WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression.

BACKGROUND AND PURPOSE: Antidepressants, which raise the CNS concentrations of 5-HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity. EXPERIMENTAL APPROACH: In this report, we present the in vitro pharmacological and in vivo pharmacokinetic and pharmacological properties of the novel, orally available and CNS penetrant inhibitor of the noradrenaline transporter (NET), WAY-318068 (1-[(1S,2R)-1-(3,5-difluorophenyl)-2-hydroxy-3-(methylamino)propyl]-7-fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one). KEY RESULTS: WAY-318068 is a potent and effective inhibitor of the NET with a K(i) of 8.7 nM in a binding assay, and an IC(50) of 6.8 nM in an assay of transporter function, without significant binding to the dopamine transporter. Furthermore, the compound has only weak activity at the 5-HT transporter, leading to a functional selectivity of greater than 2500-fold. It is orally bioavailable with substantial quantities of the compound found in the CNS after oral dosing. As measured by microdialysis in rats, the compound causes a robust and significant increase in cortical noradrenaline levels without affecting 5-HT. WAY-318068 was effective in models of acute, visceral, inflammatory, osteoarthritic, neuropathic, diabetic and bone cancer pain, as well as in traditional models of depression at doses that do not cause motor deficits. CONCLUSIONS AND IMPLICATIONS: Collectively, the present results support the conclusion that selectively increasing CNS levels of noradrenaline is sufficient for efficacy in models of depression and pain.

Cloning, localization, and functional expression of sodium channel beta1A subunits.

Auxiliary beta1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of alpha and beta1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of beta1, termed beta1A, that results from an apparent intron retention event. beta1 and beta1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. beta1A mRNA expression is developmentally regulated in rat brain such that it is complementary to beta1. beta1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of beta1 expression. In contrast, beta1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed beta1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of beta1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of alphaIIA and beta1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing alphaIIA alone. This increase in current density reflected two effects of beta1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [(3)H]Saxitoxin binding analysis revealed a 4-fold increase in B(max) with no change in K(D) in cells coexpressing alphaIIA and beta1A compared with cells expressing alphaIIA alone. beta1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of beta1A subunits on sodium channel function may be physiologically important events in the development of excitable cells.

Identification of a neuronal calmodulin-binding peptide, CAP-19, containing an IQ motif.

Neurons produce polypeptides which can bind the calcium-poor or pre-activated form of calmodulin. It is expected that this class of peptide will serve an important role in maintaining cellular homeostasis since it would modulate calcium-dependent target regulation and redirect intracellular signaling. The lack of conserved sequence has made the identification of these peptides difficult, consequently leading us to exploit their property of binding calcium-poor calmodulin as a means of finding new species. A new peptide termed Calmodulin-Associated Peptide-19 (CAP-19) was purified and characterized. The protein-sequence information was employed in order to recover a cDNA clone from rat which included the entire reading frame for the peptide. Like its counterparts, neuromodulin (GAP-43), neurogranin (RC3) and PEP-19, it contains an IQ motif although the remainder of the peptide is quite different. Northern blot analysis of ribonucleic acid (RNA) from animals of differing ages indicated that the message appears at birth and then persists into adulthood. Antibodies to synthetic peptide were employed for localizing CAP-19. The results indicated that the peptide was localized to neurons in several brain regions. CAP-19 is similar to other calmodulin-binding proteins in that the domain spanning the IQ motif was demonstrated to participate in binding to calmodulin. Database searching showed CAP-19 to be homologous to the silkworm protein, multiprotein bridging factor 1 (MBF1). This homology suggests a potential new role for calmodulin-associated proteins in cellular homeostasis.

Metabolite and matrix interference in phenytoin immunoassays.

