Analytical and biological evaluation of high throughput screen actives using evaporative light scattering, chemiluminescent nitrogen detection, and accurate mass LC-MS-MS.

In this investigation the utility of evaporative light scattering detection (ELSD) combined with HPLC-MS was demonstrated as a key component of a bioassay-guided fractionation, or "biofractionation" technique, for the evaluation of high throughput screen actives. ELSD provided on-line analyte mass information that was critical for the classification of the samples. Chemiluminescent nitrogen detection (CLND) was also evaluated for sample concentration estimation for nitrogen-containing compounds, and accurate mass LC-MS-MS analysis was employed for rapid structural confirmation and elucidation of components previously identified as active via biofractionation.

UPS on Weinreb resin: a facile solid-phase route to aldehyde and ketone derivatives of "unnatural" amino acids and peptides.

The solid-phase synthesis of "unnatural" amino aldehydes, amino ketones, peptide aldehydes, and peptide ketones was accomplished from commercially available resin in a series of room temperature reactions. The initial step involved addition of an "unnatural" side chain to the N-terminus of a benzophenone imine-activated Weinreb resin-bound amino acid or peptide derivative. The alkylated imine was hydrolyzed, and the amine was converted to the Boc-, Cbz-, or naphthoyl derivative. The resin-bound substrate was then cleaved with DIBAL-H or a Grignard reagent to give the amino aldehyde, amino ketone, peptide aldehyde, or peptide ketone products. Twenty-four reactions were carried out simultaneously using a "Billboard" reaction apparatus to give products in 27-87% (59% average) isolated yield.

Central venous catheters. An overview of Food and Drug Administration activities.

This article presents an overview of recent Food and Drug Administration issues pertaining to central venous catheters (CVCs) with emphasis on the CVC working group. Caveats for practitioners concerning CVC use are offered.

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.

Induction of immunogenicity of monoclonal antibodies by conjugation with drugs.

Human anti-mouse antibody has been a nearly consistent result of human clinical trials utilizing murine antibodies. It is generally anticipated that the problem of human anti-mouse antibody will be reduced as genetically engineered, more human ("humanized") antibodies become available. It is not clear, however, what effect chemical modification of such "humanized" antibodies will have on their immunogenicity. The present studies utilize a mouse antibody and rat host model to explore aspects of this question. Rats injected with unmodified mouse monoclonal antibodies failed to mount anti-mouse immune responses, presumably due to their phylogenetic relatedness. In contrast, rats injected with a Vinca immunoconjugate mounted strong anticonjugate antibody responses that were directed primarily against the linker portion of the conjugate. The in vivo serum pharmacokinetics of 125I-labeled antibody and conjugates were evaluated in rats with existing anticonjugate antibody. The peak serum level attained was inversely correlated with the level of reactivity of the anticonjugate antibody with the injected compound. This model provides a potentially useful tool for exploration of the immunogenicity of drug, toxin, or radionuclide monoclonal antibody conjugates.

Medical-device complication reporting. A quality assurance mechanism.

As the primary users of medical devices, nurses have a responsibility to report device-related problems to the federal government's Device Experience Network (DEN). The procedures for reporting these problems are described. Nurses wishing to conduct research into problems with medical devices may wish to access the DEN data base. A Food and Drug Administration study using DEN data to study central venous catheter complications is offered as an example of this data base's role in research.

Complications associated with central venous catheters. A survey.

The Federal Food and Drug Administration has a system for reporting problems with medical devices that requires manufacturers of medical devices to report medical complications or equipment malfunction that causes, or could cause, death or serious injury. In a two-year period, central venous catheters were associated with 170 complications: tissue perforation, loss of catheter integrity, (including: catheter separation, severance, break, rip, puncture, or leak), and other problems. Causes of the complications were related to device failure (12 percent), health care professionals (55 percent), patients (3 percent), or pathologic or physiologic aspects (3 percent); causes of 28 percent of the complications were indeterminable. Further analysis indicated that complications (especially tissue perforation) were primarily health professional technique-related. There were no reports of complications related to infection. Data support the need for more education in catheter application and the need to modify the system by which these data are reported to more reliably detect infection.

Increased plasma concentration of cross-linked fibrin polymers in acute myocardial infarction.

