Oral vaccination of dogs with a monovalent live-avirulent vaccine confers 1 year of immunity against Bordetella bronchiseptica challenge.

This experimental challenge study assessed immune protection 1 year after a single dose of live-attenuated oral Bordetella bronchiseptica (Bb) vaccine in dogs. Forty Bb-seronegative 7-9-week-old puppies were randomly assigned at Day 0 to receive a single oral dose of either Bb vaccine (n = 20; vaccinated group) or sterile water (n = 20; control group). Groups were housed separately until comingling 1 day pre-challenge (Day 365). Challenge with virulent aerosolized Bb occurred at Day 366. Clinical scores were obtained at Days 1-7, and 366-380. Bb microagglutination test (MAT) titers were obtained at Days -7, 0, monthly post-vaccination, and Days 358, 365, and 380. Nasal swabs were collected for microbiological assessment at Days -7, 0, 365, and 367-380. Oral Bb vaccination was not associated with side effects. Pre-challenge, vaccinated dogs developed persistent Bb MAT titers and control dogs remained seronegative. Post-challenge, duration of cough was longer in control dogs (least square means [LSM], 8.6 days) than vaccinated dogs (LSM, 1.5 days; P < 0.0001), with more control dogs having cough on 2 or more consecutive days (control group, n = 17/19, 89.5%; vaccinated group, n = 3/19, 15.8%; P = 0.0011). Post-challenge, Bb shedding occurred in all control dogs and 5/19 (26%) vaccinated dogs. Average duration of Bb shedding was longer in the control group (11.9 days vs. 0.6 days; P < 0.0001) and nasal Bb loads were higher in the control group (P < 0.00001). Orally administered Bb vaccine stimulated immunity that was still protective against virulent Bb challenge after 1 year.

Comparative analysis of antibody responses to outer surface protein (Osp)A and OspC in dogs vaccinated with Lyme disease vaccines.

Lyme disease (LD), the most common tick-borne disease of canines and humans in N. America, is caused by the spirochete Borreliella burgdorferi. Subunit and bacterin vaccines are available for the prevention of LD in dogs. LD bacterin vaccines, which are comprised of cell lysates of two strains of B. burgdorferi, contain over 1000 different proteins and cellular constituents. In contrast, subunit vaccines are defined in composition and consist of either outer surface protein (Osp)A or OspA and an OspC chimeritope. In this study, we comparatively assessed antibody responses to OspA and OspC induced by vaccination with all canine bacterin and subunit LD vaccines that are commercially available in North America. Dogs were administered a two-dose series of the vaccine to which they were assigned (3 weeks apart): Subunit-AC, Subunit-A, Bacterin-1, and Bacterin-2. Antibody titers to OspA and OspC were determined by ELISA and the ability of each vaccine to elicit antibodies that recognize diverse OspC proteins (referred to as OspC types) assessed by immunoblot. While all of the vaccines elicited similar OspA antibody responses, only Subunit-AC triggered a robust and broadly cross-reactive antibody response to divergent OspC proteins. The data presented within provide new information regarding vaccination-induced antibody responses to key tick and mammalian phase antigens by both subunit and bacterin LD canine vaccine formulations.

A simple bioanalytical method for the quantification of antiepileptic drugs in dried blood spots.

