Antimicrobial usage in farm animal practices in the UK: A mixed-methods approach.

Antimicrobial resistance (AMR) is a growing One Health problem. Monitoring antimicrobial usage in farm animals is crucial for tackling AMR. A cohort study using the electronic clinical records during 2019 from 23 farm animal veterinary practices across the UK belonging to two corporate groups, with a range of 2-14 veterinarians per practice, estimated the usage of antimicrobials and highest priority critically important antimicrobials (HP-CIAs). Risk factors for using HP-CIAs were evaluated using hierarchical mixed-effects logistic regression modelling with practice ID and farm ID added as random effects. Using a qualitative approach, veterinarians from one of the participating practice groups were recruited for a qualitative study to explore the barriers and facilitators in relation to antimicrobial use. Semi-structured interviews were conducted with participants and analysed thematically. During the year 2019, 98,824 antimicrobial prescribing events overall were recorded from the treatment records of the 23 participating practices. The median count of antimicrobial events per practice was 3226 (range 263-22,159). There were 17,111/98,824 (17.3%) HP-CIAs events overall, with a median of 15.4% at practice level (range 4.8-22.1%). Penicillins were the most frequently used antimicrobials 29,539/98,824 (29.9%) followed by tetracyclines 19,015/98,824 (19.2%). HP-CIA use was strongly clustered, with more clustering seen at the farm level (intraclass correlation coefficient (ICC)= 0.56) than at the practice level (ICC= 0.32). Country, route of administration, season and practice type were significantly associated with the usage of HP-CIAs. Four main themes were identified from the analysis of the veterinarians' interviews: pressure from the industry, drug-related factors, knowledge level of veterinarians and clinical factors. Supermarket contracts and farm assurance schemes were facilitators for reducing antimicrobial use and the use of HP-CIAs. Ease of administration and the withdrawal period of the antimicrobials influenced veterinarians' choice of antimicrobials. The clinical condition and clinical signs presented on farm were reported to influence participating veterinarians' prescribing decision. Participants showed a good understanding of AMR, responsible use of antimicrobials and the term 'critically important antimicrobials'. In conclusion, integrating the quantitative and qualitative findings can inform policymaking on antimicrobials stewardship in farm practice. By estimating the relative levels of clustering of antimicrobial use at the practice and farm level, as well as identifying major risk factors for using HP-CIAs, more targeted interventions can be designed to promote responsible antimicrobial use in farm practice. Furthermore, better understanding the industry pressures on farms to reduce antimicrobials usage could reduce the barriers for responsible antimicrobial use by veterinarians.

Bovine TB infection status in cattle in Great Britain in 2015.

This report, provided by the APHA, presents the key descriptive epidemiological parameters of bovine TB in cattle in Great Britain from January 1 to December 31, 2015, providing summary information on the epidemic, including key statistics and epidemiological parameters as presented in the annual surveillance report for Great Britain, with supporting detail from specific reports for England and Wales. It updates the previous annual summaries for 2012 to 2014, also published in Veterinary Record.

Longitudinal study of CTX-M ESBL-producing E. coli strains on a UK dairy farm.

The aim of this study was to investigate the bacterial strains and farm environment that may contribute to the persistence of ESBL-producing E. coli on a single UK dairy farm. A longitudinal study was conducted comprising 6 visits, between August and October 2010, followed by a further visit at approximately 69weeks after the initial visit. Faecal and environmental samples were collected from different parts of the farm. The persistence and extent of faecal shedding of ESBL E. coli by individual calves was also determined. Twenty two different PFGE types were identified. Four of these were persistent during the study period and were associated with serotypes: O98, O55, O141 and O33. The counts suggest that shedding in calf faeces was an important factor for the persistence of strains, and the data will be useful for parameterising mathematical models of the spread and persistence of ESBL strains within a dairy farm.

Farm characteristics and farmer perceptions associated with bovine tuberculosis incidents in areas of emerging endemic spread.

