Phase I study of emactuzumab single agent or in combination with paclitaxel in patients with advanced/metastatic solid tumors reveals depletion of immunosuppressive M2-like macrophages.

BACKGROUND: Emactuzumab is a monoclonal antibody against the colony-stimulating factor-1 receptor and targets tumor-associated macrophages (TAMs). This study assessed the safety, clinical activity, pharmacokinetics (PK) and pharmacodynamics (PD) of emactuzumab, as monotherapy and in combination with paclitaxel, in patients with advanced solid tumors. PATIENTS AND METHODS: This open-label, phase Ia/b study comprised two parts (dose escalation and dose expansion), each containing two arms (emactuzumab, every 2 or 3 weeks, as monotherapy or in combination with paclitaxel 80 mg/m2 weekly). The dose-escalation part explored the maximum tolerated dose and optimal biological dose (OBD). The dose-expansion part extended the safety assessment and investigated the objective response rate. A PK/PD analysis of serial blood, skin and tumor biopsies was used to explore proof of mechanism and confirm the OBD. RESULTS: No maximum tolerated dose was reached in either study arm, and the safety profile of emactuzumab alone and in combination does not appear to preclude its use. No patients receiving emactuzumab monotherapy showed an objective response; the objective response rate for emactuzumab in combination with paclitaxel was 7% across all doses. Skin macrophages rather than peripheral blood monocytes or circulating colony-stimulating factor-1 were identified as an optimal surrogate PD marker to select the OBD. Emactuzumab treatment alone and in combination with paclitaxel resulted in a plateau of immunosuppressive TAM reduction at the OBD of 1000 mg administered every 2 weeks. CONCLUSIONS: Emactuzumab showed specific reduction of immunosuppressive TAMs at the OBD in both treatment arms but did not result in clinically relevant antitumor activity alone or in combination with paclitaxel. (ClinicalTrials.gov Identifier: NCT01494688).

Identification of early proteomic markers for hepatic steatosis.

The identification of biomarkers for disease state, drug efficacy, and toxicity is becoming increasingly important for drug discovery and development. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify proteomic markers associated with hepatocellular steatosis in rats after dosing with a compound (CDA) in preclinical development. Rats were dosed daily for up to 5 days with CDA for measurement of blood biochemical parameters, histological, and proteomic analysis. Alterations in plasma glucose and liver transaminases were detected from dosing day 3 onward, and livers showed trace levels of hepatocellular vacuolation from 6 h which increased in extent and severity over the 5 day time course. The number of significantly altered protein spots increased over the 5 day time course, and Ingenuity Pathway Analysis showed that the predominant functions altered by CDA treatment were cell death and cellular assembly and organization. This included alterations in secreted proteins, endoplasmic reticulum and mitochondrial chaperones, antioxidant proteins, and enzymes involved in fatty acid biosynthesis. Comparative in vitro dosing studies showed similar alterations to the proteome, neutral lipid accumulation, and mitochondrial dehydrogenase activity in response to CDA treatment of cultured rat hepatocytes. The finding that several proteins showed significant changes in abundance before the onset of overt toxicity in vivo suggested that these could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis. These markers underwent further direct analysis in the in vitro hepatocyte toxicity model to determine their utility in the development of high throughput assays for drug-induced steatosis.

CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes.

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform enzyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)-treated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the inclusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism b both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of metabolism of all three substrates was 72 h, with no medium changes being necessary during this period. 5. The effect of treatment with 0.5-20 microM BNF, 50-2000 microM NaPB, 2-20 microM dexamethasone (DEX), 20-100 microM methylclofenapate (MCP), and 50 and 200 microM isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocytes was studied. BFC metabolism was induced by treatment with BNF, NaPB and MCP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat treated with CYP isoform inducers was also studied. BFC metabolism was induced by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C and CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 microM the greatest rates of BFC metabolism were observed with the CYP1A1, CYP1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for assessing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96-well plate format.

Rapid determination of rat hepatocyte mRNA induction potential using oligonucleotide probes for CYP1A1, 1A2, 3A and 4A1.

