Revision of the classical dopamine D2 agonist pharmacophore based on an integrated medicinal chemistry, homology modelling and computational docking approach.

The scientific advances during the 1970ies and 1980ies within the field of dopaminergic neurotransmission enabled the development of a pharmacophore that became the template for design and synthesis of dopamine D2 agonists during the following four decades. A major drawback, however, is that this model fails to accommodate certain classes of restrained dopamine D2 agonists including ergoline structures. To accommodate these, a revision of the original model was required. The present study has addressed this by an extension of the original model without compromising its obvious qualities. The revised pharmacophore contains an additional hydrogen bond donor feature, which is required for it to accommodate ergoline structures in a low energy conformation and in accordance with the steric restrictions dictated by the original model. The additional pharmacophore feature suggests ambiguity in the binding mode for certain compounds, including a series of ergoline analogues, which was reported recently. The ambiguity was confirmed by docking to a homology model of the D2 receptor as well as by pharmacological characterization of individual enantiomers of one of the analogues. The present research also addresses the potential of designing ligands that interact with the receptor in a large, distal cavity of the dopamine D2 receptor that has not previously been studied systematically. The pharmacological data indicate that this area may be a major determinant for both the dopamine D2 affinity and efficacy, which remains to be explored in future studies.

Novel 7-phenylsulfanyl-1,2,3,4,10,10a-hexahydro-pyrazino[1,2-a]indoles as dual serotonin 5-HT2C and 5-HT6 receptor ligands.

Novel 7-phenylsulfanyl-1,2,3,4,10,10a-hexahydro-pyrazino[1,2-a]indoles are synthesized using a six-step protocol. Notably, the synthesis route make use of a new and improved ring-closing methodology for the assembly of the hexahydro-pyrazino[1,2-a]indole scaffold, which is based on intramolecular C-H insertion of a carbene. The compounds act as dual serotonin 5-HT2C- and 5-HT6-ligands.

Widespread retinal degenerative disease mutation (rdAc) discovered among a large number of popular cat breeds.

The recent discovery of a mutational variant in the CEP290 gene (CEP290: IVS50+9T>G), conferring recessive retinal degeneration in Abyssinian and Somali (long-haired Abyssinian) cats (rdAc) prompted a survey among 41 cat breeds (846 individuals) to assess the incidence, frequency and clinical consequence of rdAc. The rdAc allele displayed widespread distribution, observed in 16/43 (37%) breeds, exhibiting a high allele frequency (∼33%) in North American and European Siamese populations. Clinical evaluations demonstrated high concordance between rdAc pathology and the CEP290 (IVS50+9T>G) homozygous genotype (P=1.1E-6), with clinical disease similar to affected Abyssinians/Somalis. This retinal degeneration has not been reported in breeds other than the Abyssinian/Somali and poses a significant health risk particularly in the Siamese breed group. Alertness of the veterinary community and the present availability of commercial diagnostic testing could synergistically enable breeders to reduce the incidence of rdAc blindness in pure-bred cat populations.

Derivation of germ cells from embryonic stem cells.

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine.

Apparent absolute oral bioavailability in excess of 100% for a vitronectin receptor antagonist (SB-265123) in rat. II. Studies implicating transporter-mediated intestinal secretion.

1. Transporters have been increasingly identified as a factor in limiting the oral bioavailability of certain drugs. Previously, the present authors investigated a compound (SB-265123) with an apparent absolute oral bioavailability (Fapp) consistently > 100%, and excluded likely artefactual causes for this observation, as well as standard considerations of non-stationary or non-linear pharmacokinetics. The data led the authors to believe that SB-265123 might be a transporter substrate in the rat, and it was hypothesized that transporter interactions might be responsible for the observed Fapp > 100%. 2. In the present study, a model was proposed incorporating rapid and complete absorption and elimination by a saturable intestinal secretory pathway. Intestinal secretion was demonstrated for SB-265123 using a rat single-pass intestinal perfusion technique. In addition, in a study employing both independent and simultaneous intravenous and oral administration of SB-265123, exposure to SB-265123 was greater than additive on joint intravenous and oral administration, lending further support to the hypothesis of a saturable transporter. Furthermore, in a study with co-administration of GF120918A, a transporter inhibitor, the observed Fapp for SB-265123 was only 84 +/- 17%, providing additional evidence for transporter involvement in the >100% Fapp phenomenon. 3. Experience with SB-265123 illustrates a counterintuitive impact of transporters on oral bioavailability and highlights the importance of considering transporter interactions in the systemic disposition of xenobiotics, even those not demonstrating low oral bioavailability.

