Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data.

Preliminary investigations into a novel, long-acting, injectable, intramuscular formulation of omeprazole in the horse.

BACKGROUND: Pilot investigations have suggested that a novel, long-acting, injectable i.m. formulation of omeprazole (LA-OMEP) can induce acid suppression for up to 7 days following a single injection. OBJECTIVES: To investigate the pharmacodynamics and assess the clinical efficacy of the LA-OMEP formulation. STUDY DESIGN: Part A comprised a pharmacodynamic study. Part B consisted of a pilot clinical trial. METHODS: Part A enrolled six adult Thoroughbred horses with percutaneous gastrotomy tubes. Intragastric pH was measured for continuous 23-h periods (08.00-07.00 h) for eight consecutive days (days 0-7). A single 2.0-g dose of a 100 mg/mL LA-OMEP formulation was administered at 08.00 h on day 1. In Part B, 26 horses with squamous or glandular gastric disease were enrolled based on routine gastroscopic evaluation. Once enrolled, horses received 2.0 g of the 100 mg/mL LA-OMEP formulation by i.m. injection on days 0 and 7. Repeat gastroscopy was performed on days 14 (23 horses) or 16 (one horse). RESULTS: In Part A, the percentage of time during which pH was above 4 exceeded 66% for days 1-4 in all horses and days 1-7 in four of the six horses studied. In Part B, healing was observed in all 22 (100%, 95% confidence interval [CI] 89-100%) horses with squamous disease and in nine of 12 (75%, 95% CI 47-92%) horses with glandular disease. Improvement, by at least one grade, was observed in all 22 (100%, 95% CI 89-100%) horses with squamous disease and in all 12 (100%, 95% CI 81-100%) horses with glandular disease. No worsening of lesions was observed. Lesion grade decreased over time in both the squamous (P<0.0001) and glandular (P = 0.0024) mucosa. MAIN LIMITATIONS: Small sample sizes. CONCLUSIONS: The results of the present study compare favourably with previous reports on the pharmacodynamics of omeprazole and the clinical outcomes of trials reporting response to oral omeprazole therapy.

The effects of dose and diet on the pharmacodynamics of esomeprazole in the horse.

BACKGROUND: Esomeprazole warrants further investigation as a treatment for equine gastric ulcer syndrome. OBJECTIVES: To investigate the duration of intraday acid suppression achieved with two doses of esomeprazole under two dietary conditions. STUDY DESIGN: A four way crossover design. METHODS: Six adult Thoroughbreds instrumented with percutaneous gastrotomy tubes were used. Intragastric pH was measured for continuous 23 h periods (08.00-07.00 h) for 6 consecutive days (Days 0-5). Baseline data was recorded on Day 0 and esomeprazole was administered on Days 1-5. Two doses (0.5 and 2.0 mg/kg bwt/day per os once daily) and two diets (a high grain/low fibre (HG/LF) and ad libitum hay (HAY) diet) were studied. Data for the percentage of time pH was above 4 (%tpH>4) and median intraday pH was reported for two measurement points and analysed using generalised estimating equations. RESULTS: An inconsistent effect of both diet and dose was evident with mean %tpH>4 and mean of the median intraday pHs typically higher at the 2.0 mg/kg bwt dose and in HG/LF diet. A cumulative effect of dosing was present with the magnitude of acid suppression observed on Day 5 consistently higher than that observed on Day 1. The magnitude of acid suppression, at measurement point 1, compared favourably with previous reports on omeprazole and exceeded human therapeutic breakpoints for the 0.5 mg/kg bwt dose in the HG/LF diet and 2.0 mg/kg bwt dose in the HAY diet. MAIN LIMITATIONS: Instrumentation may have modified gastric function and horses were not fasted or exercised. CONCLUSIONS: The findings of the present study suggested that both dose and diet affect the response to esomeprazole in the horse and that a cumulative effect is present over the first 5 days of treatment. Further investigation into the clinical efficacy of esomeprazole and trials directly comparing esomeprazole and omeprazole appear to be warranted.

The effects of dose and diet on the pharmacodynamics of omeprazole in the horse.

