Antitumor activity of ipatasertib combined with chemotherapy: results from a phase Ib study in solid tumors.

BACKGROUND: This phase Ib study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of the oral AKT inhibitor ipatasertib and chemotherapy or hormonal therapy in patients with advanced or metastatic solid tumors to determine combined dose-limiting toxicities (DLTs), maximum tolerated dose, and recommended phase II doses and schedules. PATIENTS AND METHODS: The clinical study comprised four combination treatment arms: arm A (with docetaxel), arm B [with mFOLFOX6 (modified leucovorin, 5-fluorouracil, and oxaliplatin)], arm C (with paclitaxel), and arm D (with enzalutamide). Primary endpoints were safety and tolerability; secondary endpoints were pharmacokinetics, clinical activity per Response Evaluation Criteria in Solid Tumors v1.1, and prostate-specific antigen levels. RESULTS: In total, 122 patients were enrolled. Common adverse events were diarrhea, nausea, vomiting, decreased appetite, and fatigue. The safety profiles of the combination regimens were consistent with those of the background regimens, except for diarrhea, hyperglycemia, and rash, which were previously observed with ipatasertib treatment. The only combination DLT across all treatment arms was one event of grade 3 dehydration (ipatasertib 600 mg and paclitaxel). Recommended phase II doses for ipatasertib were 600 mg (and mFOLFOX6) and 400 mg (and paclitaxel), respectively. The maximum assessed dose of ipatasertib 600 mg combined with docetaxel or enzalutamide was well tolerated. Coadministration with enzalutamide (a cytochrome P450 3A inducer) resulted in approximately 50% lower ipatasertib exposure. CONCLUSIONS: Ipatasertib in combination with chemotherapy or hormonal therapy was well tolerated with a safety profile consistent with that of ATP-competitive AKT inhibitors. CLINICAL TRIAL NUMBER: NCT01362374.

Sphingosine inhibits monocyte tissue factor-initiated coagulation by altering factor VII binding.

Tissue factor is a lipoprotein, expressed on the surface of cells, which binds coagulation Factor VII or VIIa, leading to activation of Factors X and IX with subsequent fibrin generation. Cellular tissue factor activity is important in pathophysiologic processes such as inflammation and disseminated intravascular coagulation. In this study, the long-chain base sphingosine inhibited coagulation initiated by lipopolysaccharide-stimulated intact human monocytes. Sphingosine (5-100 microM) also profoundly inhibited thromboplastin-initiated coagulation (greater than 90% decrease in thromboplastin activity). This inhibition was dose- and time-dependent. Sphingosine inhibited neither the intrinsic pathway of coagulation nor thrombin generation of fibrin. The sphingosine analogues sphingomyelin, ceramide, or N-acetylsphingosine did not affect thromboplastin activity, suggesting that the polar head of sphingosine was necessary for interaction of the molecule with the coagulation system. Investigation of the biochemical mechanism revealed that sphingosine (5-50 microM), but neither sphingomyelin nor ceramide, inhibited specific binding of radiolabeled Factor VII to lipopolysaccharide-stimulated intact monocytes. The results suggest that sphingosine may regulate monocyte tissue factor-initiated coagulation by modulating Factor VII binding to tissue factor. Sphingosine may represent a new class of inhibitors of hemostasis.

Human mononuclear phagocyte transglutaminase activity cross-links fibrin.

The physiologic function of the monocyte transglutaminases is not known. In this study, we detected Factor XIII A-subunit antigen and "tissue" transglutaminase antigen in human monocytes by polyacrylamide gel electrophoresis and immunoblotting techniques. Flow cytometric analysis demonstrated that 27% and 49% of the total Factor XIII antigen in monocytes and human peritoneal macrophages, respectively, are expressed on the surface of the cells. Monocytes maintained in culture for 8 days had a 4-fold increase in Factor XIIIa activity and a 3.2-fold increase in the amount of Factor XIII antigen/mg cell protein. However, there was no increase in the "tissue" transglutaminase activity or antigen levels in cultured monocytes. In addition, we identified a Factor XIII deficient individual who does not express Factor XIII activity or antigen in plasma, platelets, monocytes, lymphocytes or erythrocytes. Intact monocytes from normal donors were able to cross-link fibrin formed in the plasma from the Factor XIII deficient individual. This suggests that transglutaminase activity expressed by peripheral blood monocytes may play a physiologic role in cross-linking fibrin during blood clotting or inflammation.

Clinical trials with human tumor necrosis factor: in vivo and in vitro effects on human mononuclear phagocyte function.