Bioisosteric determinants for subtype selectivity of ligands for heteromeric GABA(A) receptors.

The potency and efficacy of a series of bioisosterically modified GABA analogues were determined electrophysiologically using heteromeric GABA(A) receptors expressed in Xenopus oocytes. These agonist parameters were shown to be strongly dependent on the receptor subunit combination. On the other hand, the antagonist potencies of the classical GABA(A) antagonists SR 95531 (7) and BMC (8) and also of 5g and the phosphinic acid bioisosteres of 5a, compounds 5f and 6, were essentially independent of the receptor subunit combinations.

Synthesis and pharmacology of seleninic acid analogues of the inhibitory neurotransmitter gamma-aminobutyric acid.

[structure: see text] The first seleninic acid analogues of gamma-aminobutyric acid (GABA) and of the specific GABA(A) agonist piperidine-4-carboxylic acid (isonipecotic acid), 1 and 2, respectively, have been synthesized and shown to be potent agonists at the GABA receptors.

Piperidinyl-3-phosphinic acids as novel uptake inhibitors of the neurotransmitter gamma-aminobutyric acid (GABA).

Piperidinyl-3-phosphinic acid 2, piperidinyl-3-methylphosphinic acid 3 and N-(4,4-diphenyl-3-butenyl)piperidinyl-3-phosphinic acid 4 have been synthesized as bioisosteres of the corresponding amino carboxylic acids, which are potent and specific GABA-uptake inhibitors. The novel amino phosphinic acids were tested for their GABA-uptake inhibitory activity and 2 and 4 were identified as the first phosphinic acid based GABA-uptake inhibitors. The methylphosphinic acid 3 was found to be inactive.

Development of comparative anchor tagged sequences (CATS) for canine genome mapping.

The development of a useful genetic map of the domestic dog would benefit by the inclusion of type I markers; coding genes that can connect the canine map to the homologous gene maps of other mammalian species. A group of 280 comparative anchor tagged sequences (CATS), and universal mammalian sequence tagged sites (UM-STS), were optimized for canine assessment. One hundred and five were screened for genetic polymorphism among nine canine breeds and three wild species of Canis in an attempt to promote gene mapping of comparative type I markers. Three categories of variation--size, restriction fragment length polymorphism (RFLP), and single-strand conformation polymorphism (SSCP)--were assessed. The data showed that 50% of the type I markers discriminate between species and 40% showed genetic variation among dog breeds. Although polymorphism incidence between nominated breeds for gene mapping is more limited than found for established reference pedigrees in other species, the concept and application of these CATS and UM-STS markers is useful in capturing the comparative information required for the full application and efficacy of the dog gene map.

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase.

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.

Determination of Pranlukast and its metabolites in human plasma by LC/MS/MS with PROSPEKT on-line solid-phase extraction.