The major phenytoin metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin glucuronide (HPPG), was primarily responsible for the positive bias noted when uremic specimens were assayed with the Abbott TDx Free Phenytoin fluorescence polarization immunoassay. The amount of bias depended on both HPPG and phenytoin concentration, increasing with increases in either concentration. The new Abbott TDx II assays for phenytoin and free phenytoin exhibited no significant cross-reactivity with HPPG and no bias in clinical specimens from uremic patients. Both assays correlated well with Emit-based assays (r >0.98), had CVs of <3.5%, and had minimum detection limits of <0.1 mg/L. Calibration curves were stable for at least 6 weeks. All of the TDx assays cross-reacted with another metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), but expected HPPH concentrations are too low to cause a clinically significant bias. The Emit-based phenytoin assay exhibited a significant matrix effect when calibrators were prepared in defibrinated plasma processed to resemble serum.

The human brain homeogene, DLX-2: cDNA sequence and alignment with the murine homologue.

A novel homeobox-containing cDNA from the developing human brain has been cloned and sequenced. The transcript is most closely related to the Distal-less (Dll) homeogene of Drosophila melanogaster and to the Dlx genes in the mouse, specifically to Dlx-2. As such, it is the first report of a human Dll-like gene.

Molecular cloning and sequencing of a cDNA for olfactory marker protein.

cDNA clones corresponding to mRNA for rat olfactory marker protein (OMP) were isolated from a cDNA library. The library was constructed from olfactory mucosa poly(A)+ RNA enriched for OMP mRNA and cloned into a pBR322-derived plasmid, pMG5. OMP cDNA clones were detected by using a 17-base oligonucleotide probe that contained all 16 possible sequences coding for a known partial amino acid sequence of rat OMP. The identity of these clones was confirmed by hybrid-selected translation and nucleotide sequencing. The sequence of one clone was determined and contained the complete OMP coding region of 486 nucleotides followed by 1630 nucleotides of the 3' untranslated region. The 3' untranslated region included the polyadenylylation signal 16 nucleotides upstream of the poly(A) tail. No other ATG-initiated open reading frame larger than 20 codons was present in register. RNA blot analysis of olfactory mucosa poly(A)+ RNA using this clone as a probe indicated that the level of OMP mRNA, but not its size, declined significantly within a few days following olfactory bulbectomy. OMP mRNA was not detected in 14 nonolfactory rat tissues. Surprisingly, a small amount of OMP mRNA was observed in olfactory bulb. The presence of OMP mRNA in olfactory bulb was confirmed by in vitro translation and immunoprecipitation. These results suggest either that a previously undescribed population of neurons in the olfactory bulb synthesize OMP or that OMP mRNA is transported to the bulb by axonal transport.

Olfactory neuron-specific protein is translated from a large poly(A)+ mRNA.

Poly(A)+ mRNA was isolated from rat olfactory mucosa and translated in a rabbit reticulocyte cell-free protein synthesizing system. Olfactory marker protein (OMP) of Mr 18,500 was faithfully produced by this system upon addition of mucosal mRNA. The protein was identified by radioimmunoprecipitation with specific anti-OMP serum and by competitive displacement of the radioactive product with authentic OMP. In addition, the immunoprecipitated product comigrated with OMP on NaDodSO4/polyacrylamide gels and on HPLC. In vitro synthesized OMP represented 0.5% of the total translational products. Total olfactory mucosal poly(A)+ mRNA is approximately 1.5-21 kilobases in size, as determined by denaturing agarose gels. Translational assays of gel-fractionated poly(A)+ mRNA demonstrated that OMP mRNA occurs in the 2.5- to 3.4-kilobase range. An mRNA of this size could code for a protein significantly larger than OMP. Since the in vitro synthesized OMP is indistinguishable in size from OMP isolated from tissue, our data indicate that OMP is synthesized directly without the intermediate formation of a larger polypeptide precursor. Thus, OMP mRNA contains untranslated regions that are four to five times larger than the coding region.

Novel mode of cytotoxicity obtained by coupling inactive anthracycline to a polymer.