BACKGROUND: Conflicting data are presented in the current literature regarding the efficacy of omeprazole for suppressing gastric acidity in the horse. OBJECTIVES: The objective of this study was to investigate the duration of intraday acid suppression achieved with two doses of omeprazole under two different dietary conditions. STUDY DESIGN: A four-way crossover design. METHODS: Six adult Thoroughbred horses instrumented with percutaneous gastrotomy tubes were used. Intragastric pH was measured for continuous 23 h periods (08.00-07.00 h) for six consecutive days (Days 0-5). Baseline data was recorded on Day 0 and omeprazole administered on Days 1-5. Two doses (1 mg/kg and 4 mg/kg bwt per os once a day) and two diets (a high grain/low fibre [HG/LF] and ad libitum hay [HAY)] diet) were studied. Data for the percent (%) time pH was above 4 (%tpH>4) and median intraday pH was reported for two measurement locations and analysed using generalised estimating equations. RESULTS: An effect of both diet and dose was evident with mean %tpH>4 and the mean of the median intraday pHs typically higher at the higher (4 mg/kg bwt) dose and in HG/LF diet. The overall efficacy of omeprazole in raising intragastric pH was good under the HG/LF conditions but relatively poor in the HAY diet. A cumulative effect of dosing, not previously reported in the horse, was observed. CONCLUSIONS: The overall efficacy of omeprazole in raising ventral gastric pH was less than previously reported. Both dose and diet may play a role in the efficacy of omeprazole in the horse. Therefore, the use of singular dosing recommendations that encompass all horse types and management conditions may not be appropriate and dosing recommendations that take into account the diet of the horse may be advantageous.

The effects of dose and diet on the pharmacokinetics of omeprazole in the horse.

This study aimed to investigate the effect of diet and dose on the pharmacokinetics of omeprazole in the horse. Six horses received two doses (1 and 4 mg/kg) of omeprazole orally once daily for 5 days. Each dose was evaluated during feeding either a high-grain/low-fibre (HG/LF) diet or an ad libitum hay (HAY) diet in a four-way crossover design. Plasma samples were collected for pharmacokinetic analysis on days 1 and 5. Plasma omeprazole concentrations were determined by ultra-high pressure liquid chromatography-mass spectrometry. In horses being fed the HG/LF diet, on day 1, the area under the curve (AUC) and maximal plasma concentration (Cmax ) were higher on the 4 mg/kg dose than on the 1 mg/kg dose. The AUC was higher on day 5 compared to day 1 with the 4 mg/kg dose on the HG/LF diet. On days 1 and 5, the AUC and Cmax were higher in horses being fed the HG/LF diet and receiving the 4 mg/kg dose than in horses being fed the HAY diet and receiving the 1 mg/kg dose. These findings suggest that both dose and diet may affect pharmacokinetic variables of omeprazole in the horse.

Pharmacokinetics and bioequivalence testing of five commercial formulations of omeprazole in the horse.

Omeprazole is widely used in the treatment of equine gastric ulcer syndrome. To date, little is known about the relative pharmacokinetics of the different formulations making comparisons between products difficult. The objectives of the study were to investigate the relative pharmacokinetics of five commercially available formulations of omeprazole in the horse and to test for bioequivalence of four of the formulations using one of the formulations as a reference standard. Twelve mature Thoroughbred horses were fasted for 16 h then administered 2 g of each formulation in a cross-over design. Serial blood samples were collected and plasma omeprazole concentration was determined by ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS). No significant differences were present between three of the formulations and the reference formulation, while the fourth formulation had a lower Cmax and longer Tmax than the reference formulation. Bioequivalence against the reference formulation could not be demonstrated for any of the formulations tested. The findings of the study suggested that the method of protection utilised by different formulations of omeprazole (enteric-coated granules vs. buffering) does not significantly alter the pharmacokinetics of the drug. Further work to establish bioequivalence is needed before direct comparisons can be drawn between different formulations.

Regional variations in percutaneous absorption of methimazole: an in vitro study on cat skin.

The use of transdermal gel medications in cats has become popular in veterinary medicine due to the ease of administration compared to oral medication. The research to support systemic absorption of drugs after transdermal gel administration and the preferred skin region to apply these drugs in cats is limited. The aim of this study was to characterize the effect of different skin regions on the percutaneous absorption pharmacokinetics of a commercially available transdermal methimazole after a finite dose was applied to feline skin in vitro. A commercial formulation of methimazole (10 mg) was applied to four skin regions (the inner stratum corneum of the ear, groin, neck, and thorax regions) from six cats. The receptor medium was sampled up to 36 h postapplication, and methimazole concentrations were measured using high-performance liquid chromatography. Methimazole was absorbed more completely across the pinnal skin, compared to the groin, neck, and thorax (P < 0.001), which justifies application to the pinna to maximize efficacy and also to minimize the effects of grooming.