An increasing number of publications on the dried blood spot (DBS) sampling approach for the quantification of drugs and metabolites have been spurred on by the inherent advantages of this sampling technique. In the present research, a selective and sensitive high-performance liquid chromatography method for the concurrent determination of multiple antiepileptic drugs (AEDs) [levetiracetam (LVT), lamotrigine (LTG), phenobarbital (PHB)], carbamazepine (CBZ) and its active metabolite carbamazepine-10,11 epoxide (CBZE)] in a single DBS has been developed and validated. Whole blood was spotted onto Guthrie cards and dried. Using a standard punch (6mm diameter), a circular disc was punched from the card and extracted with methanol: acetonitrile (3:1, v/v) containing hexobarbital (Internal Standard) and sonicated prior to evaporation. The extract was then dissolved in water and vortex mixed before undergoing solid phase extraction using HLB cartridges. Chromatographic separation of the AEDs was achieved using Waters XBridge™ C18 column with a gradient system. The developed method was linear over the concentration ranges studied with r≥0.995 for all compounds. The lower limits of quantification (LLOQs) were 2, 1, 2, 0.5 and 1 µg/mL for LVT, LTG, PHB, CBZE and CBZ, respectively. Accuracy (%RE) and precision (%CV) values for within and between day were <20% at the LLOQs and <15% at all other concentrations tested. This method was successfully applied to the analysis of the AEDs in DBS samples taken from children with epilepsy for the assessment of their adherence to prescribed treatments.

Public awareness and views on unlicensed use of medicines in children.

AIMS: To explore awareness and views of the general public on unlicensed use of medicines in children and on the participation of children in clinical trials. METHODS: Members of the public completed a questionnaire survey administered by face-to-face interview in public areas in N. Ireland. The main outcome measures were the views on unlicensed use of medicines in children and on clinical trials in children. RESULTS: One thousand participants (59.2% female) took part; 610 were parents. Most participants (86%) had no previous knowledge about unlicensed use of medicines in children. Being a parent did not influence this nor did being a parent of a child who suffered from a health problem (P > 0.05). Most participants (92%) felt that parents should be told about unlicensed use of medicines, with the doctor most frequently selected as the person who should inform parents. At the outset, only 1.8% of participants felt that the use of medicines in children was unsafe. However, having been informed about unlicensed use of medicines, this proportion increased dramatically (62.4%; P < 0.001). Views on whether participants would enter a child of their own into a clinical trial varied according to the health status of the child (P < 0.05) i.e. a child in good health (3.9%) vs a child with a life-threatening condition (41.9%). CONCLUSIONS: There is limited public knowledge of unlicensed use of medicines in children and a general reluctance to involve children in clinical trials unless the child to be involved has a life-threatening condition.

A cross-sectional study evaluating the relationship between cortisol suppression and asthma control in patients with difficult asthma.

AIM: To examine the relationship between cortisol suppression and asthma symptoms in patients with difficult asthma. METHODS: Patients, referred to a specialist difficult asthma service and who fulfilled the criteria for difficult asthma, were recruited to the study in a sequential, unselected manner. At each clinic visit, all patients completed a validated asthma control questionnaire. For measuring cortisol suppression, early morning urinary cortisol [corrected for creatinine to give urinary cortisol creatinine ratio (UCC ratio)] was used. The urine samples were collected and stored at -70 degrees C until ready for analysis. Urinary cortisol was extracted (solid-phase extraction) and analysed using high-performance liquid chromatography. The Pearson correlation coefficient was used for correlation analysis while t-tests were used for between-group differences for normally distributed data. If the data were not normally distributed, nonparametric statistics were used. A P-value < 0.05 was considered statistically significant. RESULTS: During the study period all the patients who attended the difficult asthma clinic and fulfilled the criteria for difficult asthma (n = 66) agreed to take part in the study. There were moderate to strong and significant associations between several measures of asthma control and UCC ratio. The correlation coefficient with five indicators of asthma control ranged between 0.3 and 0.5 (P < 0.05). CONCLUSIONS: We have demonstrated a relationship between cortisol suppression and asthma control in difficult asthmatics on high-dose steroid therapy. We have proposed a model based on the relationship between symptom control and cortisol suppression, whereby both adherence and therapeutic adjustments could potentially be made. A properly controlled prospective clinical trial should examine the utility of this approach in clinical practice.

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples.

An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.

Dried blood spot liquid chromatography assay for therapeutic drug monitoring of metformin.