Thrombin cleaves fibrinopeptides from fibrinogen, converting it to fibrin monomer, and activates factor XIII, which catalyzes the formation of intermolecular epsilon-(gamma-glutamyl)-lysine bonds to stabilize the fibrin polymer. The formation of factor XIIIa-catalyzed fibrin polymers during clotting of plasma and purified fibrinogen in vivo was followed by a sodium dodecyl sulfate agarose gel technique, and an increase in both amount and size of gamma-chain cross-linked polymers was demonstrated before visible clot formation. Plasma from patients presenting with acute myocardial infarction showed increases in the plasma concentration of fibrin polymer and in the proportion of total fibrinogen present as polymer, as determined by a quantitative adaptation of the electrophoretic technique. The plasma concentration in patients with subendocardial or transmural myocardial infarction showed significant (p less than .005) increases to 4.0 +/- 1.0% and 3.6 +/- .8%, respectively, as compared with the concentration in normal plasma (0.8 +/- 0.1%). There was no difference in plasma concentration in samples from patients with transmural compared with those with subendocardial myocardial infarction. This study provides the first demonstration of factor XIIIa cross-linked fibrin polymers in thrombotic disease and indicates the presence of increased activity of both thrombin and factor XIIIa in patients with acute myocardial infarction.

Specific identification of urinary fibrinogen, fibrinogen degradation products, and cross-linked fibrin degradation products in renal diseases and after renal allotransplantation.

We have applied a sensitive and discriminating electrophoretic technique that distinguishes between fibrinogen, fibrin polymers, fibrinogen degradation products (FDP), and cross-linked fibrin degradation products (XLDP) to evaluate urinary fibrinogen antigen in control subjects and in patients with a variety of renal diseases and after renal allograft transplantation. Although only one of 11 controls showed the trace presence of urinary fibrinogen, 14 of 28 patients with renal disease had urinary fibrinogen antigen, mostly as fibrinogen or fibrin monomer. Thrombin treatment failed to remove fibrinogen from urine, indicating that methods using this step to eliminate clottable protein will overestimate the quantity of urinary fibrinogen and fibrin degradation products. No association was found between the amount or type of antigen and the specific clinical diagnosis or the presence of proteinuria or hematuria, although urinary FDP and XLDP were found only with greater degrees of renal impairment. Fifteen patients were evaluated for 3 weeks after renal transplantation surgery by serial urine and serum electrophoretic analysis. Urinary FDP and XLDP were found significantly more often in the first week after surgery and in association with episodes of acute renal transplant rejection (ARTR) than at other times. This suggests that fibrin deposition and degradation is involved in the pathologic process of ARTR and that identification of specific XLDP and FDP could have diagnostic and prognostic application in such patients.

Thiorphan and analogs: lack of correlation between potency to inhibit "enkephalinase A" in vitro and analgesic potency in vivo.

The potencies of the R and S isomers of thiorphan and rigid analogs of thiorphan to produce analgesia in a mouse hot-plate assay have been compared with their potencies to inhibit enkephalin degradation by rat brain "enkephalinase A." The R and S isomers of thiorphan were equipotent as enzyme inhibitors (IC50 approximately 10(-9) M) but had significantly different analgesic profiles when injected i.c.v. Rigid analogs of thiorphan were less potent enzyme inhibitors (IC50 values of 10(-7) - 10(-4) M) but produced analgesia and potentiated Tyr-D-Ala-Gly-Phe-Met-NH2 induced analgesia at doses (i.c.v.) comparable to thiorphan. These observations suggest that inhibitors of enkephalinase A produce analgesia through a pharmacological mechanism which is not directly related to inhibition of enkephalin degradation.

Enantiomers of [R,S]-thiorphan: dissociation of analgesia from enkephalinase A inhibition.

The [R] and [S] enantiomers of the enkephalinase A inhibitor [R,S]-thiorphan have been prepared by asymmetric synthesis. The [S] isomer is principally responsible for the angiotensin converting enzyme inhibitory activity of [R,S]-thiorphan, whereas there were only small differences in the ability of the [R] and [S] isomers to inhibit enkephalinase both in vivo and in vitro. In contrast, the in vivo analgesic activity of [R,S]-thiorphan resided principally in the [R] isomer. These data indicate a surprising dissociation of enkephalinase inhibition from analgesic activity. The fact that the two enantiomers of [R,S]-thiorphan were effective inhibitors of enkephalinase, yet the [R] isomer had substantially greater analgesic activity, indicates that factors other than enkephalinase inhibition may be important for [R, S]-thiorphan's analgesic properties.

Radiologic technologist licensure. An assessment of economic considerations.

This report presents criteria by which states and professions can evaluate certain economic aspects of the credentialing of diagnostic radiologic technologists. Based on the criteria presented, the literature reviewed, and data obtained from state credentialing programs, certain outcomes are suggested. The criteria include shortages in services; availability of supplies, materials, and energy; teaching opportunities; and the effect on competition.