Protein conjugates of defined structure: synthesis and use of a new carrier molecule.

A new carrier molecule, NH2OCH2CO-(Gly)3-[Lys(H-Ser-)]5-Gly-OH, has been synthesized to facilitate the preparation of protein conjugates of defined structure. Special features are as follows: (i) (aminooxy)-acetyl as a terminal group, which reacts specifically to form an oxime bond under very mild conditions with an aldehyde group placed on a protein in a prior step; (ii) a spacer group of three Gly residues; and (iii) a set of five Lys residues, each of which is acylated with a Ser residue. A second form of the carrier molecule, HCO-m-C6H4CH = NOCH2CO-(Gly)3-[Lys(H-Ser)]5-Gly-OH, was also prepared. This form possesses a terminal aldehyde group which permits site-specific attachment by formation of a hydrazone bond to the carboxyl termini of polypeptide chains which have been modified enzymatically with carbohydrazide in a prior step. Once the carrier is linked to protein in one of the above ways, i.e. through formation of either an oxime or hydrazone bond, the Ser residues of the carrier (but not of the protein) may be oxidized by very mild periodate treatment to generate aldehyde groups. Drugs possessing a hydrazide group (e.g. methotrexate gamma-hydrazide or desacetylvincaleukoblastine hydrazide) may then be conjugated via hydrazone formation to the aldehyde groups of the carrier. A cluster of five drug molecules may thus be attached to a single site on a protein, giving a relatively homogeneous product in spite of the high drug conjugation ratio. Synthesis of the carrier, formation of a pentadrug-protein conjugate, and wider implications of the chemistry are presented.

Chemoimmunoconjugate development for ovarian carcinoma therapy: preclinical studies with vinca alkaloid-monoclonal antibody constructs.

Preclinical efficacy studies are presented in a human ovarian carcinoma model utilizing several novel conjugation strategies with the KS1/4 monoclonal antibody and derivatives of the vinca alkaloid desacetylvinblastine hydrazide. The chemoimmunoconjugates KS1/4-beta-alanine-methylenemalonic acid ethyl ester-4-decacetylvinblastine 23-hydrazide (KS1/4-BAMME-DAVLB-HY), KS1/4-beta-alanine-5-formylpyrrole-2-carboxylic acid-4-desacetylvinblastine 23-hydrazide (KS1/4-BAP-DAVLB-HY), and KS1/4-4-desacetylvinblastine 23-hydrazide were explored in the OVCAR-3 human ovarian carcinoma xenograft model. These conjugates, constructed with variable linker stability between the vinca alkaloid and the antibody, were studied by comparing the route of administration and the treatment schedule. Under these conditions a mean survival time from 28 to 35 days in untreated control animals was observed. Significant increases in survival (i.e. 3-9-fold over untreated control animals) were observed with all the immunoconjugates tested but with varying potency and efficacy dependent on linker strategy. Parallel therapy with equivalent doses of free DAVLB-HY or a non-antigen-binding immunoconjugate did not significantly increase the survival of the animals. These results suggest several chemoimmunoconjugate strategies for site-directed therapy of human ovarian cancer.

Progress in the medicinal chemistry of dopaminergic agents.

Ergot alkaloids and their derivatives have long been recognized for their potent pharmacologic activity. A number of ergot derivatives, including the dopamine agonists bromocriptine and pergolide, are currently in clinical use for the treatment of CNS and endocrine disorders. In an effort to develop more selective dopamine agonists, studies were directed to elucidate the dopaminergic pharmacophore of the ergoline nucleus. During the course of this work, it was found that the tricyclic system containing only the B-, C-, and D-rings of the ergoline skeleton (2, X = CH) possessed D-2 dopamine agonist activity. As a result of this discovery, interest was stimulated in the preparation of other heteroareno[g]quinoline systems (3, "BCD partial ergolines") for investigation of their dopaminergic properties. Factors which we found to be particularly important in determining dopaminergic activity were: (1) the nature of the heteroaromatic B ring; (2) the orientation of that heteroaromatic ring; (3) the substituents on the heteroaromatic ring; and (4) the relative and absolute stereochemistry at the CD ring fusion. We report here the synthesis and pharmacologic activity of a series of BCD partial ergolines (3) and describe how the study of these new compounds allows for the delineation of structural features important in D2 dopamine receptor activation.

In vivo antitumor activity of a panel of four monoclonal antibody-vinca alkaloid immunoconjugates which bind to three distinct epitopes of carcinoembryonic antigen.

