Christine Eberhardt

Human Lysophosphatidic Acid Acyltransferase cDNA CLONING, EXPRESSION, AND LOCALIZATION TO CHROMOSOME 9q34.3

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate (LPA)) is a phospholipid with diverse biological activities. The mediator serves as an intermediate in membrane phospholipid metabolism but is also produced in acute settings by activated platelets. LPA is converted to phosphatidic acid, itself a lipid mediator, by an LPA acyltransferase (LPAAT). A human expressed sequence tag was identified by homology with a coconut LPAAT and used to isolate a full-length human cDNA from a heart muscle library. The predicted amino acid sequence bears 33% identity with a Caenorhabditis elegans LPAAT homologue and 23–28% identity with plant and prokaryotic LPAATs. Recombinant protein produced in COS 7 cells exhibited LPAAT activity with a preference for LPA as the acceptor phosphoglycerol and arachidonyl coenzyme A as the acyl donor. Northern blotting demonstrated that the mRNA is expressed in most human tissues including a panel of brain subregions; expression is highest in liver and pancreas and lowest in placenta. The human LPAAT gene is contained on six exons that map to chromosome 9, region q34.3.

Platelet-activating factor acetylhydrolase

The present invention provides purified and isolated polynucleotide sequences encoding human plasma platelet-activating factor acetylhydrolase. Also provided are materials and methods for the recombinant production of platelet-activating factor acetylhydrolase products which are expected to be useful in regulating pathological inflammatory events.

cDNA Cloning, Expression and Chromosomal Localization of Two Human Lysophosphatidic Acid Acyltransferases

In this report we describe a pair of human LPAAT isozymes. These isozymes are encoded by distinct genes located on different chromosomes, but share sequence homology, substrate specificity, and intracellular location. The biological value of maintaining the two closely related LPAAT genes in the human genome is not clear. We find that both isozymes are widely expressed, although expression levels do diverge significantly in tissues such as the liver, placenta, testes, and pancreas. We also find that, at least in the artificial system of over-expression in COS7 cells, both isozymes localize to the ER membrane. Thus, distinct tissue-specific or subcellular compartment-specific roles for the two isozymes are not supported by the current experimental evidence. It does remain possible that induction of expression or subcellular translocation of one or the other isozyme may distinguish their functions. A survey of a limited number of acyl CoA substrates indicates that the two isozymes display similar substrate specificities, although slight differences are suggested by the data. However, extensive analysis of both isozymes with multiple substrates in the same assay system will be required to detect physiologically relevant differences in substrate specificity. LPA and PA are central intermediates in phospholipid biogenesis. Furthermore, they have the capacity to mediate signaling both between and within cells. The importance of these mediators is reflected in the growing body of literature dedicated to unraveling the mechanistic basis for their actions. Until recently, the field has been hampered by a dearth of reagents appropriate for the molecular dissection of the LPA and PA metabolic and signaling pathways in eukaryotes. However, the recent cloning of possible LPA receptors will promote further understanding of LPA signaling. Similarly, the recent appearance of LPAAT homologs in the EST database has prompted a flurry of reports describing their characterization. These clones will afford opportunity for defining the function of LPAAT in eukaryotic phospholipid metabolism.

Abstract A252: A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers.

Introduction: Within the tumor microenvironment, CSF1R signaling is thought to play an important role in recruitment and differentiation of tumor-associated macrophages and osteoclasts, promoting disease progression through suppression of anti-tumor immune response, promotion of angiogenesis, tumor cell metastasis and tumor-induced osteolysis. ARRY-382 is a potent, highly selective, oral inhibitor of CSF1R. This Phase 1 dose-escalation study was designed to determine the maximum tolerated dose (MTD) and to assess the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of ARRY-382 in patients (pts) with advanced or metastatic cancers refractory to standard treatment. Methods: Pts received ARRY-382 orally once daily (QD) in 28-day cycles. Study design included an initial accelerated titration phase, followed by 3+3 dose escalation and an expansion phase. Safety was assessed by adverse events (AEs), ECGs, clinical laboratory tests, physical exams and vital signs. PK parameters were estimated based on serial plasma PK samples. Levels of pERK in pt monocytes stimulated with CSF1 ex vivo, circulating CD14dim/CD16+ nonclassical monocytes (NCM) and CSF1 were evaluated as markers of CSF1R inhibition. Urinary collagen type 1 cross-linked N[[Unable to Display Character: ‑]]telopeptide (NTX) was evaluated as a marker of bone turnover. Results: Twenty-six pts received ARRY-382 at doses ranging from 25 to 500 mg QD. The MTD of ARRY-382 was 400 mg QD. Dose-limiting toxicities were increased creatine kinase (CK), increased AST and pyrexia (1 pt each, all Grade 3). The most common drug-related AEs were fatigue (42%), increased CK (27%), nausea (23%), decreased appetite (15%) and vomiting (12%). Grade 3/4 AEs in >1 pt were increased CK (23%), pneumonia (15%), anemia (8%) and vomiting (8%). Increases in ARRY-382 exposure were approximately dose proportional. At the MTD, a Cmax of 3.06 µg/mL was achieved with a Ctrough > 1 µg/mL after repeated dosing. Doses ≥ 200 mg QD resulted in > 80% mean reduction from Baseline in monocyte pERK, consistent with exposures above the ARRY-382 IC50 value. The PD activity of ARRY-382 was consistent with CSF1R inhibition. Reductions in NCM were observed at doses ≥ 200 mg QD, with a 96% mean decrease from Baseline observed at the MTD. Reductions in urinary NTX were observed at doses ≥ 50 mg QD. Five of 7 pts with elevated urinary NTX at Baseline experienced reductions to within the normal range in the first cycle, including 3 pts with bone metastases. Increases in CSF1 appeared dose-dependent with ∼28-fold maximal increase from Baseline observed at the MTD in the first cycle. No objective responses were observed, although 4 pts (15%) experienced stable disease which lasted > 3 months for 2 pts. Conclusions: ARRY-382 was generally well tolerated in pts with advanced or metastatic cancers. Exposure and PD activity of ARRY[[Unable to Display Character: ‑]]382 indicate that CSF1R was significantly inhibited at tolerated doses. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A252. Citation Format: Johanna C. Bendell, Anthony W. Tolcher, Suzanne F. Jones, Muralidhar Beeram, Jeffrey R. Infante, Paul Larsen, Kevin Rasor, Jennifer E. Garrus, Jinfang Li, P. Louann Cable, Christine Eberhardt, Jennifer Schreiber, Selena Rush, Kenneth W. Wood, Emma Barrett, Amita Patnaik. A phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A252.