While much is known about the risk factors for bovine tuberculosis (bTB) in herds located in high incidence areas, the drivers of bTB spread in areas of emerging endemicity are less well established. Epidemiological analysis and intensive social research identified natural and social risk factors that may prevent or encourage the spread of disease. These were investigated using a case-control study design to survey farmers in areas defined as recently having become endemic for bTB (from or after 2006). Telephone surveys were conducted for 113 farms with a recent history of a bTB incident where their officially tuberculosis free status had been withdrawn (OTFW) (cases) and 224 controls with no history of a bTB incident, matched on location, production type and the rate of endemic bTB spread. Farmers were questioned about a range of farm management strategies, farm characteristics, herd health, wildlife and biosecurity measures with a focus on farmer attitudes and behaviours such as farmers' perception of endemicity and feelings of control, openness and social cohesion. Data generated in the telephone surveys was supplemented with existing herd-level data and analysed using conditional logistic regression. Overall, herd size (OR 1.07), purchasing an animal at a cattle market compared to purchasing outside of markets (OR 2.6), the number of contiguous bTB incidents (2.30) and the number of inconclusive reactors detected in the 2 years prior to the case incident (OR 1.95) significantly increased the odds of a bTB incident. Beef herds using a field parcel more than 3.2km away from the main farm and dairy herds reporting Johne's disease in the previous 12 months were 3.0 and 4.7 times more likely to have a recent history of a bTB incident, respectively. Beef herds reporting maize growing near, but not on, their farm were less likely to be case herds. Operating a closed farm in the two years prior to the case breakdown did not reduce the odds of a bTB incident. Farmers that had recently experienced a bTB incident were more likely to have implemented badger biosecurity in the previous year, but no more likely than control farms to have implemented cattle biosecurity. Case farmers felt significantly less likely to be influenced by government, vets or other farmers compared to those with no history of bTB. This suggests that alternative methods of engaging with farmers who have recently had a breakdown may need to be developed.

Selecting, assessing and interpreting measures of function for patients with severe hip pathology: The need for caution.

INTRODUCTION: It is not always possible to use a combination of patient-reported outcome measures (PROMs), performance tests and clinician-administrated measures to assess physical function prior to hip surgery. We hypothesised that there would be low correlations between these three types of measure and that they would be associated with different patients' characteristics. MATERIALS AND METHODS: We conducted a cross-sectional analysis of the preoperative information of 125 participants listed for hip replacement. The WOMAC-function subscale, Harris Hip Score (HHS) and walk, step and balance tests were assessed by questionnaire or during a clinic visit. Participant's socio-demographics and medical characteristics were also collected. Correlations between functional measures were investigated with correlation coefficients. Regression models were used to test the association between the patient's characteristics and each of the three types of functional measures. RESULTS: None of the correlations between the PROM, clinician-administrated measure and performance tests were very high (<0.90). Associations between patient's characteristics and functional scores varied by type of measure. Psychological status was associated with the PROM (P-value<0.0001) but not with the other measures. Age was associated with the performance test measures (P-value ranging from ≤0.01 to <0.0001) but not with the PROM. The clinician-administered measure was not associated with age or psychological status. DISCUSSION: Substantial discrepancies exist when assessing hip function using a PROM, functional test or a clinician-administered test. Moreover, these assessment methods are influenced differently by patient's characteristics. Clinicians should supplement their pre-surgery assessment of function with patient-reported measure to include the patient's perspective. LEVEL OF EVIDENCE: III, observational cross-sectional study.

Assessment of physical function following total hip arthroplasty: Inertial sensor based gait analysis is supplementary to patient-reported outcome measures.

BACKGROUND: Functional outcome assessment after total hip arthroplasty often involves subjective patient-reported outcome measures whereas analysis of gait is more objective. The study's aims were to compare subjective and objective functional outcomes after total hip arthroplasty between patients with low and high self-reported levels of pre-operative physical function. METHODS: Patients undergoing total hip arthroplasty (n=36; m/f=18/18; mean age=63.9; SD=9.8 years; BMI=26.3; SD=3.5) were divided into a low and high function subgroup, and prospective measures of WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index) function score and gait were compared at baseline and 3 and 12 months post-operatively. FINDINGS: WOMAC function scores significantly improved in both low and high function subgroups at 3 months post-operatively whereas gait parameters only improved in patients with a low pre-operative function. Between 3 and 12 months post-operatively, WOMAC function scores had not significantly further improved whereas several gait parameters significantly improved in the low function group. WOMAC function scores and gait parameters were only moderately correlated (Spearman's r=0.33-0.51). INTERPRETATION: In a cohort of patients undergoing total hip arthroplasty, pre-operative differences in mean WOMAC function scores and gait parameters between low and high function subgroups disappeared by 3 months post-operatively. Gait parameters only improved significantly during the first 3 post-operative months in patients with a low pre-operative function, highlighting the importance of investigating relative changes rather than the absolute changes and the need to consider patients with high and low functions separately.

