Discovery of ONO-8590580: A novel, potent and selective GABAA α5 negative allosteric modulator for the treatment of cognitive disorders.

The identification and SAR development of a series of negative allosteric modulators of the GABAA α5 receptor is described. This novel series of compounds was optimised to provide analogues with high GABAA α5 binding affinity, high α5 negative allosteric modulatory activity, good functional subtype selectivity and low microsomal turnover, culminating in identification of ONO-8590580.

Aerial distribution of ONRAB baits as a tactic to control rabies in raccoons and striped skunks in Ontario, Canada.

During August 2006 and 2007, baits containing oral rabies vaccine, live adenovirus vector, known as ONRAB , were aerially distributed in SW Ontario, Canada. Bait acceptance during 2006 was 62 and 74% in raccoons (Procyon lotor) in areas baited at 150 baits/km(2) and 75 and 77% in plots baited at 300 baits/km(2). During 2007, bait acceptance for raccoons ranged between 59% and 80%, and 83% and 87%, in areas baited at 75 and 400 baits/km(2), respectively. Bait acceptance by skunks varied among plots (5-24%). Rabies virus-specific seroconversion during 2006 averaged 66 and 81% in raccoons in areas baited at 150 and 300 baits/km(2), respectively. During 2007, seroconversion by raccoons was 76 and 84% in areas baited at 75 and 400 baits/km(2), respectively. Seroconversion by skunks varied among plots (17-51%). Vaccine efficacy, as judged by the percentage of animals that consumed a bait and seroconverted, averaged 79 and 87% during 2006 for raccoons in areas baited at 150 and 300 baits/km(2), respectively, and 81 and 90% in areas baited during 2007 at 75 and 400 baits/km(2), respectively. Because tetracycline marking was poor in skunks, an estimate of vaccine efficacy was not possible. Aerial distribution of ONRAB vaccine baits seems to be a feasible tactic for controlling rabies in skunks and raccoons.

Prevalence of tetracycline and rabies virus antibody in raccoons, skunks, and foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario, Canada.

More than 3.6 million baits containing a recombinant vaccinia virus-rabies glycoprotein (V-RG) oral rabies vaccine were aerially or hand-distributed during 1999-2006 in an approximate 4,000-9,000 km(2) area of eastern Ontario, Canada, as part of a multitactic approach to control the raccoon variant of rabies. The efficacy of the program was assessed through the collection and testing of > 6,900 animals for bait acceptance and rabies virus-specific antibodies. Raccoon acceptance of rabies vaccine baits was significantly greater (71-83% ) in areas baited at a density of 150 baits/km(2) compared to areas baited at 75 baits/km(2) (26-58% ), and more raccoons consumed vaccine baits in areas baited with a flight line spacing of 0.75 km (45.3% [321/708]) than with a spacing of 1.5 km (33.8% [108/320]). In addition, greater numbers of raccoons consumed vaccine baits during a drop in September (52.7% [213/404]) as opposed to a June bait drop (34.6% [216/624]). Seropositivity rates for raccoons ranged between 7% and 28% in areas baited at 75/km(2) and 10% to 27% in areas baited at 150/km(2) with statistical differences varying among years and treatments. The last case of raccoon-variant rabies reported in Ontario was in September 2005. The control of raccoon rabies in Ontario has resulted in an estimated $6M to $10 M Cdn annual savings in rabies-associated costs.

Imprint cytology of needle core-biopsy specimens of breast lesions: is it a useful adjunt to rapid assessment breast clinics?

This study aimed at assessing the practicability of imprint cytology (IC) of core biopsy (CB) specimens in order to achieve one-stop diagnosis of breast lesions. In total, 199 symptomatic patients underwent free-hand CB of the suspected breast lesions. The slides were stained by Diff-QuikO and reported independently of histological reporting. For practical reasons cytology specimens were graded as follows: C1=inadequate, as less than 4 groups of epithelial cells were seen, C2=benign, C3=probably benign, C4=probably malignant and C5=positive for malignancy. The results of IC were correlated with CB histology. Absolute sensitivity of the IC was 85.0% and complete sensitivity was 89.2% when correlated with CB. Specificity (biopsy cases only) of IC was 53.1% while full specificity was 53.1%. Positive predictive value of C5 was 99.3%, C4 55.6 % and C3 was 100%. Overall suspicious rate was 5.5%. It was concluded that IC is a reliable way of diagnosing symptomatic breast lesions in one-stop breast clinic and retains the advantage of pre-operative availability of detailed pathological characteristics of tumours for treatment planning.