The use of blood spot collection cards is a simple way to obtain specimens for analysis of drugs for the purpose of therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in routine clinical setting. We describe the development and validation of a microanalytical technique for the determination of metformin from dried blood spots. The method is based on reversed phase high-performance liquid chromatography with ultraviolet detection. Drug recovery in the developed method was found to be more than 84%. The limits of detection and quantification were calculated to be to be 90 and 150 ng/ml, respectively. The intraday and interday precision (measured by CV%) was always less than 9%. The accuracy (measured by relative error, %) was always less than 12%. Stability analysis showed that metformin is stable for at least 2 months when stored at -70 degrees C. The small volume of blood required (10 microL), combined with the simplicity of the analytical technique makes this a useful procedure for monitoring metformin concentrations in routine clinical settings. The method is currently being applied to the analysis of blood spots taken from diabetic patients to assess adherence to medications and relationship between metformin level and metabolic control of diabetes.

Chiral aspects of the human metabolism of ethosuximide.

Ethosuximide is a chiral drug substance primarily indicated for the treatment of absence seizures. This drug is used clinically as the racemate. The human urinary metabolites of ethosuximide (I) have been studied using chiral gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The metabolites identified were the previously reported unchanged ethosuximide (I) enantiomers, all four stereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide (II), and the four stereoisomers of 2-ethyl-3-hydroxy-2-methylsuccinimide (III). Through chemical derivatization methodology and GC/MS (using electron impact ionization [EI] and chemical ionization [CI] techniques) two enantiomers of a previously unreported metabolite of ethosuximide, 2-ethyl-2-hydroxymethylsuccinimide (VI), have been identified.

Ratio spectra derivative spectrophotometry for the determination of furosemide and spironolactone in a capsule formulation.

The determination of furosemide and spironolactone in a capsule formulation has been investigated using techniques such as Vierordt's method and derivative spectroscopy dA/d lambda and d2A/d lambda2 applying the zero-crossing technique following reported methods. In our hands, using standard mixtures, these methods gave unreliable results. We have therefore investigated the use of ratio spectra derivative spectrophotometry for this determination. The technique of ratio spectra derivative spectrophotometry was developed in 1990, and has recently been used for a number of analyses of co-formulated products. The method was applied to the analysis of standard mixtures of the two drugs and the combined contents of 20 capsules resulting in values (mean +/- standard deviation) of 102.1 +/- 1.9% and 101.4 +/- 4.0% of the stated content for furosemide and spironolactone, respectively. Similarly, the analysis of individual capsules resulted in values of 101.5 +/- 1.6% and 102.2 +/- 1.4% of the stated content for furosemide and spironolactone, respectively.

The development and validation of liquid chromatography method for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma.

This article describes the development of SPE and HPLC methods for the simultaneous determination of metformin and glipizide, gliclazide, glibenclamide or glimperide in plasma. Several extraction and HPLC methods have been described previously for the determination of each of these analytes in plasma separately. The simultaneous determination of these analytes is important for the routine monitoring of diabetic patients who take combination medications and for studying the pharmacokinetics of the combined dosage forms. In addition this developed method can serve as a standard method for the plasma determination of these analytes therefore saving time, effort and money. The recoveries of the developed methods were found to be between 76.3% and 101.9%. The limits of quantification were between 5 and 22.5 ng/ml. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error %) was always less than 12%. Stability analysis showed that all analytes are stable for at least 3 months when stored at -70 degrees C.

Relationship between lung function and asthma symptoms in patients with difficult to control asthma.

