N-Alkyl-N-arylmethylpiperidin-4-amines: novel dual inhibitors of serotonin and norepinephrine reuptake.

A series of N-alkyl-N-arylmethylpiperidin-4-amines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.

Benzothienyloxy phenylpropanamines, novel dual inhibitors of serotonin and norepinephrine reuptake.

A series of benzothienyloxy propylamines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake.

Use of the Hmb backbone-protecting group in the synthesis of difficult sequences.

Aggregation due to hydrogen-bonded interchain association is thought to be the cause of difficult sequences in solid-phase peptide synthesis. Hmb (2-hydroxy-4-methoxybenzyl) was introduced recently as a backbone-protecting group for Fmoc/tBu strategies which inhibits this association. Hmb derivatives of four amino acids are now available commercially. This paper describes the syntheses of two difficult sequences which were achieved by judicious use of the Hmb protecting group. (Hmb)Gly, which is attractive because of the ease of coupling the following residue, did not always produce the expected long-range effect. Moving the Hmb protection to a suitable preceding position in the sequence overcame this shortcoming.

Synthesis of disulfide-bridged fragments of omega-conotoxins GVIA and MVIIA. Use of Npys as a protecting/activating group for cysteine in Fmoc syntheses.

The 3-nitro-2-pyridinesulphenyl (Npys) moiety is finding increasing utility as a protecting-activating group for cysteine, particularly in the synthesis of cyclic and unsymmetrical disulfides using the Boc strategy. This chemistry has been extended to peptides assembled by the Fmoc strategy. N-Terminal Cys(Npys) is introduced via Boc-Cys(Npys)-OPfp. Non-N-terminal Cys(Npys) is incorporated by reacting a resin-bound, fully protected Cys(Acm) peptide with NpysCl. This approach has been applied to the synthesis of four disulfide-bridged fragments of omega-conotoxins GVIA and MVIIA.

Purification of mouse haptoglobin by antibody affinity chromatography and development of an ELISA to measure serum haptoglobin levels.

Mouse haptoglobin was isolated from acute-phase serum initially by affinity chromatography on haemoglobin-Sepharose. This proved inefficient, but sufficient material was obtained for use as an immunogen. Rabbit anti-haptoglobin antibodies were used as immunoabsorbents to isolate larger quantities of haptoglobin. Subsequently, specific anti-haptoglobin antibodies were prepared by affinity chromatography on haptoglobin-Sepharose. A direct sandwich ELISA for mouse serum haptoglobin was developed, using affinity purified reagents. The working range of the haptoglobin standard curve was 0.02-0.5 microgram/ml. The reagents did not cross-react with albumin or haemoglobin and the antibody also recognised rat haptoglobin.

Drug localisation and growth inhibition studies of vindesine-monoclonal anti-CEA conjugates in a human tumour xenograft.

The distribution of tritiated vindesine (3H-VDS) was studied in the tissues and tumours of athymic mice bearing a human colorectal tumour xenograft. Selective tumour localisation was obtained when 3H-VDS was injected as a conjugate with a monoclonal anti-CEA antibody (11.285.14) but not as a conjugate with a non-binding monoclonal IgGl (Ag8) or as free succinoyl-VDS. The amounts of VDS that localised in the tumour following injections of 3H-VDS-11.285.14 increased in proportion to the amount injected, over a wide dose range. Conjugates prepared using the Fab fragments of 11.285.14 showed no evidence of selective tumour uptake in comparison with normal tissues. Various dose levels of VDS-11.285.14 conjugate and free VDS were studied for effects on the growth of the tumour xenograft. A growth inhibition of 50% was obtained at 1.5 mg/kg with free VDS and at 2.5 mg/kg with conjugated VDS. The conjugate was, however, considerably less toxic.

Experience in the preparation and experimental use of immunocytostatics.

Vindesine (VDS) was coupled directly to a monoclonal antibody (791T/36) raised against a human osteogenic sarcoma cell line, and methotrexate (MTX) was coupled to 791T/36 via an intervening human serum albumin (HSA) bridge. Both the VDS-791T/36 and MTX-HSA-791T/36 conjugates were cytotoxic in vitro specifically for tumour target cells expressing the 791T/36-defined antigen, while the free drug in each case was indiscriminately toxic to all target cells. The VDS-791T/36 conjugate retarded growth of osteogenic sarcoma xenografts in immunodeprived mice when administered in multiple doses. Free 791T/36 did not significantly affect tumour growth. VDS was tumour inhibitory, but was toxic to the mice at a total dose of 20 micrograms per kg body weight, while VDS-791T/36 conjugate was not toxic at total doses incorporating VDS at up to 45 mg per kg. It is suggested that this is due to selectivity conferred upon the conjugate by the antibody moiety, and that such conjugates may offer considerable potential as anti-cancer agents.

Selective cytotoxicity against human tumour cells by a vindesine-monoclonal antibody conjugate.