A panel of four murine monoclonal antibodies apparently directed against three distinct epitopes of carcinoembryonic antigen (CEA) was conjugated via oxidized carbohydrate groups to 4-desacetylvinblastine-3-carboxyhydrazide. The resulting antibody-vinca conjugates were evaluated for antitumor activity against 2-9-day-established LS174T human colorectal carcinoma xenografts. The antibodies (immunoglobulin G, IgG) employed in this study were 11.285.14 (IgG1), 14.95.55 (IgG2a), CEM231 (IgG1), ZCE025 (IgG1). Additive immunofluorescence studies indicated that CEM231 and ZCE025 recognized the same or a closely related epitope(s) on CEA which was distinct from the two epitopes bound by 11.285.14 and 14.95.55. The in vivo antitumor efficacy studies demonstrated that chemoimmunoconjugates prepared from 14.95.55 and ZCE025 were more active than the conjugates constructed from the 11.285.14 and CEM231 antibodies. The 14.95.55 and ZCE025 immunoconjugates were also more efficacious than free drug or drug conjugated to irrelevant murine IgG. The presence of increased carbohydrate content on the light chain of ZCE025 may have been responsible for the ability to construct ZCE025-vinca conjugates with about twice the drug content (approximately 10 mol of vinca/mol of IgG) than was achieved with the other antibodies. The highly conjugated form of ZCE025 demonstrated similar efficacy but was much less toxic than a ZCE025 conjugate containing 5 mol of vinca/mol of IgG. These data indicated that significant differences existed in the ability of monoclonal antibodies to target a cytotoxic agent for effective antitumor activity even when the immunoconjugates recognized the same antigen or even the same or closely related antigen epitope(s). Furthermore, these differences could not have been identified without extensive in vivo evaluation for antitumor efficacy.

Preclinical studies on quinelorane, a potent and highly selective D2-dopaminergic agonist.

Quinelorane (LY163502) has the endocrine, neurochemical and behavioral profile of a potent and highly selective D2-dopaminergic agonist. The administration of quinelorane produced dose-related decreases in serum prolactin concentration of reserpinized, male rats and increases in serum corticosterone concentration of male rats. The minimum effective doses (MED) for these effects were 10 and 30 micrograms/kg i.p., respectively. Quinelorane induced increases in 3-methoxy-4-hydroxyphenylglycol-sulfate levels in the brain stem (MED, 30 micrograms/kg i.p.) and decreases in hypothalamic epinephrine levels (MED, 100 micrograms/kg i.p.) in male rats as determined by high-pressure liquid chromatography with electrochemical detection methods. Quinelorane induced increases in extracellular ascorbic acid as determined by in vivo voltammetry in the nucleus accumbens and striatum of male rats. Quinelorane produced concentration-dependent suppression of K+-evoked release of acetylcholine from superfused caudate slices, with an IC50 of approximately 10(-8)M. Quinelorane administration produced dose-related increases in compulsive, contralateral turning in male rats with unilateral nigrostriatal lesions and increases in locomotor activity and stereotypic behavior in male rats. In dogs, quinelorane administration produced dose-related increases in emetic response with an ED50 of 7 micrograms/kg i.v. Quinelorane administration also produced dose-related decreases in the striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic (MED, 1 microgram/kg i.p. for both metabolites) as determined by high-pressure liquid chromatography with electrochemical detection methods and decreases in extracellular concentrations of homovanillic acid in the nucleus accumbens and striatum as determined by in vivo voltammetry., Quinelorane produced concentration-dependent decreases in K+-evoked dopamine release from superfused striatal slices (IC50 = 3 X 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity.

A method has been developed to allow the direct coupling of the cytotoxic vinca alkaloid 4-desacetylvinblastine-3-carbohydrazide (DAVLB hydrazide) to a variety of murine monoclonal antibodies directed against human solid tumors. Periodate oxidation of carbohydrate residues on the antibodies, followed by reaction with DAVLB hydrazide in aqueous acid affords, in most cases, conjugates with conjugation ratios of 4-6 vincas per antibody in high yield without significantly impairing antigen binding or solubility. The outcome of the conjugation reaction is highly dependent on the concentration of, and time of exposure of the protein to, the oxidant. These conjugates exhibit potent antitumor activity in vivo against a number of human solid tumor-nude mouse xenografts, with efficacy and safety increased over unconjugated DAVLB hydrazide. This antitumor activity is also superior to that of similarly prepared but nontarget tumor binding antibody-DAVLB hydrazide conjugates. MoAb-DAVLB hydrazide conjugates release DAVLB hydrazide in solution in a temperature- and pH-dependent manner. Hydrolytic release of unmodified DAVLB hydrazide from tumor-localized MoAb-DAVLB hydrazide conjugates in vivo may be an important factor in their antitumor activity.