Several studies have demonstrated a poor relationship between measures of asthma control and lung function in patients with asthma. We sought to examine this relationship in a cohort of difficult to control asthmatics attending a hospital outpatient clinic. FEV1 % and asthma control scores (ACSs) were measured at the first clinic visit and at a follow-up visit. A total of 59 patients took part in the study. At the initial visit, FEV1 % correlated with limitation of activity (p = 0.002), shortness of breath (p = 0.02), wheezing (p = 0.029), and ACS (p = 0.014). However, at follow-up, there was no correlation between FEV1 % and any measured index of asthma control. When patients with severe fixed airflow obstruction were excluded from the analysis (n = 16), FEV1 % at follow-up became significantly correlated with night waking (p = 0.02), wheezing (p = 0.05), and ACS (p = 0.036). The improvement in asthma control score at follow-up was significantly and strongly associated (r = 0.51 for total asthma control, p < 0.001) with the improvement in lung function in patients without severe fixed airflow obstruction. Lung function was not associated with any measure of asthma control in patients with severe fixed airflow obstruction. FEV1 % correlates well with asthma symptoms in difficult asthma patients with poor control but not when control improves. This loss of relationship is due to subjects with severe fixed airflow obstruction where good subjective control does not exclude the presence of significant obstruction. How severe fixed airflow obstruction should be prevented, delayed, or managed in asthma requires further research.

Intravenous indometacin in preterm infants with symptomatic patent ductus arteriosus. A population pharmacokinetic study.

AIMS: To characterize the population pharmacokinetics of indometacin in preterm infants with symptomatic patent ductus arteriosus and to investigate the influence of various factors on the response to treatment. METHODS: Data were collected from 35 infants (gestational age 25-34 weeks; postnatal age 1-77 days) in neonatal units in Belfast and Copenhagen. Infants received an initial course of up to three doses of intravenous indometacin (0.1-0.2 mg kg(-1)) as considered appropriate by the treating physician. For those infants who did not respond to therapy or in whom the ductus reopened, a second course was sometimes given. Population analysis of the 185 plasma concentrations obtained was conducted using NONMEM and pharmacokinetic and demographic differences between responders and nonresponders were compared. RESULTS: The concentration-time course of indometacin was best described by a one-compartment model. The final population parameter estimates of clearance (CL) and volume of distribution (V) (standardized to the median weight of 1.17 kg) were 0.00711 l h(-1) and 0.266 l, respectively. CL increased from birth by approximately 3.38% per day and V by approximately 1.47% per day. Concomitant digoxin therapy resulted in a 30% decrease in V. Interindividual variability in CL and V was 41% and 21%, respectively. Interoccasion variability for CL was 43%. Residual variability corresponded to a standard deviation of 0.148 mg l(-1). Closure occurred in 75% of infants with a plasma concentration > or = 0.4 mg l(-1) 24 h after the last dose. CONCLUSIONS: Dosing regimens for indometacin should take into account the weight and postnatal age of the infant and any concomitant digoxin therapy. The population estimates can be used to determine typical values of CL and V allowing the prediction of individualized doses of indometacin that should increase the probability of achieving a 24 h plasma concentration > or = 0.4 mg l(-1). Although the pharmacokinetic estimates will be affected by both interindividual and within-individual variation, it is anticipated that this approach will decrease the variability of exposure and optimize treatment outcome.

Liquid chromatographic determination including simultaneous "on-cartridge" separation of ranitidine cisapride drug combinations from paediatric plasma samples using an automated solid-phase extraction procedure.

HPLC methodology was investigated for the simultaneous determination of cisapride and ranitidine in small volume paediatric plasma samples. Such a simultaneous determination proved difficult due to the small sample volumes, the low concentrations of the drugs and the different log P values of the two compounds. The two drugs and their respective internal standards were separated "on-cartridge" using HLB Solid Phase Extraction cartridges and the samples quantified by individual HPLC methodologies. The technique has been applied successfully to 60 paediatric plasma samples containing both cisapride and ranitidine.

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges.

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 microm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography.

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70 degrees C. The method has been applied to 150 patient samples as part of a medication adherence study.

Characterization of a cytosolic aminopeptidase from Encephalitozoon intestinalis.

Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70.8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4.8. The enzyme was both heat (> 37 degrees C) and cold (< 0 degrees C) labile with an optimal activity at pH 7.2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

The pharmacokinetics of ethosuximide enantiomers in the rat.