Farming on the edge: farmer attitudes to bovine tuberculosis in newly endemic areas.

Defra's recent strategy to eradicate bovine tuberculosis (bTB) establishes three spatial zones: high-risk areas (HRAs) and low-risk areas, and an area referred to as 'the edge', which marks the areas where infection is spreading outwards from the HRA. Little is known about farmers in the edge area, their attitudes towards bTB and their farming practices. This paper examines farmers' practices and attitudes towards bTB in standardised epidemiologically defined areas. A survey was developed to collect data on farmer attitudes, behaviours, practices and environmental conditions as part of an interdisciplinary analysis of bTB risk factors. Survey items were developed from a literature review and focus groups with vets and farmers in different locations within the edge area. A case-control sampling framework was adopted with farms sampled from areas identified as recently endemic for bTB. 347 farmers participated in the survey including 117 with bTB, representing a 70per cent response rate. Results show that farmers believe they are unable to do anything about bTB but are keen for the government intervention to help control the spread of bTB.

A novel approach to mapping and calculating the rate of spread of endemic bovine tuberculosis in England and Wales.

A mathematical method for estimating the endemic status of bovine tuberculosis (bTB) in cattle in England and Wales has been developed. 6.25km(2) hexagonal cells were used as the base resolution. Maps were produced for overlapping two-year periods spanning 2001/03 to 2009/11. Distance from a farm to the ten nearest 'Officially Tuberculosis Free status - Withdrawn' incidents within the same time period was measured. Endemic areas were defined as those hexagons containing farms where the 3rd nearest incident occurred within 7km. Temporal spread of endemic bTB was estimated by creating a contour map displaying the spread of endemic bTB over the two-year periods, and using boundary displacement to calculate the rate of spread across each hexagon. A rate was obtained for ∼2300 cells and varied between 0.04km and 15.9km per year (median=3.3km per year). This work will enable further analysis of the factors associated with this expansion.

Cross-sectional survey of antibiotic resistance in Escherichia coli isolated from diseased farm livestock in England and Wales.

Between 2005 and 2007, E. coli obtained from clinical diagnostic submissions from cattle, goats, pigs and sheep to government laboratories in England and Wales were tested for sensitivity to 16 antimicrobials. Resistance was most commonly observed against ampicillin, streptomycin, sulphonamides and tetracyclines. Resistance levels varied significantly between species, with isolates from cattle frequently showing the highest levels. Verocytotoxigenic E. coli (VTEC) expressed less resistance than non-VTEC. Only 19·3% of non-VTEC and 43·5% of VTEC were susceptible to all antimicrobials, while 47·1% and 30·4%, respectively, were resistant to ⩾5 antimicrobials. The resistance phenotype SSuT was commonly observed, and isolates resistant to third-generation cephalosporins were also identified. We recommend judicious antimicrobial usage in the livestock industry in order to preserve efficacy.

Degradation of cefquinome in spiked milk as a model for bioremediation of dairy farm waste milk containing cephalosporin residues.

AIMS: The aims of this work were to develop a model of dairy farm waste milk and to investigate methods for the bioremediation of milk containing cefquinome residues. METHODS AND RESULTS: Unpasteurized milk and UHT milk that had both been spiked with cefquinome at a concentration of 2 µg ml(-1) were used as a model for waste milk containing cephalosporin residues. Adjustment of the spiked UHT milk to pH 10 or treatment with conditioned medium from bacterial growth producing cefotaximase, were the most effective methods for decreasing the cefquinome concentrations within 24 h. A large-scale experiment (10 l of cefquinome-spiked unpasteurized milk) suggested that fermentation for 22 h at 37°C followed by heating at 60°C for 2 h was sufficient to decrease cefquinome concentrations to below the limit of quantification (<125 µg kg(-1) ) and to kill the majority of the enriched bacterial population. CONCLUSIONS: One or a combination of the bioremediation methods described may have potential as a practical treatment for dairy farm waste milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of waste milk to decrease cephalosporin residue concentrations and also to kill bacteria prior to feeding to dairy calves could decrease the risk of selection for ESBL bacteria on dairy farms.