A highly sensitive and selective liquid chromatography/ionspray tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of Pranlukast and its oxidative metabolites (SB 240103, SB 241484 and SB 218663) in human plasma in order to support pharmacokinetic studies. The method employed direct injection of human plasma into an on-line solid phase extraction (SPE) PROSPEKT instrument for isolation of the analytes followed by column switching to the LC/MS/MS. The use of on-line SPE resulted in reduced sample preparation time and cleaner extracts, therefore minimizing ion suppression and HPLC back-pressures issues. The use of a 20 mM ammonium acetate-methanol system and a step gradient yielded intense ion species, excellent separation between the polar metabolites and the parent drug and sufficient selectivity for baseline resolution of the two positional isomers, SB 240103 and SB 218663. Pranlukast, its metabolites and the internal standard (SK&F 108566) were quantified using a turbo-ionspray interface by negative ion selected reaction monitoring (SRM). The lower limit of quantification (LLQ) for the assay was 10.0 ng ml-1 for Pranlukast and 1.00 ng ml-1 for its metabolites based on a 100 microliters plasma aliquot. The calibration curves were linear for analyte concentrations ranging from 10.0 to 2000 ng ml-1 for Pranlukast and 1.00 to 200 ng ml-1 for the metabolites. The calculated intra- and inter-assay precision from quality control (QC) samples resulted in mean variability values of less than 12% for all analytes. Pranlukast and its metabolites were shown to be stable under routine analysis conditions for clinical trial samples. The method provides automated sample analysis in a total cycle time of 5 min with improved robustness, sensitivity, selectivity, accuracy and reproducibility compared to the existing methodology.

RXRA and HSPA5 map to the telomeric end of dog chromosome 9.

Previous results showed that loci from human chromosome 17q (HSA17q) map to the centromeric two-thirds of dog chromosome 9 (CFA9). In these studies fluorescence in situ hybridization (FISH) using a human total chromosome 17 painting probe, indicated that the telomeric one-third of CFA9 must have homology to one or more human chromosomes other than HSA17. Here we report that this distal part of CFA9 contains a segment syntenic to the telomeric end of HSA9q and mouse chromosome 2 (MMU2). The gene loci encoding retinoid X receptor, alpha (RXRA) and heat shock protein 5 (HSPA5 or GRP78), which are found on HSA9q34 and MMU2, occupy a region on CFA9 distal to NF1 and CRYBA1. FISH of a canine specific genomic cosmid clone for RXRA demonstrated the more telomeric localization of this locus to NF1 on CFA9. A linkage map developed for the distal region of CFA9 included: NF1-(2.7 CM)-CRYBA1-(6.5 CM)-RXRA-(22CM)-HSPA5. The next best order, RXRA-NF1-CRYBA1-HSPA5 with a difference in the log odds of 1.43 does not correspond to our findings with FISH. The most probable map order places HSPA5 distal to RXRA on CFA9 whereas in humans it lies centromeric of RXRA on HSA9q34.

Complementation of a fibre mutant adenovirus by packaging cell lines stably expressing the adenovirus type 5 fibre protein.

Adenovirus-based gene therapy vectors now in use cannot be targeted to specific cell types in vivo and are immunogenic, properties which limit their clinical utility. Improved vectors lacking the genes for viral structural proteins may overcome these limitations. We have developed cell lines which stably express the adenovirus type 5 (Ad5) fibre protein in its native trimeric form. These cells can complement an Ad5 mutant with a defect in the fibre gene, and are capable of incorporating the Ad5 fibre into particles of a different Ad serotype. As the fibre protein is responsible for the initial binding of virus to cells, packaging cell lines expressing different or modified fibre proteins will be useful in studying the mechanism by which adenovirus infects different cell types.

Synthesis and hybridization properties of an acyclic achiral phosphonate DNA analogue.

Protected N-(2-hydroxyethyl)-N-(nucleobase-acetyl)aminomethanephosphonic+ ++ acid (6a-d) of all four DNA nucleobases have been prepared and oligomerized by solid-phase synthesis. Four DNA decamers containing 1-10 of these 'PPNA' monomers were prepared and evaluated by Tm measurements (medium salt) for binding to their DNA and RNA complements. One central modification reduced the binding strongly (delta Tm = -10 degrees C), but contiguous PPNA monomers gave smaller effects, and the all-PPNA decamer bound to RNA with a delta Tm of -1.2 degrees C per modification. Thus PPNA oligomers are inferior DNA and RNA binders compared to the closely related and strongly binding PNA oligomers.

Severe intraoperative coagulopathy following routine aortobi-iliac bypass with knitted Dacron graft.