The inactive anthracycline analog 4-demethoxy-7,9-di-epi-daunorubicin was covalently coupled to polyglutaraldehyde microspheres. The polymer-bound analog acquired significant cytostatic activity as evaluated with doxorubicin resistant and sensitive murine L1210 leukemia cells. A suggested multiple membrane interaction at the cell surface may represent a novel mechanism of cytotoxicity.

Cell surface-mediated cytotoxicity of polymer-bound Adriamycin against drug-resistant hepatocytes.

Growth inhibition properties of Adriamycin-coupled polyglutaraldehyde microspheres have been assessed using a highly resistant rat liver cell line, RLC. Covalent attachment of Adriamycin to microspheres increased the cytostatic activity of the drug 1000-fold for this cell line as measured by 50% inhibitory concentration determinations. The effects of Adriamycin-polymer complexes were investigated and compared to free drug, using trypan blue dye exclusion and 51Cr release as indicators of cell viability. When carcinogen-altered drug-resistant rat hepatocytes were tested at an Adriamycin concentration of 10(-6) M, the polymer-bound drug killed 32% of the cells, while the free drug had no detectable cytotoxic effect. However, with normal rat hepatocytes at the same drug concentration, the Adriamycin-coupled microspheres were shown to be less toxic than free drug at 24 hr. It was demonstrated that greater than 99.5% of the drug remains covalently coupled to the microspheres throughout the experiments. Scanning electron micrographs are presented for both cell types, which demonstrate the effects of free and bound Adriamycin on the ultrastructure of the cell surface. The cells lose their microvilli, exhibit numerous blebs, and develop holes and pits in the surface. Transmission electron microscopy demonstrates that less than 1% of the microspheres is internalized by either cell type. Multiple interactions of the drug-polymer complexes with the cell surface are presented as the most probable explanation for the results.

Endoscopic cytology and biopsy in the diagnosis of gastroesophageal malignancy.

Direct-vision endoscopic cytologies and biopsies were performed on 1,437 patients during a five-year period, and the incidence, correct typing and diagnosis of gastroesophageal cancer were studied. At the first cytology, malignant cells were diagnosed in 80 cases, all of which were confirmed by the endoscopic biopsy; in 30 cases, despite suspicious cytology, no malignancy was seen at the first biopsy. At repeat endoscopic cytology and biopsy, however, 21 of these 30 cases were correctly correlated for malignancy, resulting in a final correct correlation in 101 of the 110 cases (91.8%) and a diagnostic discrepancy in 9 cases (8.2%). It thus appears that the procedure is useful in diagnosing gastroesophageal cancer in the majority of cases. In the cases of discrepancies, gastric ulcers were found later; the repeatedly suspicious cytologies were due to the cytologic atypia of the cells from regenerating hyperplastic and/or metaplastic epithelium at the margins of the ulcers. Such cells showed a wide spectrum of changes, ranging from mild atypia to severe atypia mimicking adenocarcinoma of the upper gastrointestinal tract.

Synthesis of adriamycin-coupled polyglutaraldehyde microspheres and evaluation of their cytostatic activity.

Adriamycin was coupled to polyglutaraldehyde microspheres having an average diameter of 4500 A. The coupled microspheres remained stable during incubation with cells. Full cytostatic activity was observed when the coupled adriamycin was tested with murine or human leukemia and murine sarcoma cell lines. A 10-fold increase in sensitivity was obtained with drug-resistant human leukemia cell lines. Repeated use of the coupled microspheres in the cytostatic assays did not increase their activity, indicating that these complexes can be recycled. The results suggest that coupled adriamycin sufficiently perturbs the plasma membrane to lead to cytostatic activity. It is proposed that this mode of drug delivery provides multiple and repetitious sites for drug-cell interactions. In addition, the drug-polymer complexes may overcome those forms of resistance that are the result of decreased drug binding at the cell surface.