The purpose of this investigation was to understand the biological effects of recombinant human tumor necrosis factor used as therapy for cancer. We studied changes in mononuclear phagocyte function following exposure to this cytokine in vitro or in vivo. Tumor necrosis factor increased phorbol myristate acetate-induced hydrogen peroxide production 8- to 20-fold in peripheral blood monocytes and peritoneal macrophages in vitro in a dose-dependent manner. Similarly, tumor necrosis factor increased phorbol myristate acetate-induced peroxide production 2.3-fold in monocytes isolated from nine patients following an i.v. infusion of this cytokine (40 to 200 micrograms/m2). In addition, tumor necrosis factor induced a 2.3-fold increase in tissue factor-like activity in mononuclear phagocytes in vitro. In vivo, tumor necrosis factor induced a trend toward higher procoagulant activity in monocytes, although this change was not statistically significant. We also noted a trend toward increased activated partial thromboplastin times and the presence of fibrin D-dimer in patients treated with tumor necrosis factor, demonstrating activation of the coagulation and fibrinolytic systems. Thus, in vivo treatment of humans with i.v. recombinant human tumor necrosis factor induced functional changes in mononuclear phagocytes similar to those noted with in vitro treatment.

Tumor necrosis factor induces tissue factor-like activity in human leukemia cell line U937 and peripheral blood monocytes.

The induction of procoagulant activity (PCA) by human recombinant tumor necrosis factor (rTNF) was studied in human monoblastic leukemia cell line U937 and human peripheral blood monocytes. Using a one-step recalcificating clotting assay, PCA in cell lysates or whole cell preparations was measured by comparison to a rabbit brain thromboplastin standard. There was a dose- and time-dependent increase in PCA when U937 cells were cultured with rTNF. The effect of rTNF was not enhanced by recombinant human interferon-gamma (rIFN gamma). Cycloheximide inhibited the expression of PCA by U937 cells, showing that protein synthesis was necessary to mediate the effects of rTNF. Whole cell preparations demonstrated that greater than 80% of the PCA was expressed on the surface of the cells. The PCA functioned as a tissue factor-like substance, since it required coagulation factor VII and factor X. rTNF also increased PCA in human monocytes in a dose- and time-dependent manner. This effect was abrogated by boiling the rTNF for ten minutes, and was not inhibited by adding polymyxin-B to the cultures, making it unlikely that endotoxin accounted for the observed effects. These results suggest that TNF-induced expression of tissue factor by mononuclear phagocytes may modulate immunologic, inflammatory, and hemostatic processes.

Identification of normal human peripheral blood monocytes and liver as sites of synthesis of coagulation factor XIII a-chain.

Factor XIII is the fibrin-stabilizing factor that covalently cross-links fibrin monomers to form a highly organized, stable fibrin clot. The plasma form of factor XIII is a heterodimer, a2b2, consisting of two a-chains and two b-chains; the intracellular form, such as in platelets and placenta, is a dimer, a2, consisting of a-chains only. The catalytic function of factor XIII, a transglutaminase, resides in the a-chain. To address questions regarding sites of synthesis of factor XIII a-chain, an EcoRI restriction fragment from the protein-coding region of the factor XIII a-chain cDNA was used as a probe for Northern blot analysis. The cDNA probe showed hybridization with a single approximately 4.0-kilobase (kb) message in poly (A)+ mRNA prepared from normal human peripheral blood monocytes and normal human liver. The results demonstrate conclusively that factor XIII a-chains are actively synthesized in circulating monocytes and in liver. To our knowledge, these data represent the first demonstration of synthesis of any blood coagulation factor in primary uncultured and unstimulated monocytes or macrophage cells.

Activation of monocyte and granulocyte antibody-dependent cytotoxicity by phorbol myristate acetate.

We have characterized the effects of phorbol myristate acetate (PMA) on human monocyte and neutrophil oxidative metabolism and antibody-dependent cytotoxicity toward anti-D sensitized human erythrocytes (RBC) and a human lymphoblastoid cell line (CEM). Hexose monophosphate shunt activity was measured by [1-(14)C]glucose oxidation and target lysis by (51)Cr release. PMA produced a dose-dependent stimulation of hexose monophosphate shunt activity. Neutrophils responded with higher hexose monophosphate shunt activity and at a lower PMA concentration than did monocytes. PMA increased monocyte lysis of antibody-sensitized RBC by two-thirds, but did not affect lysis of CEM targets. Neutrophils were unable to lyse either antibody-sensitized or nonsensitized RBC without the addition of PMA. When PMA was added, lysis of both targets increased markedly. Neutrophils without PMA were able to lyse a small number of both antibody-sensitized and nonsensitized CEM targets. PMA also increased neutrophil lysis of these targets. Target lysis by neutrophils from a patient with chronic granulomatous disease, cells unable to produce reactive oxygen species, was not increased by PMA. Chronic granulomatous disease monocytes, however, responded to PMA by more than doubling lysis of antibody-sensitized RBC. Hypoxia inhibited PMA augmentation of antibody-sensitized RBC lysis by neutrophils, but not by monocytes. Generation of reactive oxygen species by the xanthine-xanthine oxidase system inhibited CEM growth, but did not cause lysis, indicating that in some cases oxidative injury may be nonlytic. We suggest that PMA augments neutrophil cytotoxicity to tumor and RBC targets by stimulating reactive oxygen species-mediated lysis, but in monocytes augmentation of lysis is due to activation of a nonoxidative mechanism of lysis.