Assessing the impact of diagnosis and the related supportive care needs in patients with cutaneous melanoma.

PURPOSE: Despite the large number of people affected by melanoma, little is known about the specific needs of melanoma patients. Understanding the effects of melanoma diagnosis and the specific supportive care needs of this group of patients is a necessary step towards provision of effective psychosocial care. METHODS: Semi-structured interviews were carried out with 15 patients with malignant melanoma of the skin. The sample size, which was purposive, included 8 females and 7 males from 27 to 78 years old. Data were analysed using the NVIVO 8 software and principles of thematic analysis. RESULTS: Four major areas were identified: (a) Emotional effects due to body image, fear of the sun and uncertainty for the future; (b) Effects on Relationships, with some patients in need of more support than others from family and work colleagues; (c) Functional effects due to on-going symptoms such as pain and lymphedema; and (d) Health System and Information Needs, around the clarity, quality and timing of the information received from the health care professionals. CONCLUSIONS: The findings suggest that we often fail to pick up melanoma patients' health and psychosocial needs and fail to refer them appropriately, rather than the services not being available. Interventions should focus on patient and carer education about melanoma and sun protection, psychosocial support and effective information giving. Patient-reported outcome measures should routinely be collected to identify issues of specific concerns to the patients and directing them to the right services based on their individual needs.

Prevalence and correlates of unmet supportive care needs in patients with resected invasive cutaneous melanoma.

BACKGROUND: Knowledge about supportive care needs in patients with cutaneous invasive melanoma is scarce. We examined the unmet needs of melanoma patients treated with surgery and factors associated with these needs to assist health professionals identify areas needing clinical attention. PATIENTS AND METHODS: Cross-sectional multisite survey of UK patients ascertained 3 months to 5 years after complete resection of stage I-III cutaneous melanoma. Participants completed the following validated questionnaires: Supportive Care Needs Survey (SCNS-SF34 with melanoma module), Hospital Anxiety and Depression Scale and 51-item Functional Assessment of Cancer Therapy-Melanoma quality-of-life scale. RESULTS: A total of 472 participants were recruited [319 (67%) clinical stage I-II). Mean age was 60 years (standard deviation = 14) and 255 (54%) were female. One hundred and twenty-three (27%) participants reported at least one unmet need (mostly 'low' level). The most frequently reported unmet needs were fears of cancer returning (n = 138, 29%), uncertainty about the future (n = 119, 25%), lack of information about risk of recurrence (n = 112, 24%) and about possible outcomes if melanoma were to spread (n = 91, 20%). One hundred and thirty-eight (29%) participants reported anxiety and 51 (11%) depression at clinical or subclinical levels. Patients with nodal disease had a significantly higher level of unmet supportive care needs (P < 0.001) as did patients with anxiety or depression (P < 0.001). Key correlates of the total SCNS-SF34 score for unmet supportive care needs were younger age (odds ratio, OR = 2.23, P < 0.001) and leaving school early (OR = 4.85, P < 0.001), while better emotional (OR = 0.89, P < 0.001) and social well-being (OR = 0.91, P < 0.001) were linked with fewer unmet needs. Neither patients' sex nor tumour thickness was associated with unmet needs. CONCLUSIONS: Around a quarter of melanoma patients may have unmet support needs in the mid to long term after primary treatment. In particular, patients who are younger, less educated, distressed or socially isolated could benefit from more support.

A longitudinal field trial assesing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves.

A longitudinal field trial was carried out on a farm known to harbour cefotaximase (CTX-M)-positive Escherichia coli, in order to assess the impact of feeding waste milk containing antibiotic residues (WM+AR) on the prevalence of these bacteria in the faeces of calves. Fifty calves were alternately assigned to one of two groups at birth and fed either milk replacer (control group) or WM+AR (treatment group). Faecal samples were collected from all calves daily for the first week after enrolment, twice weekly until weaning, then weekly for a further six weeks. Environmental samples from the calf housing were collected weekly. WM+AR and powdered milk samples were examined for antibiotic residues and CTX-M-positive E. coli. Total E. coli and CTX-M-positive E. coli in faecal samples were enumerated using selective media. Regression analyses were performed on the bacterial count data using a population-averaged approach based on generalised estimating equations (GEE) to account for repeated measurements on individual calves over time. Cefquinome, a fourth generation cephalosporin, was detected in 87% of WM+AR samples at a mean concentration of 0.746 mg/l. All environmental sampling locations yielded CTX-M-positive E. coli. Significantly more pen floor samples were positive in the treatment group. Calves in the treatment group shed greater numbers of CTX-M-positive E. coli than calves in the control group throughout the study, and shedding decreased at a slower rate in the treatment group. CTX-M-positive E. coli persisted in a larger number of calves fed WM+AR compared with calves fed milk replacer where the prevalence in the treatment group declined significantly slower over time. There was no difference between calves fed WM+AR or calves fed milk replacer in the proportion of E. coli isolates that were CTX-M-positive. These findings indicate that feeding WM+AR increased the amount of resistant bacteria shed in the faeces. Shedding of CTX-M-positive E. coli persisted for longer in calves fed WM+AR, and persisted after weaning.