1. A new assay to quantify mRNA levels in small numbers of rat hepatocytes has been developed for cytochrome P450 (CYP) isoforms 1A1, 1A2, 3A and 4A1. The assay uses sets of oligonucleotide probes end-labelled with [35S]-dATP to hybridize to mRNA in control- or drug-treated rat hepatocytes cultured on Cytostar-T 96-well scintillating microplates. 2. The rat hepatocyte induction potential (RHIP) assays for CYP3A, 1A1, 1A2 and 4A1 are sensitive and selective and have an excellent qualitative relationship with CYP induction data ex vivo. The robustness of the CYP3A assay was determined following a run of > 40 plates. The variation of the dexamethasone (DEX) response on each plate, calculated as %coefficient of variation, showed that there was no significant difference between the variability of the response to DEX. 3. Assay specificity for each CYP isoform was achieved by designing probes (four per isoform) antisense to coding regions of each CYP gene sequence. In the CYP3A RHIP assay, pregnenalone 16alpha-carbonitrile (PCN), DEX, clotrimazole (CLOT) and miconazole (MIC) were all good inducers of CYP3A mRNA; beta-napthoflavone (BNF) and methylclofenapate (MCP), however, did not induce CYP3A mRNA, further defining the specificity of this methodology. Specificity was similarly confirmed for the other CYP isoforms. 4. Ind50, the concentration of inducer required to elicit a 50% induction of CYP-specific mRNA, was derived for prototypical CYP inducers: BNF 0.54 and 0.17 microM (CYP1A1 and 1A2 respectively), 3-methylcholanthrene (3MC) 0.11 and 0.04 microM (CYP1A1 and 1A2 respectively), PCN 0.03 microM, DEX 0.17 microM, CLOT 0.48 microM, MIC 3 microM, TAO 3 microM (CYP3A), MCP 1.8 microM, clofibrate (CLOF) 65 microM and ciprofibrate (CIP) 1.9 microM (CYP4A1). Ind50 for BNF and 3MC at CYP1A2 was 3-fold lower than that at CYP1A1 indicating a subfamily difference in inducer potency. 5. Reducing the numbers of animals and the amount of compound required to study CYP induction is an important advantage of the RHIP assays over conventional evaluations in vivo. Typically four rats are dosed for 4 days using oral doses in the range 50-500 mg kg(-1) day(-1). In comparison, the amount of hepatocytes required to carry out all the studies reported herein may be obtained from a single animal (< 2 x 10(8) viable cells) and CYP induction investigated using microg rather than g quantities of drug substance. 6. With appropriately designed oligonucleotide probes, the RHIP technology can assess CYP induction in human hepatocytes, which together with preclinical data can contribute to improving the quality of compounds progressing into the expensive process of drug development.

High throughput ribonuclease protection assay for the determination of CYP3A mRNA induction in cultured rat hepatocytes.

1. A rapid 96-well plate based method for the determination of CYP3A mRNA induction in primary rat hepatocytes has been developed which has substantial advantages over current technologies including the ability to test the effect of relatively large numbers of new chemical entities on the expression of CYP3A mRNA in hepatocytes. 2. The ribonuclease protection assay detects changes in mRNA levels in small numbers of hepatocytes by the utilization of a radiolabelled antisense riboprobe that will hybridize CYP3A1 and CYP3A23. Using in situ hybridization techniques in conjunction with Amersham 96-well Cytostar-T scintillating microplates, there is no need for isolation of mRNA. A simple ribonuclease digestion step allows quantitative data to be generated easily within 1 week of hepatocyte isolation. 3. Rat hepatocytes were cultured for 48 h post-isolation on the Cytostar plates coated with a basal matrix of Matrigel. Prototypical CYP3A inducers (dexamethasone and pregnenolone 16alpha-carbonitrile) have been studied using various treatment periods from 0.5 to 24 h. Methylclofenapate and beta-naphthoflavone, prototypical inducers of CYP4A and CYP1A respectively, have been used as controls to show specificity of the [33P]-labelled riboprobe for the CYP3A family. 4. Time-dependent increases in CYP3A mRNA were demonstrated following exposure of hepatocytes to prototypical CYP3A inducers, but not for methylclofenapate or beta-naphthoflavone, so demonstrating specificity for CYP3A mRNA over CYP1A and CYP4A. Analysis of the 24-h induction data demonstrates that significant differences from controls can be determined and that induction potential can be assessed. The system has the potential to screen for overall CYP3A mRNA induction in response to compounds at an early stage in drug research.