The anti-mitotic drug vindesine was coupled chemically to a monoclonal antibody raised originally against the human osteogenic sarcoma cell line, 791T. The cytotoxicity of the conjugate in vitro was tested, in comparison with free vindesine, against sarcoma 791T and other antigenically cross-reactive osteogenic sarcoma-cell lines, and also against tumour cell lines which have no detectable reaction with the monoclonal antibody. Continuous exposure of cultured 791T cells indicated that the vindesine was partially inactivated following conjugation since the conjugate was less toxic than the free drug. However, antibody-binding activity was essentially preserved following conjugation. Despite diminished drug activity in the conjugate, assays designed to mimic antibody binding to tumour in which target cells were treated with conjugate and washed before culture, showed selective cytotoxicity for osteogenic sarcoma lines with little or no effect on non-cross reactive control cells. In comparison, free vindesine was toxic equally for all cell lines and free antibody was non-toxic. These studies indicate that conjugation of a cytotoxic agent to a monoclonal antibody can confer on that agent selectivity for a particular target cell type which is recognised by the antibody.

Localisation and toxicity study of a vindesine-anti-CEA conjugate in patients with advanced cancer.

Safety of administration of a vindesine (VDS)-anti-CEA conjugate and its ability to localise after radiolabelling were investigated in patients with advanced metastatic carcinoma (4 colorectal and 4 ovarian). For imaging, patients received between 230 and 520 micrograms of 131I labelled antibody. In 5, localisation of conjugate was demonstrated, in another it was equivocal and in 2 patients, undetectable. For assessment of safety each patient also received a single dose of conjugate increasing from 1.2 to 42 mg antibody linked to 24 to 1800 micrograms VDS. The in vitro activity of the anti-CEA antibody and its ability to localise in vivo were preserved after conjugation. There was no obvious toxicity or hypersensitivity attributable to either the radiolocalisation or escalated doses of conjugate in any of the patients. The feasibility of the preparation and administration to patients of a vindesine-antibody conjugate has been demonstrated.

3-phenylazo-4-hydroxyphenylisothiocyanates: reagents for efficient haptenation of immunoglobulin and other carrier molecules.

The synthesis of 2 non-cross-reacting 3-(substituted-phenyl)-azo-4-hydroxyphenylisothiocyanates is described. These compounds are designed as alternatives to the corresponding hydroxybenzimidates for use in hapten-sandwich double labelling of cell surfaces. The new reagents rapidly and controllably haptenate immunoglobulin and other biological carrier molecules. Haptenated immunoglobulins present the appropriate hapten determinant with little concomitant loss of antigen binding activity.

Positive and negative selection of cells by hapten-modified antibodies.

A method for simultaneous positive and negative selection of cells identified by an antibody is described. It is an adaption of the panning technique using anti-benzene arsonate (Ars)-coated Petri dishes to select the Ars-antibody-coated cells. The adherent cell population is recovered in a suitable state by hapten elution with Ars-azo-tyrosine.

The relationship between hyperglycaemia and renal immune complex deposition in mice with inherited diabetes.

Kidney lesions were studied by light microscopy and immunofluorescence in diabetic (db/db) and obese (ob/ob) mutant mice. The db/db mutation was studied both on the C57Bl/KsJ genetic background (where it produces severe hyperglycaemia) and on the C57Bl/6J background (where hyperglycaemia is only mild). In all cases, more IgG, IgM and C3 were deposited in the renal glomeruli of mutant mice than in the glomeruli of normal (+/?) mice of equivalent age. First signs of immunoglobulin deposition occurred at a slightly younger age than first signs of C3 deposition or histological change (mainly mesangial thickening). Insulin deposits were occasionally seen in the glomeruli of older mutant mice and immunoglobulin eluted from diabetic mouse kidneys had anti-insulin activity. Increased anti-DNA activity was present in the serum of older mutants. In those mutants with severe hyperglycaemia, the macula densa and distal convoluted tubules also contained immunoglobulin deposits, probably derived from the glomerular mesangium. Urine from diabetic mice contained high molecular weight material reacting with antisera to Fab or kappa but not the Fc portion of immunoglobulin. We conclude that diabetic mice have immune complexes in the kidney containing antibodies against insulin and possibly other antigens. We find no evidence that hyperglycaemia itself is the direct cause of glomerular immune complex deposition, although there may be a link between hyperglycaemia and tubular dysfunction.

Stereospecific inversion of (R)-(-)-benoxaprofen in rat and man.

(R)-(-)-benoxaprofen is stereospecifically inverted to the (S)-(+)-enantiomer by rats and humans. The rate of inversion is much faster in rats (t 1/2 ca. 2.5 h) than in humans (t 1/2 108 h). Inversion in rats apparently does not occur in the liver, but can be brought about in vitro by an everted intestinal sac preparation, suggesting that the transformation takes place while passing through the gut wall.