Does measuring the range of motion of the hip and knee add to the assessment of disability in people undergoing joint replacement?

BACKGROUND AND HYPOTHESIS: Range of motion (ROM) is a core component of some commonly used measures of disability, such as the American Knee Society Score and Harris Hip Score. However, the relationship between ROM and function is contested. The aim of this cross-sectional analysis was to investigate the relationship between pre-operative range of motion (ROM) and disability in patients undergoing hip and knee joint replacement. PATIENTS AND METHODS: Two hundred and forty-nine patients recorded on NHS records as listed for joint replacement completed a range of measures prior to surgery. Pre-operative hip or knee ROM was measured by a trained research nurse using a hand-held goniometer. Joint pain severity was assessed using the WOMAC Pain Scale. Self-report activity limitations and participation restrictions were measured with the WOMAC Function Scale and the Aberdeen Impairment, Activity Limitation and Participation Restriction Measure. Observed activity limitations were assessed through three performance tests: 20-metre timed walk, sit-to-stand-to-sit, and 20-cm step tests. RESULTS: Pre-operative hip and knee ROM correlated weakly with self-report activity limitations (0.11 to 0.43), observed activity limitations (0.09 to 0.39) and self-report participation restrictions (-0.32 to 0.06). In comparison to ROM, correlations between joint pain and self-report activity limitations and participation restrictions were consistently moderate-high (-0.53 to 0.80). However, patients with restricted knee joint flexion (<110°) had significantly worse pain, activity limitations and participation restrictions than patients with non-restricted flexion (≥110°). Patients with restricted hip joint flexion (<95°) had greater activity limitations on some measures than patients with non-restricted flexion (>95°). DISCUSSION: This study suggests that modest restrictions of ROM are of little relevance to functional ability but that a certain amount of flexion is required for adequate function. We recommend that ROM is not the best means of assessing patients' disability prior to surgery. LEVEL OF EVIDENCE: III - cohort study.

A survey of antimicrobial usage on dairy farms and waste milk feeding practices in England and Wales.

The cause for the high prevalence of cefotaximase-producing Escherichia coli reported in dairy calves is unknown but may be partly due to the selective pressure of antimicrobial residues in waste milk (milk unfit for human consumption) fed to the calves. Antimicrobial use and waste milk feeding practices were investigated in 557 dairy farms in 2010/2011 that responded to a randomised stratified postal survey. The mean number of cases of mastitis per herd in the previous year was 47, and 93 per cent of respondents used antibiotic intra-mammary tubes to treat mastitis. The most frequently used lactating cow antibiotic tubes contained dihydrostreptomycin, neomycin, novobiocin, and procaine penicillin (37 per cent), and cefquinome (29 per cent). Ninety-six per cent of respondents used antibiotic tubes at the cessation of lactation ('drying off'). The most frequently used dry cow antibiotic tube (43 per cent) contained cefalonium. Frequently used injectable antibiotics included tylosin (27 per cent), dihydrostreptomycin and procaine penicillin (20 per cent) and ceftiofur (13 per cent). Eighty-three per cent of respondents (413) fed waste milk to calves. Of these 413, 87 per cent fed waste milk from cows with mastitis, and only one-third discarded the first milk after antibiotic treatment. This survey has shown that on more than 90 per cent of the farms that feed waste milk to calves, waste milk can contain milk from cows undergoing antibiotic treatment. On some farms, this includes treatment with third- and fourth-generation cephalosporins. Further work is underway to investigate the presence of these antimicrobials in waste milk.