Regional intravenous limb perfusion compared to systemic intravenous administration for marimastat delivery to equine lamellar tissue.

Pharmaceutical agents with potential for laminitis prevention have been identified. Many of these, including the MMP inhibitor marimastat, are impractical for systemic administration. This study compared local delivery of marimastat by regional limb perfusion (RLP) to systemic intravenous bolus dosing (SIVB), and established whether RLP results in local lamellar drug delivery. Six adult horses received 0.23 mg/kg of marimastat by RLP followed by 0.23 mg/kg marimastat by SIVB, with a 24-h washout period. Lamellar ultrafiltration probes sampled lamellar interstitial fluid as lamellar ultrafiltrate (LUF). LUF and plasma marimastat concentrations (LUF[M] and P[M] respectively) were measured for 24 h after each treatment. Regional pharmacokinetic parameters were calculated using noncompartmental analyses. The LUF C(max) following RLP was 232 [34-457] times that following SIVB. LUF[M] after RLP were higher than those obtained after SIVB for 18 h (P < 0.03). Median LUF[M] were > IC(90) of equine lamellar MMP-2 and MMP-9 for 9 h after tourniquet removal. RLP appeared superior to SIVB for lamellar marimastat delivery (higher LUF C(max),, AUC and T > IC(90) of lamellar MMPs). However, frequent dosing is necessary to achieve therapeutic lamellar concentrations. RLP could be used to investigate whether marimastat prevents experimentally induced laminitis. Further refinement of the technique and dosing interval is necessary before clinical application.

Percutaneous absorption of methimazole: an in vitro study of the absorption pharmacokinetics for two different vehicles.

The use of transdermal medications in cats has become popular in veterinary medicine due to the ease of administration compared to oral medication. However, the research to support systemic absorption of drugs applied to the pinna after transdermal administration in cats is limited. The aim of this study was to characterize the percutaneous absorption pharmacokinetics of methimazole in a lipophilic vehicle compared to methimazole in Pluronic(®) lecithin organogel (PLO) using a finite dose applied to feline ear skin in an in vitro Franz cell model. The two formulations of methimazole (10 mg) were applied to the inner stratum corneum of six pairs of feline ears. The receptor medium was sampled up to 30 h post-administration, and methimazole concentrations were measured using high-performance liquid chromatography (HPLC). Histological examination of all ears was undertaken as small differences in the thickness of ear skin may have contributed to inter-individual differences in methimazole absorption between six cats. Methimazole was absorbed more completely across the pinnal skin when administered in the lipophilic vehicle compared to administration in the PLO gel (P < 0.001).

The effect of feeding on the pharmacokinetic variables of two commercially available formulations of omeprazole.

The objectives of this study were to investigate the impact of formulation (enteric coated and buffered) and feeding on pharmacokinetic variables associated with the oral administration of omeprazole in the horse. Six thoroughbred racehorses were studied in a crossover design. Each received 2 g of an enteric coated or buffered formulation in both the fed and fasted state. Plasma omeprazole concentrations were determined by UHPLC-MS. The effects of feeding or formulation on AUC0-inf_obs, half-life, Tmax or Cmax were not statistically significant. However, a wider-than-expected degree of variation was present and examination of the raw data suggests that an effect of feeding, wherein the bioavailability of omeprazole may be reduced in the fed animal, may be present. Further investigation in a larger population of animals to assess the factors that contribute to the wide degree of absorption observed is warranted.

Myocardial collagen deposition and inflammatory cell infiltration in cats with pre-clinical hypertrophic cardiomyopathy.

The histological features of feline hypertrophic cardiomyopathy (HCM) have been well documented, but there are no reports describing the histological features in mild pre-clinical disease, since cats are rarely screened for the disease in the early stages before clinical signs are apparent. Histological changes at the early stage of the disease in pre-clinical cats could contribute to an improved understanding of disease aetiology or progression. The aim of this study was to evaluate the histological features of HCM in the left ventricular (LV) myocardium of cats diagnosed with pre-clinical HCM. Clinically healthy cats with normal (n = 11) and pre-clinical HCM (n = 6) were identified on the basis of echocardiography; LV free wall dimensions (LVFWd) and/or interventricular septal wall (IVSd) dimensions during diastole of 6-7 mm were defined as HCM, while equivalent dimensions <5.5 mm were defined as normal. LV myocardial sections were assessed and collagen content and inflammatory cell infiltrates were quantified objectively. Multifocal areas of inflammatory cell infiltration, predominantly lymphocytes, were observed frequently in the left myocardium of cats with pre-clinical HCM. Tissue from cats with pre-clinical HCM also had a higher number of neutrophils and a greater collagen content than the myocardium of normal cats. The myocardium variably demonstrated other features characteristic of HCM, including arteriolar mural hypertrophy and interstitial fibrosis and, to a lesser extent, myocardial fibre disarray and cardiomyocyte hypertrophy. These results suggest that an inflammatory process could contribute to increased collagen content and the myocardial fibrosis known to be associated with HCM.

