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.

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.

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.

Modified peptide antagonists of interleukin 5 exhibit extended in vivo persistence but restricted species specificity.

AF18748 is disulphide-linked homodimeric peptide with 19 amino acids in each chain that antagonises the action of the eosinophil-specific cytokine, interleukin 5 (IL-5). We have generated a set of N-terminally truncated peptides derived from AF18748 and demonstrated that the first five amino acids of the peptide do not contribute to receptor binding activity. The shortened peptide blocked IL-5-dependent adhesion of eosinophils with an IC(50)of 350 pM, and had no effect on stimulation by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF)-alpha or fMet-Leu-Phe. The peptides were rapidly broken down in mouse plasma through cleavage of a single chain of the dimer. However, this breakdown did not correlate with loss of biological activity, indicating that the asymmetric peptide fragment retains full receptor binding capacity. The activity of AF18748 disappeared rapidly from the blood following intravenous injection into mice. Coupling of polyethylene glycol to the N-terminus of AF18748 resulted in a moderate loss in biological potency (IC(50)30 nM), but the resulting conjugate persisted in the circulation for more than 8 h after injection. Despite its high potency at the human IL-5 receptor, AF18748 was unable to antagonise the activity of IL-5 on murine B13 cells, or on canine eosinophils, indicating that the peptide is highly specific for the human IL-5 receptor.

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.

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.

Cytochrome P4501A (CYP1A) induction in rat and man by the benzodioxino derivative, fluparoxan.

1. Fluparoxan is an alpha 2-adrenoceptor antagonist that has a relatively planar, tricyclic structure and was considered a potential substrate and inducer of cytochrome P4501A (CYP1A) enzymes. 2. Structure-activity analysis indicated some potential for CYP1A interaction, although its greater log P and molecular depth, compared with many CYP1A inducers, suggested fluparoxan would be a weak ligand for the aryl hydrocarbon (Ah) receptor and only a weak inducer. 3. In vitro, fluparoxan showed little affinity for the CYP1A enzymes. The compound was not metabolized by human CYP1A1 or 1A2 heterologously expressed in yeast and its rate of metabolism in rat and human microsomes was unaffected by the addition of the 1A inhibitor alpha-naphthoflavone. Furthermore, Ki's for fluparoxan against EROD activity were > 4000-fold higher than those of alpha-naphthoflavone. 4. In vivo, however, fluparoxan did show some capacity for CYP1A induction. In rat, hepatic EROD activity increased approximately 40-fold with seven once-daily oral doses of fluparoxan (50 mg/kg, solution), and immunoblotting studies confirmed induction of CYP1A2, though not of 1A1. In man, administration of 11 twice-daily oral doses of fluparoxan (8 mg tablet) produced some reduction in plasma levels of orally administered phenacetin and in the ratio of phenacetin AUC/urinary paracetamol, consistent with increased O-deethylation.

Expression of human cytochrome P4501A1 (CYP1A1) in Saccharomyces cerevisiae inhibits cell division.

1. Saccharomyces cerevisiae cells, genetically engineered to express human cytochrome P4501A1 (CYP1A1), have a mean doubling time of 5.8 h, which is considerably slower than that of control yeast cells that have undergone the same transformation process but with a plasmid lacking CYP1A1 cDNA (3.3 h). 2. A smaller reduction in the rate of cell division is observed in yeast cells expressing the closely related human P450, CYP1A2. No reduction is seen with plaice CYP1A, despite similar levels of P450 expression and enzyme activity (ethoxyresorufin O-deethylation) and no inhibition of growth is observed with yeast cells expressing higher levels of human CYP2D6. 3. Repeated culture of cells from a single CYP1A1 transformant colony results in a gradual loss of P450 expression and of CYP1A1-associated enzymatic activity over a 5-6 week period. In contrast, expression of human CYP2D6 by a single transformant colony is stable for at least 6 months. 4. The loss of CYP1A1 activity from transformed cells is accompanied by a return to normal growth rate, similar to that of control cells. 5. Inhibition of CYP1A1 enzyme activity during culture, by either type I (alpha-naphthoflavone), type II (ellipticine) or mechanism-based (1-(1'propynyl)pyrene) CYP1A inhibitors, does not affect growth rate, suggesting that some other property of human CYP1A1 protein is responsible for the growth inhibition observed.

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.

The impact of the new Mental Health Act 1989 on admissions to the Psychiatric Hospital in Barbados

Although the new Mental Health Legislation appears to have facilitated hospital admission procedures, it has raised some serious concerns about the rights of patients under the Act. A retrospective case-note study using a questionnaire analysed 200 admissions under the old Mental Health Act and compared them with 200 admissions under the new Act. The results were interpreted along specific parameters of admission status, place from which the patient was brought to hospital, who accompanied the patient and others. The new admission status of Hospital order-emergency (HO-E) accounted for 20 percent of admissions. These HO-E patients appear to be those involuntary admissions that would have been admitted as temporary patients under the old Act. Admissions (56 percent) under the temporary involuntary status of the old Act were significantly reduced to (27 percent) under the new Act. The new Act made no mention of certification by Magistrates, however 5 percent of the 1993 admissions were admitted on certified status. In addition certified patients already in hospital are still detained on certified admission status and this is obviously an area that needs urgent attention. Further studies will be needed to fully evaluate the effects of the new Act. (AU)

The impact of the new Mental Health Act 1989 on admissions to the Psychiatric Hospital in Barbados

Although the new Mental Health Legislation appears to have facilitated hospital admission procedures, it has raised some serious concerns about the rights of patients under the Act. A retrospective case-note study using a questionnaire analysed 200 admissions under the old Mental Health Act and compared them with 200 admissions under the new Act. The results were interpreted along specific parameters of admission status, place from which the patient was brought to hospital, who accompanied the patient and others. The new admission status of Hospital order-emergency (HO-E) accounted for 20 percent of admissions. These HO-E patients appear to be those involuntary admissions that would have been admitted as temporary patients under the old Act. Admissions (56 percent) under the temporary involuntary status of the old Act were significantly reduced to (27 percent) under the new Act. The new Act made no mention of certification by Magistrates, however 5 percent of the 1993 admissions were admitted on certified status. In addition certified patients already in hospital are still detained on certified admission status and this is obviously an area that needs urgent attention. Further studies will be needed to fully evaluate the effects of the new Act.

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.

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.

Biotransformation of amlodipine. Identification and synthesis of metabolites found in rat, dog and human urine/confirmation of structures by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry.

Metabolism of the dihydropyridine calcium antagonist (R,S)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-3-ethoxycarbony l-5- methoxycarbonyl- 6 -methyl- 1,4-dihydropyridine (amlodipine) has been studied in animals and man using 14C-labelled drug. The metabolite patterns are complex; 18 metabolites have been isolated from rat, dog and human urine. Based on chromatographic and mass-spectral evidence, structures have been proposed for the main metabolites and confirmed by synthesis of unambiguous reference compounds. Comparison of all reference compounds and isolated metabolites was made by gas chromatography-mass spectrometry pressure liquid chromatography on-line thermospray-mass spectrometry of underivatised compounds directly in urine. The metabolites are largely pyridine derivatives. The methods used in structure designation are presented, along with the proposed route of metabolism, which indicates that the metabolic pattern for amlodipine in man has features in common with those of both rat and dog.