Symptoms experienced by cancer patients during the first year from diagnosis: patient and informal caregiver ratings and agreement.

OBJECTIVE: The aim of this study was to explore the symptom experience of patients with cancer, identify changes in symptoms over time, and explore the congruence of symptom reports between patients and their informal caregivers. METHOD: This was a prospective longitudinal evaluation of symptoms over 1 year from start of treatments (T1) using the Memorial Symptom Assessment Scale. Assessments and follow up took place at 3 months (T2), 6 months (T3) and 12 months (T4). A heterogeneous sample of 100 patients with cancer participated, providing 325 assessments over time. Furthermore, 82 caregivers also participated, providing 238 dyadic patient-caregiver assessments over the same time. RESULTS: The most commonly occurring, and by far most distressing, symptom was "lack of energy." Common symptoms reported were lack of concentration, difficulties sleeping, shortness of breath, cough, pain, dry mouth, and feeling drowsy. Symptom occurrence and distress improved over time, particularly from T2 to T3 (p < 0.05), but the "chronicity" of some generic symptoms was notable. Caregivers tended to overestimate occurrence and distress compared to patients, particularly in symptoms of psychological nature; κ statistics had a highest coefficient of 0.45, suggesting moderate agreement between patients and caregivers at best. SIGNIFICANCE OF RESULTS: More attention needs to be paid to the commonly reported symptoms by patients, as they have the potential of impacting on quality of life (QOL). As patient-caregiver reports had moderate agreement, effort should be directed to improving this agreement, as caregivers are often communicating patient symptoms to clinicians.

Mapping patients' experiences from initial change in health to cancer diagnosis: a qualitative exploration of patient and system factors mediating this process.

Delays in the diagnosis of cancer are common, and they are attributed to both patient and healthcare system factors. Minimizing such delays and improving early detection rates is a key goal of the new cancer reform strategy in England, in light of recent data showing that survival rates in the UK are low. The aim of this study was to explore the pathway from initial persistent change in health to diagnosis of cancer in a sample of patients from seven diagnostic groups in the UK and the factors mediating this process. Qualitative interviews with patients diagnosed with cancer were carried out. Seventy-five cancer patients discussed their pre-diagnosis experience as part of a broader exploration of their symptom experience for a larger study. Data were analysed by using content analysis and chart events. A broader range of mediating factors affecting and extending the patient pathway to diagnosis were reported in relation to lung, gastrointestinal and head and neck cancers and lymphoma, compared with breast, gynaecological and brain cancer patients. Many of the mediating factors were patient-related (e.g. misattribution of symptoms to common ailments, underestimation of the seriousness of the symptoms, self-medication or monitoring of symptoms, etc.). Primary care practitioner-factors were also prominent, including the exploration of firstly more common possibilities for treating the presenting symptoms without follow-up of persisting symptoms. Public health education about common cancer signs and symptoms, educational approaches in primary care to improve early diagnoses of cancer and updated guidelines for referral of suspected cancers should be enhanced before we can see any improvements in survival rates from cancer in the UK.

Preclinical pharmacokinetics and metabolism of a novel prototype DNA-PK inhibitor NU7026.

In this study we investigated the in vitro time dependence of radiosensitisation, pharmacokinetics and metabolism of NU7026, a novel inhibitor of the DNA repair enzyme DNA-dependent protein kinase (DNA-PK). At a dose of 10 muM, which is nontoxic to cells per se, a minimum NU7026 exposure of 4 h in combination with 3 Gy radiation is required for a significant radiosensitisation effect in CH1 human ovarian cancer cells. Following intravenous administration to mice at 5 mg kg(-1), NU7026 underwent rapid plasma clearance (0.108 l h(-1)) and this was largely attributed to extensive metabolism. Bioavailability following interperitoneal (i.p.) and p.o. administration at 20 mg kg(-1) was 20 and 15%, respectively. Investigation of NU7026 metabolism profiles in plasma and urine indicated that the compound undergoes multiple hydroxylations. A glucuronide conjugate of a bis-hydroxylated metabolite represented the major excretion product in urine. Identification of the major oxidation site as C-2 of the morpholine ring was confirmed by the fact that the plasma clearance of NU7107 (an analogue of NU7026 methylated at C-2 and C-6 of the morpholine ring) was four-fold slower than that of NU7026. The pharmacokinetic simulations performed predict that NU7026 will have to be administered four times per day at 100 mg kg(-1) i.p. in order to obtain the drug exposure required for radiosensitisation.