Squamous cell carcinoma in situ involving mesonephric remnants. A potential diagnostic pitfall.

A case of uterine cervical squamous cell carcinoma in situ (CIS) in which there was extensive endocervical glandular involvement was found to have, in addition, a deep-seated squamous epithelial lesion within the cervical stroma. Because of the deep location of this lesion, which was composed of nests of pleomorphic squamous epithelial cells, found at a site not usually occupied by endocervical glands, it was initially thought to be an invasive squamous cell carcinoma (SCC). However, on review it was found that there was CIS in the deep cervical stroma that appeared to involve small tubular structures and dilated ducts that were lined by mucin-free cuboidal epithelial cells. There was no stromal reaction to this lesion. Tubules had a poorly defined lobular arrangement and had intraluminal bright eosinophilic hyaline material. Tubular epithelial cells demonstrated immunohistochemical staining for low-molecular-weight keratin (LMWK) and vimentin but showed no staining for carcinoembryonic antigen (CEA) and high-molecular-weight keratin (HMWK). It was apparent that these tubular structures and ducts were mesonephric remnants and that this lesion represented involvement of mesonephric remnants by CIS. Although involvement of endocervical glands by CIS is well recognized, a similar lesion involving mesonephric remnants has not been previously described. Familiarity with the histological features of these lesions is essential to avoid a misdiagnosis and potential mismanagement.

Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 1. Discovery, synthesis, biological activity, and structure-activity relationships of 4-guanidino- and 4-amino-4H-pyran-6-carboxamides.

4-Amino- and 4-guanidino-4H-pyran-6-carboxamides 4 and 5 related to zanamivir (GG167) are a new class of inhibitors of influenza virus sialidases. Structure--activity studies reveal that, in general, secondary amides are weak inhibitors of both influenza A and B viral sialidases. However, tertiary amides, which contain one or more small alkyl groups, show much greater inhibitory activity, particularly against the influenza A virus enzyme. The sialidase inhibitory activities of these compounds correlate well with their in vitro antiviral efficacy, and several of the most potent analogues displayed useful antiviral activity in vivo when evaluated in a mouse model of influenza A virus infection. Carboxamides which were highly active sialidase inhibitors in vitro also showed good antiviral activity in the mouse efficacy model of influenza A infection when administered intranasally but displayed modest activity when delivered by the intraperitoneal route.

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102.

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes' active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.

Transfusion reaction due to Yersinia enterocolitica and review of other reported cases.

A 26 yr old woman was transfused after her baby was delivered by Caesarian section. During transfusion of the second pack of concentrated erythrocytes, she became acutely febrile. She then became shocked and gravely ill. Yersinia enterocolitica serogroup O:3 was recovered by blood culture from the patient and from the remnant of the bag of blood (but not the segments). The blood donor had suffered no illnesses. The patient received intensive treatment including antibiotics and made a slow recovery. Yersinia enterocolitica contaminated blood is a rare cause of potentially fatal post transfusion septicemia. Prompt recognition of the endotoxemia with cessation of the transfusion of the contaminated blood, although desirable does not seem to alter the outcome. There is no known effective measure to prevent such reactions.

Protection of laying hens against infectious bronchitis with inactivated emulsion vaccines.

