Philip Marder

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells.

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.

Blockade of human neutrophil activation by 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid (LY293111), a novel leukotriene B4 receptor antagonist

Leukotriene B4 (LTB4), a naturally occurring pro-inflammatory product of arachidonic acid metabolism, has been associated with human inflammatory disease. This study compares the abilities of two LTB4 receptor antagonists, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]- propoxy]phenoxy]benzoic acid (LY293111) and 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), to displace LTB4 binding and their functional blockade of human neutrophil activation. LY293111 inhibited the binding of [3H]LTB4 with a Ki of 25 nM; SC-41930 displayed a similar potency (Ki = 17 nM). In contrast, LY293111 prevented LTB4-induced calcium mobilization with an IC50 = 20 nM, or 40 times more effectively than SC-41930 (IC50 = 808 nM). LY293111 was 300 times more potent than SC-41930 in blocking LTB4-induced CD11b up-regulation on isolated neutrophils. LY293111 also arrested LTB4-induced up-regulation of CD11b on neutrophils in whole human blood. LY293111 was not effective in blocking human neutrophil activation responses induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), platelet-activating factor (PAF), human recombinant endothelial interleukin-8 (IL-8) or human recombinant complement component 5a (C5a).

Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-β Inhibitor

Purpose: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. Experimental Design: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. Results: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. Conclusion: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy

Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors. [Mol Cancer Ther 2007;6(9):2600–7]

Identification of functional subpopulations of murine natural killer cells based on their cell surface asialo GM1 phenotype

HSV-1 infection renders a mouse fibroblast cell line (MCN) sensitive to murine splenic NK killing which is independent of interferon (IFN) induction during the assay. This NK (HSV-1) activity is distinctive from conventional NK (YAC-1) in that they cannot be aborted by anti-asialo GM1 (anti-ASGM1) antibody plus complement treatment as NK (YAC-1) does. Further characterization of these two subpopulations was carried out by fluorescence-activated cell sorting (FACS) technique based on their cell surface asialo GM1 (ASGM1) phenotype. While almost all NK (YAC-1) activity resides within FACS-positive population, both ASGM1 positive and negative cell populations can kill the virally infected MCN equally well. One interesting observation is that only the ASGM1 positive cells respond significantly to IL-2 NK boosting. Five different mouse strains (CD-1, C57BL/6J, C57BL/6J-BG, SM/J, and SJL) were compared on their FACS profile with anti-ASGM1 antibody as well as their NK function. The differences observed are discussed.

Comparative analysis of monoclonal antibody-drug conjugate binding by flow cytometry.

Flow cytometric methods for the evaluation of the cell surface binding properties of monoclonal antibody (MoAb)-drug/toxin conjugates defining tumor-associated antigens are presented. In these techniques, suspension cultures of solid human tumor cell lines are incubated with either varying dilutions of MoAb or MoAb-drug conjugates followed by FITC-conjugated anti-mouse immunoglobulin antibodies in an indirect assay or with FITC-conjugated MoAbs specific for the tumor target cell line in a competition assay. The amount of fluorescent probe bound is measured by flow cytometry and the mean fluorescence intensity determined. The relative binding capacity is quantified by linear regression of the mean fluorescence versus the concentration of primary antibody or antibody conjugate. The application of these techniques to several drug and toxin conjugates of MoAb KS1/4, which defines a human adenocarcinoma-associated antigen, demonstrates that these assays can be effectively utilized to monitor the effects of covalent chemical modification on a MoAbs antigen binding reactivity.

Potentiation of Leu 11 + natural cytotoxicity by human interleukin 2.

Human interleukin 2 was examined for its effect on potentiation of human natural cell-mediated cytotoxicity. Cells were isolated into Leu 11a + or Leu 11a- on a cell sorter. The data suggest that interleukin 2 potentiates cytotoxicity of the Leu 11a + population.

-alkoxyphenol leukotriene B4 receptor antagonists: effect of a chroman carboxylic acid.

Abstract Several ortho -alkoxyphenols containing a chroman carboxylic acid sidechain have been prepared as antagonists of leukotriene B4 receptors. These antagonists were compared to their parent alkoxyphenols containing the tetrazole acid sidechain. These chroman containing antagonists retained their binding potency for human neutrophil receptors; however, showed enhanced potency against guinea pig receptors in both in vitro and in vivo systems.

Structural analogues of LY292728, a highly potent xanthone dicarboxylic acid leukotriene B4 receptor antagonist: spatial positioning of the secondary acid group

Abstract We report the preparation and pharmacologic activity of three spatial analogues of LY292728, a highly potent xanthone dicarboxylic LTB4 receptor antagonist. Molecular modeling of these compounds has helped to further elucidate the nature of the secondary acid binding site of the LTB4 receptor.

Murine Thymocytes Mediate a Natural Killer‐Like Activity against Herpes Virus‐Infected Target Cells but Not YAC‐1 Target Cells

In this report, we demonstrated a natural killer (NK)‐like activity against HSV‐1 infected ceils mediated by CD‐1 mouse thymocytes. This cytolytic activity is specific for HSV‐1‐infected MCN cells, since both uninfected MCN and Y AC‐1 target cells are not susceptible to thymocyte lysis. Antibody plus complement depletion experiments indicate that a portion of the activity is associated with the Lyt 2 /L3T4− thymocyte subpopulation. This NK‐like activity cannot be enhanced by addition of interleukin 2 in vitro.