A chiral gas chromatographic assay previously developed for quantitative analysis of ethosuximide and its major metabolites in rat urine has been adapted for the analysis of the drug in plasma. Ethosuximide, both as a racemic mixture and as the individual enantiomers, was administered to conscious rats by the intravenous (i.v.) and intraperitoneal (i.p.) routes. Pharmacokinetic parameters were estimated using standard non-compartmental methods. Comparison of the pharmacokinetic parameters of (S)-ethosuximide and (R)-ethosuximide showed that total body clearance of (R)-ethosuximide was significantly larger than that of (S)-ethosuximide and that elimination half-life was significantly shorter following administration of both 40 mg i.v. and i.p. doses, indicating that there is stereoselective elimination of ethosuximide. However, no significant differences were found between apparent volumes of distribution. In addition, no significant differences were found for either enantiomer between the estimates of the pharmacokinetic parameters obtained following administration as the individual enantiomer and as a constituent of the racemic mixture. This indicates that, at the doses studied, the preferential faster elimination of (R)-ethosuximide is not dependent upon the presence of the (S)-enantiomer. Also, for each enantiomer, the lack of any significant difference between estimates of clearance when administered as part of a racemic mixture and when administered separately indicates that neither enantiomer affects the clearance of the other.

Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat.

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 microg/ml) and 0.51% for (R)-ethosuximide (50 microg/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 microg/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72 h after drug administration. The appearance of all detected metabolites occurred within 24 h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs.

The use of polymeric solid phase extraction and HPLC analysis for the determination of ranitidine in routine plasma samples obtained from paediatric patients.

A sensitive HPLC method for the determination of ranitidine in small-volume (0.5 mL) paediatric plasma samples is described. Plasma samples were extracted using a simple, rapid solid phase extraction (SPE) technique developed using disposable copolymer packed SPE cartridges. Chromatographic separation was achieved by reverse-phase HPLC with isocratic elution using a microBondapak C18 column and a phosphate buffer (10 mM, pH 3.75)-acetonitrile (87:13 v/v) mobile phase with UV detection at 313 nm. The HPLC system exhibited linearity in the range 8-800 ng mL(-1). Intraday % CV and % bias values were in the range 1.28-8.09% (% bias -4.33 to -0.87) and interday % CV and % bias values were in the range 0.73-15.28% (% bias -1.80 to + 1.65). The limits of detection and quantitation obtained were 2 ng mL(-1) and 8 ng mL(-1), respectively, and ranitidine extraction recoveries from plasma ranged from 92.30 to 103.88%. In this study, the developed HPLC and SPE methodologies have been successfully applied to the determination of ranitidine concentrations in 68 paediatric plasma samples. The sampled population was drawn from patients already receiving the study drug therapeutically. Patients recruited had received ranitidine by two main routes - oral and intravenous. The plasma concentrations of ranitidine encountered in paediatric samples following oral or intravenous administration of a range of prescribed doses are presented graphically. These profiles are based on analysis of the first 68 plasma samples obtained from the first 35 patients recruited to the study receiving ranitidine by the oral or intravenous route.

The use of hydrophilic lipophilic balanced (HLB) copolymer SPE cartridges for the extraction of diclofenac from small volume paediatric plasma samples.

The extraction of diclofenac from spiked aqueous and plasma samples by liquid-liquid extraction (LLE) and solid phase extraction (SPE) methods is compared. The SPE methodology utilised a hydrophilic lipophilic balanced (HLB) copolymer as the extraction phase. Using a literature HPLC method, a calibration curve for diclofenac was constructed in the range 1.0-50.0 microg/ml. Diclofenac spiked samples (aqueous and plasma) were extracted by LLE and SPE methodologies. The SPE resulted in higher extraction efficiencies (mean 94.9%) than the LLE (mean 78.9%) with %R.S.D.s similar in both methods (3.2 vs. 2.1%, respectively). The SPE method was suitable for the extraction of diclofenac from small volume paediatric plasma samples.