Commercially-reared laying chickens were challenged at 31 weeks of age with a virulent infectious bronchitis (IB) virus. They showed a sharp drop in egg production, despite having been vaccinated at four and eight weeks old with live attenuated IB vaccines to a recommended schedule. In contrast, similar birds that had been further immunised at point-of-lay with inactivated oil emulsion IB vaccine, or with a combined IB/Newcastle disease (ND) emulsion vaccine, showed no detectable fall in egg production after the same challenge. Unvaccinated susceptible specific pathogen-free birds challenged at the same time stopped laying almost completely. In the birds revaccinated with emulsion vaccine, measurement of haemagglutination inhibition antibody levels to IB showed their geometric mean titres to be raised from less than 5 log2 at the time of vaccination to over 10 log2 four weeks later. Their antibody levels did not rise further followining the IB challenge whereas in the birds that had not been revaccinated antibody rises to nearly 10 log2 were detected after the same challenge. For pullets vaccinated earlier with live IB vaccine, revaccination with inactivated IB or IB/ND oil emulsion vaccine at point-of-lay provides a safe and effective way of protecting their egg production against IB infection.

Continence, friends, marriage and children in 51 adults with spina bifida.

Fifty-one adults with spina bifida (28 men and 23 women aged between 17 and 56 years) were interviewed in their own homes in South Wales. 17 were completely continent, 10 were incontinent and the remainder were continent to some degree. Seven of the 51 had no close friends but almost half the total had five or more close friends. Of the 29 who were unmarried, nine had a steady relationship but 13 felt that they were disqualified from such a relationship because of their disabilities. 11 men and 11 women were married and a total of 39 pregnancies had come to term, resulting in 32 normal children, five stillbirths (two with anencephaly) and two children with spina bifida. The risk of having an affected offspring when one of the parents has spina bifida (based on the combined London, Munster and Cardiff series) is 1 in 23. It is suggested that the children now growing up who have received 'total care' will be more handicapped than the adults in this series.

An assessment of ONRAB oral rabies vaccine persistence in free-ranging mammal populations in Ontario, Canada.

ONRAB is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB in captive animals, we sought to examine persistence and shedding of ONRAB in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB vaccine baits. Samples were screened for the presence and quantity of ONRAB DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB greater than 10(3)EU/mL of ONRAB DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB in animals is short-lived and the likelihood of horizontal transmission to other organisms is low.

High-density baiting with ONRAB® rabies vaccine baits to control Arctic-variant rabies in striped skunks in Ontario, Canada.

The Arctic variant of rabies virus has been maintained in striped skunks in small foci in southwestern Ontario, Canada, despite the control of the disease in red foxes. To control the disease in skunks, high-density baiting with ONRAB(®) oral rabies vaccine baits was conducted by air and by hand distribution of baits in the vicinity of skunk cases. During 2009, antibody prevalences in skunks were higher in areas baited at a density of 300 baits/km(2) and flight-line spacing of 0.25 km than at 0.5-km spacing. Once an area containing Arctic-variant cases was treated with high densities of ONRAB baits, the disease did not reoccur in skunks in those areas. During 2009, only eight skunks were diagnosed with the Arctic variant of rabies virus in Ontario.

Degree of physical handicap, education, and occupation of 51 adults with spina bifida.

51 adults with spina bifida, aged between 18 and 56 years, resident in South Wales, were interviewed in their home. Although only four had obvious hydrocephalus, one-third of them were severely handicapped and a further 40% had moderate handicap. Over half of them had had their secondary education in a normal school, with the remainder having special schooling or home tuition. Seventy per cent of the series was in normal, full-time occupation, including half those severely handicapped. Those in work were largely in managerial/technical, clerical, and light manual occupations. It is concluded that extendance and training, followed by special job placement, would help to integrate them into the community. These patients show that, in the absence of mental retardation, even severe physical handicap is no bar to normal occupation and that paralysis and incontinence alone are probably not valid selection factors for or against 'aggressive' treatment for spina bifida.

Metabolism of amlodipine in the rat and the dog: a species difference.

1. Following oral and i.v. doses of 14C-amlodipine to rat and dog, 40-50% of the dose was excreted in the urine indicating that the oral dose was well absorbed. Urinary and faecal excretion in rat was essentially complete within 48 h but was prolonged during 168 h in dog. 2. Metabolite patterns were dissimilar for rat and dog for both urine and faeces. The majority (about 95%) of the urinary metabolites were identified for both species; unchanged drug accounted for 10% and 2% of the urinary radioactivity in rat and dog respectively. 3. In rat, the principal route of metabolism involved cleavage of the 5-methoxy-carbonyl group of both the parent dihydropyridine and its pyridine analogue. In contrast, metabolism in dog involved oxidative deamination of the 2-aminoethoxy-methyl side-chain. 4. Secondary metabolism in both rat and dog was similar to that of other calcium channel blockers of the dihydropyridine class, with oxidation to the pyridine form being followed by aliphatic hydroxylation in the 6-position or O-dealkylation in the 2-position and lactonization.