In vivo efficacy of monoclonal antibody-drug conjugates of three different subisotypes which bind the human tumor-associated antigen defined by the KS1/4 monoclonal antibody.

A panel of three hybridomas has been isolated each of which secretes a single species of monoclonal antibody (MoAb) directed against the KS1/4 tumor-associated antigen originally described by Varki et al. (Cancer Res 44: 681, 1984). These MoAbs were designated L1-(IgG2b), L2-(IgG1), and L4-(IgG2a)KS. Binding specificity, immuno-precipitation, and competitive binding analyses indicated that these MoAbs each recognize the same epitope of the KS1/4 antigen. The immunoprecipitation studies indicated that the MoAbs recognized a major antigenic component of 42 kDa and a minor component of 35 kDa. The L-KS antibodies were evaluated as MoAb-drug conjugates against a variety of human tumor targets grown in vivo as nude mouse xenografts. The MoAb-drug conjugates were constructed using protein-A-purified MoAbs conjugated to 4-desacetyl-vinblastine-3-carbohydrazide. Efficacy was determined using various dosing protocols on 2-14 day established tumors of lung, pharynx, colon, and skin origin. Control experiments included the use of dual-flank antigen-positive and negative tumors, free MoAbs, free drug, and mixtures of MoAbs and drug. These studies indicated that significant tumor growth suppression and actual tumor regression could be achieved by the MoAb-vinca conjugates and that this activity was antigen-mediated. The drug conjugates were more efficacious than free drug or free MoAbs administered either singly or in combination with each other.

Elevation of acetylcholine levels in striatum of rat brain by LY163502, trans-(-)-5,5a,6,7,8,9a,10-octahydro-6-propylpyrimido less than 4,5-g greater than quinolin-2-amine dihydrochloride, a potent and stereospecific dopamine (D2) agonist.

LY163502, a partial ergoline and a trans-levorotatory enantiomer, does not stimulate adenylate cyclase in striatal membranes but inhibits 50% binding of 3H-apomorphine, 3H-pergolide and 3H-spiperone at 10, 13 and 151 nM (IC50), respectively. The racemic mixture (LY137157) is less effective, with 3, 2.7 and 1.4 times higher IC50 values, respectively, whereas the dextrorotatory isomer (LY175877) is inactive. LY163502 inhibits binding of 3H-clonidine with an IC50 value of 2600 nM, but not the binding of 3H-WB4101, 3H-dihydroalprenolol, 3H-serotonin, 3H-quinuclidinyl benzilate and 3H-pyramilamine or the uptake of serotonin, norepinephrine or dopamine, suggesting selective affinity toward dopamine receptors in vitro. Both LY163502 and LY137157 elevate striatal acetylcholine (Ach) levels. The elevation of Ach levels by LY163502 is reversed by dopamine antagonists haloperidol, cis-flupenthixol and metoclopramide. Therefore, the levorotatory enantiomer exhibits pharmacology of a D2 type of dopamine agonist.

Monitoring personnel exposure to stainless steel welding fumes in confined spaces at a petrochemical plant.

This paper documents exposure to stainless steel welding fumes at a petrochemical plant. The situation evaluated may be separated into three categories: 1) evaluation of background concentration levels in a maintenance shop (area monitoring), 2) evaluation of personnel exposures during open air welding in a maintenance shop (personal monitoring), 3) evaluation of personnel exposures in confined space welding (personal monitoring). Thirty-five area samples and seventy-six personal samples were collected and analyzed for chromium (VI), total chromium, nickel, iron, copper, and total particulates. The background concentrations in the maintenance shop were found to be far below those felt to be harmful. Personnel exposures found in the maintenance shop during open air welding were also low, especially when compared to current OSHA permissible exposure levels. Contaminant levels found during confined space welding, as in distillation towers, were considered excessive. After several possible solutions to the problem were considered, it was decided to implement a mandatory air-line respirator program for all employees entering confined spaces during stainless steel welding operations.