Pharmacokinetic/pharmacodynamic study of ZD9331, a nonpolyglutamatable inhibitor of thymidylate synthase, in a murine model following two curative administration schedules.

ZD9331 is a nonpolyglutamatable antifolate inhibitor of thymidylate synthase currently in clinical development. This enzyme is crucial for DNA synthesis and catalyzes the reductive methylation of dUMP to form thymidylate, which is subsequently converted to dTTP. The pharmacokinetics of two curative antitumor doses of ZD9331 administered by either a single i.p. bolus injection (50 mg/kg) or by 24-h s.c. infusion (3 mg/kg) have been measured in a thymidine salvage-incompetent murine lymphoma model (L5178Y) using a sensitive and specific ELISA. To gain an understanding of the relationship between the pharmacokinetics of ZD9331 and antitumor activity perturbations in tumor, dTTP and dUMP concentrations were also determined. After bolus administration, ZD9331 was eliminated from plasma and tissues relatively rapidly, with terminal elimination (lambda(z) 0-24 h) of 4-6 h. Liver concentrations were 8-fold higher than those measured in the plasma. Kidney and lymphoma drug concentrations were similar to those of plasma, although there was evidence of a slower overall elimination of drug at later time points. Steady-state concentrations of ZD9331 were obtained 4-5 h after the start of the 24 h s.c. infusion. At the end of infusion, elimination rates were similar for plasma and tissues (approximately 3.5 h) but appeared to be slower in the tumor at later time points. Liver concentrations were approximately 4-fold higher, and kidney and tumor concentrations were similar to those in the circulation. Depletion of dTTP and elevation in dUMP in the tumor were consistent with inhibition of thymidylate synthase after both administration schedules, although the time for which dTTP was decreased was longer (approximately 24 h) for the infusional route than for the bolus injection (<16 h). The results suggest that antitumor activity is dependent on attaining adequate drug concentrations to affect dTTP pools as well as on the duration of effective drug levels.

Cloning, expression, signaling mechanisms, and membrane targeting of P2Y(11) receptors in Madin Darby canine kidney cells.

The P2Y(11) receptor is hypothesized to link to both G(s) and G(q), although this proposition is based on expression and separate assays of G(s) and G(q) function in different cell types [J Biol Chem 1997;272:31969-31973]. We have cloned and characterized a canine P2Y(11)-like (cP2Y(11)) receptor from cultured Madin Darby canine kidney (MDCK-D1) cells. When cP2Y(11) receptors are expressed in canine thymocyte (CF2Th) cells that normally lack functional purinergic responses, ADP beta S stimulates phosphatidylinositol (PI) hydrolysis, Ca(2+) mobilization, and cAMP accumulation. Pharmacologic analysis indicates that the stimulation of cAMP production is direct and not a result of eicosanoid synthesis, activation of PKC, or elevation of cell Ca(2+). The rank order of potency for stimulation of PI hydrolysis by cP2Y(11) receptors (adenosine 5'-(2-O-thio) diphosphate = 2-methylthio-ADP >/= 2-methylthio-ATP >> ADP > ATP) differs from that of hP2Y(11) receptors. Microscopic examination of MDCK-D1 cells expressing carboxyl-terminal green fluorescent protein (GFP)-tagged cP2Y(11) (cP2Y(11)-GFP) receptors indicates primarily basolateral (BL) targeting. BL addition of 200 microM ADP beta S to confluent monolayers of MDCK-D1 cells produces an increase in short circuit current (I(sc)) (11.6 +/- 1.6 microA/cm(2)) whereas apical addition of agonist has no effect, confirming targeting of functional endogenous P2Y(11) receptors to the BL surface. In contrast, when either cP2Y(11) or cP2Y(11)-GFP is overexpressed in MDCK-D1 cells, the sensitivity of I(sc) to BL agonist increases by nearly 2 orders of magnitude, as if receptor density normally limited agonist potency; moreover, apical addition of ADP beta S now produces an increase in I(sc) but with low potency. The data support the BL localization of cP2Y(11) receptors and receptor coupling to changes in I(sc) in MDCK-D1 cells except in cases in which receptors are overexpressed; receptor overexpression leads to altered sensitivities and sites of coupling to physiologic responses.