Pharmacokinetics of intravenous, plain oral and enteric-coated oral omeprazole in the horse.

The objectives were to document the pharmacokinetics of intravenous, enteric-coated oral and plain oral omeprazole in fasted horses and to investigate the impact of feeding on the bioavailability of an enteric-coated omeprazole. Twelve horses received four treatments: intravenous omeprazole (0.5 mg/kg) in the fasted state (IV-Fasted), enteric-coated omeprazole (4 mg/kg) orally in the fasted state (ECO-Fasted), enteric-coated omeprazole (4 mg/kg) orally in the fed state (ECO-Fed) and plain omeprazole (4 mg/kg) orally in the fasted state (PL-Fasted). Plasma omeprazole concentrations were determined by UHPLC-MS. Bioavailability was higher (P = 0.038) in the ECO-Fasted group (21.5 [9.0-27.7]%) than the PL-Fasted group (10.1 [7.7-13.3]%). Similarly, AUC0-∞ was higher in the ECO-Fasted group than the PL-Fasted group (P = 0.027). No significant differences were present between the ECO-Fasted and ECO-Fed groups with regards to bioavailability, Cmax , Tmax or AUC0-∞ . When the half-life data from the oral formulations was pooled, it was longer than that observed in the IV-Fasted group (100 [73-118] min) and 35 [34-39] min, respectively; P < 0.0001). Bioavailability of enteric-coated omeprazole was higher than previously reported and feeding had minimal impact. Bioavailability of plain omeprazole was approximately half that of enteric-coated omeprazole. The longer half-life observed following oral administration was consistent with the flip-flop effect and has not previously been described for omeprazole in the horse.

Effects of eight vehicles on transdermal lidocaine penetration in sheep skin in vitro.

This study investigated the effects of vehicles on penetration and retention of lidocaine applied to sheep skin in vitro. Thoracic skin from two sheep was clipped of wool and stored at -20 °C, until used. Skin samples were defrosted and mounted in Franz-type diffusion cells, and then one of the following formulations, each saturated with lidocaine, was added: sodium lauryl sulphate (SLS) 0.5% in water, SLS 1% in water, dimethyl sulphoxide (DMSO) 50% in water (wt/wt), DMSO 100%, isopropyl myristate 100% (IPM), water alone, diethylene glycol monoethyl ether (DGME) 50% in water (wt/wt) and DGME 100%. The penetration of lidocaine in each skin sample was measured over 8 h. Significantly greater lidocaine skin concentrations and flux (J(SS)) were achieved with the nonaqueous vehicles, DMSO 100% (P < 0.00001 and P < 0.01, respectively), followed by DGME 100% and IPM (P < 0.00001 and P < 0.01, respectively). The lag time (t(lag)) for lidocaine penetration in the DMSO 100% vehicle was significantly shorter (P < 0.01) compared with all other vehicles except water. Improved transdermal penetration of lidocaine in the DMSO 100% vehicle was likely due to skin barrier disruption, as determined by differences in pre- and post-treatment transepidermal water loss (TEWL). This study has shown that nonaqueous vehicles enhanced penetration of lidocaine in sheep skin to a greater extent than aqueous vehicles, which has implications for topically applied local anaesthesia in sheep.

Indications, durations and outcomes of mechanical ventilation in dogs and cats with tick paralysis caused by Ixodes holocyclus: 61 cases (2008-2011).