Coagulopathy associated with aortic operation is generally described in patients undergoing supraceliac aortic clamping or ruptured abdominal aortic aneurysm repair. This intraoperative complication is managed with component blood replacement and occasional use of aminocaproic acid. The presence of intraoperative coagulopathy associated with aortic reconstruction for occlusive disease is not described. We report a case of severe intraoperative coagulopathy associated with aortobi-iliac artery bypass with a knitted Dacron prosthesis. Management of this complication was accomplished with administration of blood components, aminocaproic acid and explant of the Dacron prosthesis with polytetrafluoroethylene graft placement. The possibility of fibrinolysis induced by the knitted Dacron prosthesis is suggested.

Preparation of oligodeoxyribonucleoside phosphorodithioates by a triester method.

A method to prepare thymidine phosphorodithioate dimers (ref. 1) has been extended to allow the preparation of oligo-2'-deoxyribonucleotide phosphorodithioates containing all four bases. The method is suitable for large-scale synthesis and gives phosphorodithioates without phosphorothioate impurities (31P nmr, detection limit 0.5 to 1%). Oligonucleotides up to octamers which contain -0-(PS2-)-0- linkages at all positions have been prepared by block synthesis in solution. The phosphorodithioate linkage is introduced by the reaction of a 5'-O, N-protected nucleoside (or oligonucleotide) with a dithiophosphorylating agent RSP(S)(ODhbt)2, R = 2,4-dichlorobenzyl, Dhbt = 3,4-dihydro-4-oxo-benzotriazin-3-yl, followed by coupling of the product to a 3'-O,N-protected nucleoside (or oligonucleotide). This method gives pure protected oligodeoxyribonucleoside phosphorodithioates, and phosphorothioate linkages are only introduced if contact with conc. aqueous ammonia during or after deblocking is unduly prolonged.

A differential synaptic input to the motor nuclei of triceps surae from the caudal and lateral cutaneous sural nerves.

1. Postsynaptic potentials (PSPs) were recorded in 115 triceps surae motoneurons of 10 chloralose-anesthetized adult cats (spinal cord intact), upon electrical stimulation of the caudal and lateral cutaneous sural nerve branches (CCS and LCS, respectively). 2. With twice threshold (2T) stimulation of CCS, excitatory PSPs (EPSPs) were the predominant effect in 95% of all medial gastrocnemius (MG) motoneurons tested (min. central latency 1.5 ms; mean 2.4 ms). In only a few MG cells was the EPSP followed by an inhibitory postsynaptic potential (IPSP) and in only one cell was an IPSP the sole effect. Increasing the stimulus intensity to 5T tended to enhance both the later EPSP and IPSP components, with less change in the amplitude or latency of the earliest EPSPs. 3. In lateral gastrocnemius (LG) and soleus (SOL) motoneurons, 2T CCS stimulation led to either inhibition or no potential change in the majority of cells tested: EPSPs were the predominant effect in only 15 and 30% of LG and SOL cells, respectively (min. central latency 2.5 ms; mean 3.0 ms) and rarely occurred without subsequent inhibition. Again, increasing the stimulus intensity to 5T had more of an effect on later rather than earlier PSP components. 4. A predominance of depolarization in MG motoneurons but not in SOL motoneurons is in agreement with previous findings that CCS excitation is more powerful in "fast type" triceps surae motoneurons. However, the strong predominance of hyperpolarizing effects of CCS stimulation in the present LG population is evidence that such an organization does not transcend triceps surae motor nuclei as a whole. 5. Postsynaptic effects of LCS stimulation at 2T were frequently weak or absent but increasing the stimulus intensity to 5T produced predominant inhibition in 71% of all triceps surae motoneurons studied (n = 107). Of the few cells which did receive excitation from this nerve, most were MG, a few were SOL, and none were LG. These EPSPs occurred more frequently at 5T than at lower stimulation strengths. 6. The results indicate that excitation produced by electrical stimulation of the ipsilateral CCS nerve occurs preferentially in the MG portion of triceps surae and with the shortest central latencies. Effects of LCS stimulation are largely inhibitory throughout the motor nuclei comprising triceps surae but even here, the presence of excitation occurs more frequently in MG. A comparison of these results with those in other reports is discussed.