, phenobarbital as the template, 4-vinylpyridine as a functional monomer, ethylene glycol dimethacrylate as a cross-linker and 2-O-meth-acryloyloxyethoxyl-(2,3,4,6- tetra-O-acetyl-ß-d-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-ß-d-glucopyranoside as a surface-modifying glycomonomer, respectively. After deprotecting the glycopolymer, a surface sugar moiety-modified, hydrophilic, molecularly imprinted polymer for phenobarbital (glyco-MIP) was obtained. The resulting polymer beads were packed into a stainless steel column to evaluate their chromatographic characteristics by high-performance liquid chromatography (HPLC). Good selectivity for phenobarbital was obtained with the glyco-MIP compared to the unmodified molecularly imprinted polymer, which revealed that the recognition sites of phenobarbital were unchanged with sugar moiety surface modification. Furthermore, bovine serum albumin was almost completely recovered from the glyco-MIP column, which indicates that the glyco-MIP materials can be used to separate and analyze drugs in complex samples, such as biological samples. The results of pretreatment with and analysis of phenobarbital in serum suggest that this material can be used to analyze phenobarbital in serum through a pretreatment and reverse-phase HPLC analysis process.

Growth, shrinking, and breaking of pluronic micelles in the presence of drugs and/or beta-cyclodextrin, a study by small-angle neutron scattering and fluorescence spectroscopy.

The associative structures between F127 Pluronic micelles and four drugs, namely, lidocaine (LD), pentobarbital sodium salt (PB), sodium naproxen (NP), and sodium salicylate (SAL), were studied by small-angle neutron scattering (SANS). Different outcomes for the micellar aggregates are observed, which are dependent on the chemical nature of the drug and the presence of charge or otherwise: the micelles grow with LD, are hardly modified with PB, and decrease in size with both NP and SAL. The partition coefficient, determined by fluorescence spectroscopy, is directly correlated to the amount of charge, following NP approximately SAL < PB < LD. All drugs are found to lie at the interfacial layer, with a slightly deeper localization of LD and more superficial for PB. All drugs can form inclusion complexes with heptakis(2,6-di-O-methyl) beta-cyclodextrin (hep2,6 beta-CD). Hep2,6 beta-CD, as shown in previous studies (Joseph, J.; Dreiss, C. A.; Cosgrove, T. Langmuir, 2008, 24, 10005-10010; Dreiss, C. A.; Nwabunwanne, E.; Liu, R.; Brooks, N. J. Soft Matter, 2009, 5, 1888-1896), is also able to form a complex with F127, resulting in micellar breakup. In the ternary mixtures, a fine balance of forces is involved, which results in drastic micellar changes, as observed from the SANS patterns. Depending on the ratio of drug, polymer, and hep2,6 beta-CD and the nature of the interactions (which is directly linked to the drug chemical structure), the presence of drug either hinders micellar breakup by beta-CD (at high enough concentration of LD or PB) or leads to micellar growth (NP). These effects are mainly attributed to a preferential drug/beta-CD interaction (except for PB), which, at least in the conditions studied here, explains the higher beta-CD concentration needed for micellar breakup to occur.

Influence of the chirality of (R)-(-)- and (S)-(+)-carvone in the central nervous system: a comparative study.

Many terpenes are used therapeutically, and as flavor and fragrance materials. (R)-(-)-Carvone, the main constituent of spearmint oil, and (S)-(+)-carvone, found as major component of caraway and dill seed oils, have several applications and are used in cosmetic, food, and pharmaceutical preparations. In this study, the effect of enantiomers of carvone on the central nervous system (CNS) was evaluated in mice. The LD50 value was 484.2 mg/kg (358.9-653.2) for (S)-(+)-carvone, and 426.6 (389.0-478.6) mg/kg for (R)-(-)-carvone. Both enantiomers caused depressant effects, such as decrease in the response to the touch and ambulation, increase in sedation, palpebral ptosis, and antinociceptive effects. (S)-(+)- and (R)-(-)-carvone caused a significant decrease in ambulation. (R)-(-)-Carvone appeared to be more effective than its corresponding enantiomer at 0.5 and 2.0 h after administration. However, (S)-(+)-carvone was slightly more potent at 1 h. In potentiating pentobarbital sleeping time, (R)-(-)-carvone was more effective than (S)-(+)-carvone at 100 mg/kg, but was less potent at 200 mg/kg compared to the (+)-enantiomer, indicating a sedative action. (S)-(+)-Carvone at the dose of 200 mg/kg increased significantly the latency of convulsions induced by PTZ and PIC, but (R)-(-)-carvone was not effective against these convulsions. These results suggest that (S)-(+)-carvone and (R)-(-)-carvone have depressant effect in the CNS. (S)-(+)-Carvone appears to have anticonvulsant-like activity.

The physical characteristics of lyophilized tablets containing a model drug in different chemical forms and concentrations.

Orodispersible tablets, usually prepared using freeze-drying method, are becoming a popular drug formulation for patients who have difficulties swallowing solid dosage forms. The influence of drug solubility and concentration on the physical characteristics of lyophilized tablets composed of mannitol and gelatin was investigated. Phenobarbital and phenobarbital sodium were studied as model drugs. The tablets were analyzed for mechanical strength using a new method employing Instron, a material testing apparatus. For tablets containing phenobarbital in the form of sodium salt, better mechanical strength was demonstrated than for tablets prepared with water insoluble phenobarbital--acid form. Besides, the mechanical characteristics of the tablets indicate that plasticity and porosity were reduced when sodium phenobarbital was incorporated at a higher dose. Lyophilized tablets were not hygroscopic and only a small increase of tablet mass by 1% and 3% was observed after 4 weeks of storage at 40% and 60% RH, respectively. All formulations disintegrated in seconds in water, at a temperature of 37 degrees C.

Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene.

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.

Investigation of artificial biomembrane systems in biopartitioning micellar chromatography by method of mathematical design.

An attempt to create and study an artificial membrane system was realized via biopartitioning micellar liquid chromatography. Towards this end the known formula of membrane permeability (on the basis of Fick's diffusion equation) was modified so that membrane permeability may be estimated in terms of chromatographic characteristics. The two-factoral experiments on the basis of mathematical design of second order were carried out. The regression equations are derived which describe the dependence of membrane permeability on the concentration of polyoxyethylene (23) lauryl ether in the mobile phase and its flow-rate for compounds with biomedical significance. Some regularities were revealed, which characterize the permeability of compounds of the different nature through membranes. The extremal dependence (with passing through minimum) of permeability on the concentration of non-ionic surfactant was observed for anionic compounds. The increasing character of permeability in relation with flow-rate of mobile phase was recognized for cationic samples. Both dependences were basically fulfilled for zwitterionic compounds.

The inhibitory effect of pentobarbitone on reverse transcription-PCR.

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.

Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.

Characterization of the stereoselective metabolism of thiopental and its metabolite pentobarbital via analysis of their enantiomers in human plasma by capillary electrophoresis.

Using capillary zone electrophoresis (CZE) with a 75 mM phosphate buffer at pH 8.5 containing 5 mM hydroxypropyl-gamma-cyclodextrin (OHP-gamma-CD) as chiral selector, the separation of the enantiomers of thiopental and its oxybarbiturate metabolite, pentobarbital, is reported. Enantiomer assignment was performed via preparation of enantiomerically enriched fractions using chiral recycling isotachophoresis (rITP) processing of racemic barbiturates and analysis of rITP fractions by chiral CZE and circular dichroism spectroscopy. Thiopental and pentobarbital enantiomers in plasma were extracted at low pH using dichloromethane and extracts were reconstituted in acetonitrile or 10-fold diluted, achiral running buffer. The stereoselectivity of the thiopental and pentobarbital metabolism was assessed via analysis of 12 plasma samples that stemmed from patients undergoing prolonged or having completed long-term racemic thiopental infusion. The data obtained revealed a modest stereoselectivity with R-(+)-thiopental/S-(-)-thiopental and R-(+)-pentobarbital/S-(-)-pentobarbital plasma ratios being < 1 (P < 0.05 compared to data obtained with racemic controls) and > 1 (P < 0.001), respectively. The total S-(-)-thiopental plasma concentration was found to be on average about 24% higher compared to the concentration of R-(+)-thiopental, whereas the total R-(+)-pentobarbital plasma level was observed to be on average 29% higher compared to the S-(-)-pentobarbital concentration.

Capillary electrophoresis stereoselective determination of R-(+)- and S-(-)-pentobarbital from serum using hydroxypropyl-gamma-cyclodextrin, solid-phase extraction and ultraviolet detection.

Pentobarbital is a sedative hypnotic which is used in the treatment of people suffering from anxiety related conditions and to manage elevated intracranial pressures and cerebral ischemia due to neurosurgical procedures. This paper develops and validates a rapid and sensitive method for the determination of S-(-)- and R-(+)-enantiomers in serum using capillary electrophoresis (CE) and UV detection. Stereoselective resolution was accomplished using 40 mM hydroxypropyl-gamma-cyclodextrin contained in 50 mM phosphate buffer pH 9.0. The method involves a solid-phase extraction of both the enantiomers and the internal standard, aprobarbital, from serum using C18 Bond-Elut cartridges. The CE system consists of a 52 cmX75 microm I.D. fused-silica capillary maintained at a run voltage of 15 kV with sample detection performed at 254 nm. The detection and quantitation limits for S-(-)- and R-(+)-pentobarbital are 1 microg/ml from serum. Linear calibration curves from 1 to 60 microg of both S-(-)- and R-(+)-enantiomers show a coefficient of determination of more than 0.999. The precision and accuracy of the method calculated as R.S.D. and error are 0.20-2.20% and 0.00-4.40%, respectively for R-(+)-pentobarbital and 0.30-2.19% and 0.30-6.40%, respectively for S-(-)-pentobarbital.

Do barbiturates and their solutions suppress FMLP-induced neutrophil chemiluminescence?

The dose-response relationship of four commercially available barbiturates (methohexitone, pentobarbitone, phenobarbitone and thiopentone) and of their drug-free solutions on the production of oxygen radicals by neutrophils were tested by N-formylmethionyl-leucyl-phenylalanine (FMLP)-induced granulocyte chemiluminescence and in a cell-free chemiluminescence system. Methohexitone had no effect on neutrophil chemiluminescence. Pentobarbitone, phenobarbitone and thiopentone dose-dependently decreased FMLP-induced chemiluminescence and cell-free chemiluminescence. Suppression of neutrophil chemiluminescence by pentobarbitone and phenobarbitone was due to effects of the drug-free solutions and an osmolality greater than 360 mosmol kg-1. Only thiopentone suppressed granulocyte chemiluminescence drug-specifically. The physicochemical properties of commercially available barbiturate preparations and their solutions, as well as free radical scavenging capacity, have to be considered if these preparations are used to evaluate drug-specific effects on the production of oxygen radicals by neutrophils.