OBJECTIVES: The primary objectives of this research were to describe the indications for mechanical ventilation, the duration of mechanical ventilation and probability of survival in dogs and cats with respiratory failure induced by the Australian paralysis tick (Ixodes holocyclus). METHODS: A retrospective case series and a retrospective single cohort study were conducted using dogs and cats with tick paralysis requiring mechanical ventilation. An index of oxygenating performance of the lung (PF ratio of partial pressure of oxygen in arterial blood to fraction of inspired oxygen) was derived from arterial blood gas analysis; patients euthanased because of veterinary costs were identified and Kaplan-Meier survival analyses performed. RESULTS: In total, 36.6% of patients were ventilated because of hypoxaemia refractory to oxygen therapy, 38.3% because of hypoventilation, 18.3% because of unsustainable respiratory effort and 6.6% because of respiratory arrest. Median duration of mechanical ventilation was 23 h, median time hospitalised was 84 h and 63.9% of all patients requiring mechanical ventilation survived to discharge from the hospital. Survival probability increased to 75% when cases of cost-based euthanasia were right-censored rather than treated as deaths. The survival probability of patients ventilated because of hypoxaemia (52.6%) was significantly less than for those ventilated because of hypoventilation (90.5%). The first measured PF ratio after commencing mechanical ventilation was not significantly associated with survival probability. CONCLUSIONS: Dogs and cats with tick paralysis requiring mechanical ventilation to manage respiratory failure have reasonable survival probability. Dogs and cats requiring mechanical ventilation because of hypoventilation have a higher survival probability than those with oxygenation failure.

The feral horse foot. Part B: radiographic, gross visual and histopathological parameters of foot health in 100 Australian feral horses.

BACKGROUND: It has been proposed that the feral horse foot is a benchmark model for foot health in horses. However, the foot health of feral horses has not been formally investigated. OBJECTIVES: To investigate the foot health of Australian feral horses and determine if foot health is affected by environmental factors, such as substrate properties and distance travelled. METHODS: Twenty adult feral horses from five populations (n = 100) were investigated. Populations were selected on the basis of substrate hardness and the amount of travel typical for the population. Feet were radiographed and photographed, and digital images were surveyed by two experienced assessors blinded to each other's assessment and to the population origin. Lamellar samples from 15 feet from three populations were investigated histologically for evidence of laminitis. RESULTS: There was a total of 377 gross foot abnormalities identified in 100 left forefeet. There were no abnormalities detected in three of the feet surveyed. Each population had a comparable prevalence of foot abnormalities, although the type and severity of abnormality varied among populations. Of the three populations surveyed by histopathology, the prevalence of chronic laminitis ranged between 40% and 93%. CONCLUSIONS: Foot health appeared to be affected by the environment inhabited by the horses. The observed chronic laminitis may be attributable to either nutritional or traumatic causes. Given the overwhelming evidence of suboptimal foot health, it may not be appropriate for the feral horse foot to be the benchmark model for equine foot health.

The feral horse foot. Part A: observational study of the effect of environment on the morphometrics of the feet of 100 Australian feral horses.

AIM: To better understand the morphology of, and the effect of different travel patterns and varying substrate environments on, the feral horse foot to better manage the feet of domestic horses. METHODS: The left forefeet of 20 adult feral horses from each of five geographically separated populations in Australia (n = 100) were investigated. Populations were selected on the basis of substrate hardness under foot and the amount of travel typical for the population. Feet were radiographed and photographed and 40 morphometric measurements of each foot were obtained. RESULTS: Of the 40 parameters, 37 differed significantly (P < 0.05) among the populations, which suggested that substrate hardness and travel distance have an effect on foot morphology. Harder substrates and longer travel distances were associated with short hoof walls and minimal hoof wall flaring. Softer substrates and moderate travel distances were associated with long flared walls, similar to that of typical untrimmed feet of domestic horses. CONCLUSIONS: The morphology of the feral horse foot appeared to be affected by the distance travelled and by the abrasive qualities and mechanical properties of the substrate under foot. There were marked differences in some conformation parameters between the feral horses in the current study and domestic horses in previous studies. Although the conformation of the feral horse foot may have some prescriptive value, concerns regarding abnormal foot anatomy warrant further investigation.

Quantification of tapentadol in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology.

Tapentadol (TAP) is a novel opioid pain reliever drug that is unusual in its possession of dual mechanism of action (mu opioid-receptor agonist and noradrenaline reuptake inhibitor), this feature makes the active ingredient an attractive potential progenitor of a new pharmacological class. A liquid chromatography-mass spectrometry (LC-MS) method exists to measure TAP in urine and saliva, but the aim of the present study was to develop and validate a simple HPLC-FL based method to quantify TAP in plasma. Several parameters both in the extraction and detection method were evaluated. The applicability of the method was determined by administering TAP orally to two dogs; the protocol yielded the expected pharmacokinetic results and plasma collected by jugular venipuncture at regular intervals. The mobile phase consisted of acetonitrile (A):acetic acid (B) (33 mM), delivered in gradient mode (5-95% B [0-20 min], 95-5% B [20-25 min] and finally 5% B isocratically [25-32 min]) with a flow rate of 1 mL min⁻¹. Excitation and emission wavelengths were of 273 and 298 nm, respectively. TAP was extracted from the plasma using a mixture of Et2O:CH2Cl2 (7:3, v/v), which gave a recovery of 98.0-107.8% and a limit of quantification of 1 ng mL⁻¹. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte and IS (O-desmethyltramadol), as confirmed by HPLC-DAD experiments. In conclusion, this was a simple and effective method using HPLC-FL to detect TAP in plasma, which may be useful for future pharmacokinetic studies.