Evidence of liver damage by toxin from a bloom of the blue-green alga, Microcystis aeruginosa.

Examination of the results of routine assays for hepatic enzymes in plasma has shown a significant elevation of the levels of gamma-glutamyl-transpeptidase, and a lesser elevation of those of alanine aminotransferase, in plasma specimens from a population who obtained drinking water from a reservoir containing a heavy bloom of toxic Microcystis aeruginosa. Such elevations did not occur in the adjacent population who did not use water from this source. They coincided with the occurrence of the algal bloom, and were not present in the corresponding assays during the periods before and after the occurrence of the bloom. Increases in the levels of these enzymes indicate toxic liver injury and could not be attributed to clinical changes in the frequency of acute liver conditions or to variations in the number of plasma samples taken from patients with alcoholism. Therefore, it is concluded that the toxic algae in the water-supply reservoir cause the observed elevation of the levels of hepatic enzymes in the plasma of the consuming population.

Advantages of achiral h.p.l.c. as a preparative step for chiral analysis in biological samples and its use in toxicokinetic studies.

1. Achiral reverse-phase h.p.l.c. with semi-automated post-column fraction collection and solid-phase sample reconcentration, has been applied as the purification procedure during the enantiomeric quantification of two widely differing experimental drugs; an HMG-CoA reductase inhibitor (I) and an alpha 2-adrenoceptor antagonist (II). 2. The robust and specific achiral methodologies were available prior to the need for chiral analyses and recovery of drug from the fractions provided clean samples from a variety of biological matrices, without the need to develop compatible achiral/chiral mobile phases. 3. Compared with direct chiral chromatography of plasma extracts, this approach decreased the potential for metabolites and endogenous components to interfere or impair the performance of the chiral stationary phase. 4. The availability of quantitative data from achiral analysis of samples negated the need for internal standardization of the chiral analyses, helped confirm assay specificity and provided potential to determine enantiomeric ratios where only one isomer could be accurately measured. 5. Routine enantiomeric analyses were successfully carried out on samples taken from animals dosed orally with the racemic drugs, providing important data on the possible levels of exposure to individual enantiomers during toxicity testing.

Metabolism and kinetics of amlodipine in man.

1. The disposition of amlodipine, R,S,2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbonyl- 5- methoxycarbonyl-6-methyl-1,4-dihydropyridine has been studied in two human volunteers using single oral and intravenous doses of 14C-amlodipine. The drug was well absorbed by the oral route while the mean oral bioavailability for unchanged drug was 62.5%. 2. Renal elimination was the major route of excretion with about 60% of the dosed radioactivity recovered in urine. Mean total recovered radioactivity in urine and faeces amounted to 84% for both the oral and intravenous routes. 3. Apart from a small amount of unchanged amlodipine (10% of urine 14C), only pyridine metabolites of amlodipine were excreted in urine. The majority (greater than 95%) of the metabolites excreted in the 0-72 h post-dose period were identified; the major metabolite was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl- 2-pyridyl]methoxy) acetic acid and this represented 33% of urinary radioactivity. The data indicate that oxidation of amlodipine to its pyridine analogue is the principal route of metabolism with subsequent metabolism by oxidative deamination, de-esterification and aliphatic hydroxylation. 4. For the two volunteers, amlodipine concentrations in plasma declined with a mean half-life of 33 h, while slower elimination of total drug-related material from plasma was observed, consistent with prolonged excretion (up to 12 days) of metabolites in urine and faeces. Only amlodipine and pyridine metabolites were found in the circulation. As these pyridine derivatives have minimal calcium antagonist activity the efficacy of amlodipine in man can most probably be attributed to the parent drug.