Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785).

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.

Ring distraction technique for measuring surface tension of sputum: relationship to sputum clearability.

Poor sputum clearance has been related to sputum adhesion tension. In this study, we describe a modified du Noüy ring method for measuring the surface tension (gamma) of small samples of sputum and for comparinge the calculated work of adhesion (Wad) for sputum specimens with the measured mucociliary transportability (MCTR) and cough transportability (CTR). The gamma, as measured by this method, correlates with gamma measured by sputum contact angle on a low-surface-energy solid (R2 = 0.368, P = 0.03). There is a small but significant difference in measurements made by these two methods (P = 0.03). Wad calculated from the surface tension ring method is inversely correlated with CTR (R2 = 0.181, P = 0.004) but has no correlation with MCTR in this study. The miniaturized ring method gives accurate and reproducible measurements of the surface tension of small amounts of respiratory secretions. Because sputum behaves enough like a liquid that the assumptions made in using the Young equation to calculate Wad appear valid, we also showed that the Neumann equation can be used to determine the surface tension of sputum by its contact angle on tetrafluoroethylene (Teflon).

alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6,7-dimethoxy-alpha- [(4-methylphenyl)thio]-2(1H)-isoquinolineheptanenitrile (CL 329,753): a novel chemosensitizing agent for P-glycoprotein-mediated resistance with improved biological properties compared with verapamil and cyclosporine A.

Agents that inhibit P-glycoprotein may restore sensitivity to some antitumor drugs in cancer patients. Optimization of the specificity and potency of one class of chemosensitizing agents related to verapamil has led to the identification of alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6, 7-dimethoxy-alpha-[(4-methylphenyl) thio]-2(1H)-isoquinolineheptanenitrile, designated CL 329,753. In vitro, 0.1 to 2.0 microM CL 329,753 restored sensitivity to drugs in the multidrug resistance (MDR) phenotype in cell lines that overexpress P-glycoprotein. CL 329,753 was greater than 10-fold more potent and efficacious than cyclosporine A or verapamil in vitro, particularly in cells that express high levels of P-glycoprotein. The enhanced activity of CL 329,753 may be related to its inability to be transported by P-glycoprotein, since low drug accumulation of cyclosporine or verapamil but not CL 329,753 was found in P-glycoprotein-containing cells, yet all three agents inhibited vinblastine binding to membranes containing P-glycoprotein and inhibited photoaffinity labeling of P-glycoprotein. In vivo, CL 329,753 resensitized drug-resistant tumors to vinblastine or doxorubicin in an ascitic or solid tumor model, respectively. No alteration in the plasma pharmacokinetic profile of doxorubicin by CL 329,753 has been found. Furthermore, the compound had 70-fold less calcium channel antagonistic activity compared with verapamil.

An investigation of the foetal rat spinal cord. I. Ultrastructural observations on the onset of synaptogenesis.

Ultrastructural studies on the foetal rat spinal cord show that during synaptogeneis there is difficulty in recognizing true synaptic precursors. Symmetric densities are found at unusual sites forming, for example, somato-dendritic and somato-somatic junctions. They are also found between neurons and possible glial processes. Symmetric densities occur between nerve cells but may be confused with desmosomes. Profiles exhibiting membrane density, cleft material and 50 nm vesicles, which are the most reliable indicators of presumptive synapses, are found between neurones, but also at junctions between neurons and what may be glial processes. The picture is further confused by the presence of degeneration axodendritic synapses at early foetal stages. Caution must be exercised in defining an apparent synapse or precursor in foetal cord as that of a presumptive functional synapse because of the observed degenerating profiles and because of our knowledge, somato-somatic, somato-dendritic, and neurono-glial synapses have not been observed in adult cord. It is not known whether these structures are an unwanted consequence of development or play a role in guiding development.

An investigation of the foetal rat spinal cord. II. An ultrastructural study on the development of synapses with the aid of observations on some electrophysiological properties.

Electrophysiological and ultrastructural studies were carried out on foetal rat spinal cord. The electrophysiological observations allowed certain identification of the site of second order sensory neurones, regions of the most functionally mature ventral horn cells and the adequacy of reflex conduction at 18 days. In the ultrastructural studies we made use of these identifications. No definitive synapses were found at 13-14.5 days in dorsal and ventral horn neuropil though some possible precursors were seen. Immature axodendritic synapses are found first in both dorsal and ventral marginal zones at 14.5 days and in both dorsal and ventral neuropil regions at 15-16 days. At 17 days there is an abrupt increase in frequency and maturity of synaptic profiles in all regions; synapses containing pleomorphic populations of vesicles are first seen in the ventral horn neuropil at this age as rare axo-somatic synapses. At 18 days the synapses population increases and multiple contacts involving axons or dendrites commonly occur. Furthermore, axo-somatic synapses are seen for the first time in the dorsal horn. From 20 days onwards mature synapses were commonplace and all earlier stages can be found. In addition axo-dendritic synapses with pleomorphic populations of vesicles were first seen in the dorsal horn. Axo-somatic synapses in the dorsal horn remained immature in appearance at this time. These findings are discussed particularly in relationship to previous studies by others on the development of motility in the rat. It appears that in the rat lumbar cord, onset of formation of different synapse types in specific locations precedes the onset of possible related functions by 1-2 days.