Complement C5a: impact on the field of veterinary medicine.

Complement C5a is a pro-inflammatory polypeptide produced during activation of the complement cascade in response to foreign antigens or tissue damage secondary to physical or chemical injury. C5a, via activation of the C5a receptor (C5aR or CD88), is a major inflammatory mediator involved in a number of diseases, including some of veterinary relevance. Greater understanding of the role of C5a has been possible with the availability of gene knockout mice, specific antibodies and peptide agonists/antagonists. This review outlines the functions of C5a and its role in the development of disease, including neoplastic conditions and sepsis, in animals of veterinary importance. The application of C5aR agonist and antagonist analogues to combat those conditions is also discussed.

Comparative pharmacokinetics and pharmacodynamics of tablet, suspension and paste formulations of atenolol in cats.

This study compared the pharmacokinetic and pharmacodynamic profiles of an extemporaneously prepared (compounded) atenolol paste and suspension for oral administration, against the commercially available divided tablet in healthy cats. Eleven healthy cats (mean: age 4 ± 0.4 year, weight 5.0 ± 0.7 kg) were dosed twice-daily with 12.5 mg atenolol (tablet, paste or suspension) for 7 days in a randomized cross-over design with a 7-day wash-out period. On day 7, an electrocardiogram was performed before and immediately after stress provocation (jugular venipuncture) at prestudy screening, and at 2, 6 and 12 h after morning dosing. Systolic arterial blood pressure (BP) was assessed following the second electrocardiogram. Plasma was collected at prestudy screening, and at 1, 2, 6 and 12 h to measure atenolol plasma concentrations. Mean atenolol dose was 2.5 mg/kg (range: 2.1-3.3 mg/kg). Stress-induced rise in heart rate was attenuated (P < 0.05) at every time point compared to baseline for all formulations. Although the paste significantly attenuated stress-induced elevation in heart rate at all time points, the effect was not consistently equivalent to the tablet. The BP was not altered (P > 0.05) at any time point by any formulation. In conclusion, there were no significant differences (P > 0.05) in any of the pharmacokinetic parameters or pharmacodynamic profiles of the paste and suspension compared to the commercially available tablet.

Blastocystis subtypes in symptomatic and asymptomatic family members and pets and response to therapy.

BACKGROUND: Blastocystis is a common, enteric parasite. The pathogenicity of the organism is uncertain, but subtypes (ST) 1 and 3 have been reported more likely to cause irritable bowel-like symptoms. AIMS: We treated symptomatic patients positive for Blastocystis with conventional therapy and analysed 16 small-subunit (SSU) rDNA to assess clearance and carriage rates and ST prevalence of the parasite in the asymptomatic household members. METHODS: In a longitudinal, prospective case study, 11 symptomatic patients positive for Blastocystis underwent outpatient clinical assessment to exclude other diagnoses before 14 days of either metronidazole 400 mg three times daily or trimethoprim/sulfamethoxazole 160/800 mg twice-daily therapy. Faecal specimens were collected from patients at baseline, day 15, 28 and 56 after therapy and from 17 family members and eight pets at day 15. Specimens were analysed using faecal smear, culture and polymerase chain reaction analysis of 16SSU rDNA. RESULTS: No patient cleared the organism following therapy. ST 1 (45%), 3 (36%), 4 (36%) and 6 (9%) were found in the symptomatic Blastocystis patients, and ST identified before and after therapy were identical in each individual. All household contacts were positive for Blastocystis and 16/17 (94%) contacts showed identical Blastocystis ST to the symptomatic family member. All pets were positive for Blastocystis with polymerase chain reaction testing, 7/8 (88%) demonstrating ST concordance with the symptomatic Blastocystis patients. CONCLUSIONS: Conventional therapy is ineffective for symptomatic Blastocystis infection. The high prevalence of Blastocystis infection within households suggested transmission between humans and their pets. Subtyping analysis of SSU rDNA alone in Blastocystis does not appear to predict pathogenicity.